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Computational Drug Discovery and Design 1st Edition
Anthony Ivetac Digital Instant Download
Author(s): Anthony Ivetac, J. Andrew McCammon (auth.), Riccardo Baron
(eds.)
ISBN(s): 9781617794643, 1617794643
Edition: 1
File Details: PDF, 11.29 MB
Year: 2012
Language: english
METHODS MOLECULAR BIOLOGY
TM
IN

Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes:

http://www.springer.com/series/7651
.
Computational Drug Discovery
and Design
Edited by

Riccardo Baron
Department of Medicinal Chemistry, College of Pharmacy, The Henry Eyring Center
for Theoretical Chemistry, University of Utah, Salt Lake City, UT, USA
Editor
Riccardo Baron
Department of Medicinal Chemistry
College of Pharmacy
The Henry Eyring Center for Theoretical Chemistry
University of Utah
Salt Lake City, UT, USA
[email protected]

ISSN 1064-3745 e-ISSN 1940-6029

ISBN 978-1-61779-464-3 e-ISBN 978-1-61779-465-0
DOI 10.1007/978-1-61779-465-0
Springer New York Dordrecht Heidelberg London

Library of Congress Control Number: 2011942922

# Springer Science+Business Media, LLC 2012

All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the
publisher (Humana Press, c/o Springer ScienceþBusiness Media, LLC, 233 Spring Street, New York, NY 10013,
USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of
information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology
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The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified
as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights.

Printed on acid-free paper

Humana Press is part of Springer Science+Business Media (www.springer.com)

Preface

Assisted by the rapid and steady growth of available low-cost computer power, the use of
computers for discovering and designing new drugs is becoming a central topic in modern
molecular biology and medicinal chemistry. New effective methods provide access to an
always-increasing level of complexity in biomolecular recognition, thus expanding the
variety and the predictive power of approaches for drug development based on computa-
tional chemistry (Fig. 1).

Fig. 1. From medicinal alchemy to modern medicinal chemistry. Left: the Liber de Arte Distillandi de Compositis by
Hieronymus Brunschwig described emerging methods to extract drugs through alchemical distillation (Johann Gr€uninger
Publisher & Printer, 1512 circa; courtesy of the National Academy of Medicine, U.S.A.). Right: five hundred years later the
same long-standing problem is attacked by in silico distillation of large compound databases. Computers help
experiments along all phases of the extraction funnel: from preliminary molecule screening, trough drug discovery
and refinement, to inhibitor design based on statistical mechanics

In this volume of Methods in Molecular Biology we present robust methods for Compu-
tational Drug Discovery and Design, with a particular emphasis on method development
for biomedical applications. The goal is to offer an overview of highly promising themes
and tools in this highly interdisciplinary research field, together with the challenges calling
for new solutions in future research: from binding sites prediction to the accurate inclusion
of solvent and entropic effects, from high-throughput screening of large compound
databases to the expanding area of protein–protein inhibition, toward quantitative free-
energy approaches in ensemble-based drug design using distributed computing.
The application of physics-based methodologies—strongly coupled to molecular dynamics
simulation—is leading to a novel, dynamic view of receptor-drug recognition.
These concepts are progressively modifying the old dogma of single-structure-based
drug design into the concept of ensemble-based drug design, where conformational
diversity and selection play key roles. In this scenario, the current scientific literature is

v
vi Preface

often highlighting success stories and happy-end examples. However, the basis of this
success is often the back-stage, everyday research filled with ingenious and creative strate-
gies to bypass critical obstacles. Thus, this volume has the goal of presenting as well such
obstacles and practical guidance for the use of computational resources for researchers new
to these topics. Finally, this volume includes recent, successful examples of applications in
the description of receptor-drug interactions and computer-based discovery of new drugs
against human-lethal diseases, opening to future computer-based drug patents.
The reader will hopefully use this volume as an introductory manual for state-of-the-art
concepts and methodologies, as well as an advanced, specialized tool to design novel and
original research for public health.

Salt Lake City, UT, USA Riccardo Baron

Acknowledgments

I would like to warmly thank Andy McCammon for his support during the whole course of
this work; Sara Nichols for preparing the cover illustration; Nadeem Vellore for a critical
reading of the final proofs. Startup funding from The University of Utah is also greatly
acknowledged for the last part of their publication process.

vii
Contents

Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

PART I DRUG BINDING SITE PREDICTION, DESIGN, AND DESCRIPTORS

1 A Molecular Dynamics Ensemble-Based Approach for the Mapping
of Druggable Binding Sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Anthony Ivetac and J. Andrew McCammon
2 Analysis of Protein Binding Sites by Computational Solvent Mapping . . . . . . . . . . . 13
David R. Hall, Dima Kozakov, and Sandor Vajda
3 Evolutionary Trace for Prediction and Redesign of Protein Functional Sites . . . . . . 29
Angela Wilkins, Serkan Erdin, Rhonald Lua, and Olivier Lichtarge
4 Information Entropic Functions for Molecular Descriptor Profiling . . . . . . . . . . . . . 43
Anne Mai Wassermann, Britta Nisius, Martin Vogt,
and J€
u rgen Bajorath

PART II VIRTUAL SCREENING OF LARGE COMPOUND LIBRARIES:

INCLUDING MOLECULAR FLEXIBILITY
5 Expanding the Conformational Selection Paradigm
in Protein-Ligand Docking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Guray Kuzu, Ozlem Keskin, Attila Gursoy, and Ruth Nussinov
6 Flexibility Analysis of Biomacromolecules with Application
to Computer-Aided Drug Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Simone Fulle and Holger Gohlke
7 On the Use of Molecular Dynamics Receptor Conformations
for Virtual Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Sara E. Nichols, Riccardo Baron, and J. Andrew McCammon
8 Virtual Ligand Screening Against Comparative Protein Structure Models . . . . . . . . 105
Hao Fan, John J. Irwin, and Andrej Sali
9 AMMOS Software: Method and Application. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
Tania Pencheva, David Lagorce, Ilza Pajeva, Bruno O. Villoutreix,
and Maria A. Miteva
10 Rosetta Ligand Docking with Flexible XML Protocols . . . . . . . . . . . . . . . . . . . . . . . . 143
Gordon Lemmon and Jens Meiler
11 Normal Mode-Based Approaches in Receptor Ensemble Docking. . . . . . . . . . . . . . . 157
Claudio N. Cavasotto
12 Application of Conformational Clustering in Protein–Ligand Docking. . . . . . . . . . . 169
Giovanni Bottegoni, Walter Rocchia, and Andrea Cavalli
13 How to Benchmark Methods for Structure-Based Virtual Screening
of Large Compound Libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
Andrew J. Christofferson and Niu Huang

ix
x Contents

PART III PREDICTION OF PROTEIN-PROTEIN DOCKING AND INTERACTIONS

14 AGGRESCAN: Method, Application, and Perspectives for Drug Design . . . . . . . . . 199
Natalia S. de Groot, Virginia Castillo, Ricardo Graña-Montes,
and Salvador Ventura Zamora
15 ATTRACT and PTOOLS: Open Source Programs
for Protein–Protein Docking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
Sebastian Schneider, Adrien Saladin, Sébastien Fiorucci,
Chantal Prévost, and Martin Zacharias
16 Prediction of Interacting Protein Residues
Using Sequence and Structure Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
Vedran Franke, Mile Šikić, and Kristian Vlahoviček

PART IV RESCORING DOCKING PREDICTIONS

17 MM-GB/SA Rescoring of Docking Poses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
Cristiano R.W. Guimarães
18 A Case Study of Scoring and Rescoring in Peptide Docking . . . . . . . . . . . . . . . . . . . . 269
Zunnan Huang and Chung F. Wong
19 The Solvated Interaction Energy Method for Scoring Binding Affinities . . . . . . . . . 295
Traian Sulea and Enrico O. Purisima
20 Linear Interaction Energy: Method and Applications in Drug Design . . . . . . . . . . . 305
Hugo Guitiérrez-de-Terán and Johan Åqvist

PART V CRUCIAL NEGLECTED EFFECTS: ENTROPY,

SOLVENT, AND PROTONATION
21 Estimation of Conformational Entropy in Protein–Ligand Interactions:
A Computational Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327
Anton A. Polyansky, Ruben Zubac, and Bojan Zagrovic
22 Explicit Treatment of Water Molecules in Data-Driven Protein–Protein
Docking: The Solvated HADDOCKing Approach. . . . . . . . . . . . . . . . . . . . . . . . . . . . 355
Panagiotis L. Kastritis, Aalt D.J. van Dijk, and Alexandre M.J.J. Bonvin
23 Protein–Water Interactions in MD Simulations: POPS/POPSCOMP Solvent
Accessibility Analysis, Solvation Forces and Hydration Sites . . . . . . . . . . . . . . . . . . . . 375
Arianna Fornili, Flavia Autore, Nesrine Chakroun,
Pierre Martinez, and Franca Fraternali
24 Computing the Thermodynamic Contributions of Interfacial Water . . . . . . . . . . . . . 393
Zheng Li and Themis Lazaridis
25 Assignment of Protonation States in Proteins and Ligands:
Combining pKa Prediction with Hydrogen Bonding Network Optimization. . . . . . 405
Elmar Krieger, Roland Dunbrack, Rob Hooft,
and Barbara Krieger
 Contents xi

PART VI TOWARD THE USE OF ROBUST FREE ENERGY

METHODS IN DRUG DESIGN
26 Best Practices in Free Energy Calculations for Drug Design . . . . . . . . . . . . . . . . . . . . 425
Michael R. Shirts
27 Independent-Trajectory Thermodynamic Integration: A Practical
Guide to Protein-Drug Binding Free Energy Calculations
Using Distributed Computing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 469
Morgan Lawrenz, Riccardo Baron, Yi Wang, and J. Andrew McCammon
28 Free Energy Calculations from One-Step Perturbations . . . . . . . . . . . . . . . . . . . . . . . 487
Chris Oostenbrink
29 Using Metadynamics and Path Collective Variables to Study Ligand
Binding and Induced Conformational Transitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 501
Neva Bešker and Francesco L. Gervasio
30 Accelerated Molecular Dynamics in Computational Drug Design . . . . . . . . . . . . . . . 515
Jeff Wereszczynski and J. Andrew McCammon

PART VII BIOMEDICAL APPLICATIONS

31 Molecular Dynamics Applied in Drug Discovery:
The Case of HIV-1 Protease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 527
Yi Shang and Carlos Simmerling
32 Decomposing the Energetic Impact of Drug-Resistant Mutations:
The Example of HIV-1 Protease–DRV Binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 551
Yufeng Cai and Celia Schiffer
33 Guide to Virtual Screening: Application to the Akt Phosphatase PHLPP . . . . . . . . . 561
William Sinko, Emma Sierecki, César A.F. de Oliveira,
and J. Andrew McCammon
34 Molecular-Level Simulation of Pandemic Influenza Glycoproteins . . . . . . . . . . . . . . 575
Rommie E. Amaro and Wilfred W. Li
35 Homology Modeling of Cannabinoid Receptors: Discovery
of Cannabinoid Analogues for Therapeutic Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 595
Chia‐en A. Chang, Rizi Ai, Michael Gutierrez, and Michael J. Marsella
36 High-Throughput Virtual Screening Lead to Discovery of Non-Peptidic
Inhibitors of West Nile Virus NS3 Protease. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 615
Danzhi Huang
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 625
Contributors

RIZI AI  Department of Chemistry, University of California, Riverside,

CA, USA
ROMMIE E. AMARO  Departments of Pharmaceutical Sciences, Computer Science,
and Chemistry, University of California, Irvine, CA, USA
JOHAN ÅQVIST  Department of Cell and Molecular Biology, Uppsala University,
Uppsala, Sweden
FLAVIA AUTORE  Randall Division for Cellular and Molecular Biophysics,
King’s College London, London, UK
uRGEN BAJORATH  Departments of Chemical Biology and Medicinal Chemistry,
J€
University of Bonn, Bonn, Germany
RICCARDO BARON  Department of Medicinal Chemistry, College of Pharmacy,
The Henry Eyring Center for Theoretical Chemistry, University of Utah,
Salt Lake City, UT, USA
NEVA BEŠKER  Spanish National Cancer Research Centre, Madrid, Spain
ALEXANDRE M.J.J. BONVIN  Bijvoet Center for Biomolecular Research,
Utrecht University, Utrecht, The Netherlands
GIOVANNI BOTTEGONI  Department of Drug Discovery and Development,
Istituto Italiano di Tecnologia, Genova, Italy
YUFENG CAI  Department of Biochemistry and Molecular Pharmacology,
University of Massachusetts Medical School, Worcester, MA, USA
VIRGINIA CASTILLO  Department de Bioquı́mica i Biologia Molecular
and Institut de Biotecnologia i de Biomedicina,
Universitat Autónoma de Barcelona, Barcelona, Spain
ANDREA CAVALLI  Department of Drug Discovery and Development,
Istituto Italiano di Tecnologia, Genova, Italy; Department
of Pharmaceutical Sciences, University of Bologna, Bologna, Italy
CLAUDIO N. CAVASOTTO  School of Biomedical Informatics, The University of Texas
Health Center, Houston, TX, USA
NESRINE CHAKROUN  Randall Division for Cellular and Molecular Biophysics,
King’s College London, London, UK
CHIA-EN CHANG  Department of Chemistry, University of California,
Riverside, CA, USA
ANDREW J. CHRISTOFFERSON  National Institute of Biological Sciences, Beijing,
People Republic of China
NATALIA S. DE GROOT  Department de Bioquı́mica i Biologia Molecular
and Institut de Biotecnologia i de Biomedicina, Universitat Autónoma
de Barcelona, Barcelona, Spain
CÉSAR A.F. DE OLIVEIRA  Department of Chemistry and Biochemistry,
Center for Theoretical Biological Physics, Howard Hughes Medical Institute,
University of California, La Jolla, CA, USA
ROLAND DUNBRACK  Institute for Cancer Research, Fox Chase Cancer Center,
Philadelphia, PA, USA
xiii
xiv Contributors

SERKAN ERDIN  Department of Molecular and Human Genetics,

Baylor College of Medicine, Houston, TX, USA; W. M. Keck Center
for Interdisciplinary Bioscience Training, Houston, TX, USA
HAO FAN  Departments of Bioengineering & Therapeutic Sciences and
Pharmaceutical Chemistry and California Institute for Quantitative Biosciences,
University of California, San Francisco, CA, USA
SEBASTIEN FIORUCCI  UMR-CNRS, Université de Nice-Sophia Antipolis,
Nice Cedex 2, France
ARIANNA FORNILI  Randall Division for Cellular and Molecular Biophysics,
King’s College London, London, UK
VEDRAN FRANKE  Department of Molecular Biology, University of Zagreb,
Zagreb, Croatia
FRANCA FRATERNALI  Randall Division for Cellular and Molecular Biophysics,
King’s College London, London, UK
SIMONE FULLE  Institute of Pharmaceutical and Medicinal Chemistry,
Department of Mathematics and Natural Sciences, Heinrich-Heine-University,
D€usseldorf, Germany
FRANCESCO L. GERVASIO  Spanish National Cancer Research Centre, Madrid, Spain
HOLGER GOHLKE  Institute of Pharmaceutical and Medicinal Chemistry,
Department of Mathematics and Natural Sciences, Heinrich-Heine-University,
D€usseldorf, Germany
RICARDO GRAÑA-MONTES  Department de Bioquı́mica i Biologia Molecular
and Institut de Biotecnologia i de Biomedicina, Universitat Autónoma
de Barcelona, Barcelona, Spain
CRISTIANO R.W. GUIMARÃES  Worldwide Medicinal Chemistry Department,
Pfizer Inc., Groton, CT, USA
HUGO GUITIÉRREZ-DE-TERÁN  Fundación Pública Galega de Medicina Xenómica,
Santiago University Hospital, Santiago de Compostela, Spain
ATTILA GURSOY  Center for Computational Biology and Bioinformatics
and College of Engineering, Koc University Rumelifeneri Yolu, Istanbul, Turkey
MICHAEL GUTIERREZ  Department of Chemistry, University of California,
Riverside, CA, USA
DAVID R. HALL  Departments of Biomedical Engineering and Chemistry,
Biomolecular Engineering Research Center, Boston University, Boston, MA, USA
ROB HOOFT  Center for Molecular and Biomolecular Informatics,
Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands
DANZHI HUANG  Department of Biochemistry, University of Zurich,
Zurich, Switzerland
NIU HUANG  National Institute of Biological Sciences, Beijing,
People Republic of China
ZUNNAN HUANG  Department of Chemistry and Biochemistry,
University of Missouri-St. Louis, St. Louis, MO, USA
JOHN J. IRWIN  Department of Pharmaceutical Chemistry, California Institute
for Quantitative Biosciences, University of California, San Francisco, CA, USA
ANTHONY IVETAC  Department of Chemistry and Biochemistry, Center for Theoretical
Biological Physics, University of California, San Diego, La Jolla, CA, USA
 Contributors xv

PANAGOTIS L. KASTRITIS  Bijvoet Center for Biomolecular Research, Utrecht University,

Utrecht, The Netherlands
OZLEM KESKIN  Center for Computational Biology and Bioinformatics
and College of Engineering, Koc University Rumelifeneri Yolu, Istanbul, Turkey
DIMA KOZAKOV  Departments of Biomedical Engineering and Chemistry,
Biomolecular Engineering Research Center, Boston University, Boston, MA, USA
BARBARA KRIEGER  YASARA Biosciences GmbH, Vienna, Austria
ELMAR KRIEGER  Center for Molecular and Biomolecular Informatics,
Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands
GURAY KUZU  Center for Computational Biology and Bioinformatics and College
of Engineering, Koc University Rumelifeneri Yolu, Istanbul, Turkey
DAVID LAGORCE  Molécules Thérapeutiques in Silico, Université Paris Diderot,
Paris, France
MORGAN LAWRENZ  Department of Chemistry and Biochemistry, Center for Theoretical
Biological Physics, University of California, San Diego, La Jolla, CA, USA
THEMIS LAZARIDIS  Department of Chemistry, City College of New York,
New York, NY, USA
GORDON LEMMON  Department of Chemistry, Vanderbilt University, Nashville,
TN, USA
WILFRED W. LI  National Biomedical Computation Resource,
University of California, San Diego, La Jolla, CA, USA
ZHENG LI  Department of Chemistry, City College of New York, New York,
NY, USA
OLIVIER LICHTARGE  Department of Molecular and Human Genetics,
Verna and Marrs Mclean Department of Biochemistry and Molecular Biology,
Baylor College of Medicine, Houston, TX, USA; W. M. Keck Center
for Interdisciplinary Bioscience Training, Houston, TX, USA
RHONALD LUA  Department of Molecular and Human Genetics,
Baylor College of Medicine, Houston, TX, USA
MICHAEL J. MARSELLA  Department of Chemistry, University of California
at Riverside, Riverside, CA, USA
PIERRE MARTINEZ  Randall Division for Cellular and Molecular Biophysics,
King’s College London, London, UK
J. ANDREW MCCAMMON  Howard Hughes Medical Institute, Departments
of Chemistry and Biochemistry and Pharmacology, Center for Theoretical
Biological Physics, University of California, San Diego, La Jolla, CA, USA
JENS MEILER  Department of Chemistry, Vanderbilt University, Nashville, TN, USA
MARIA A. MITEVA  Molécules Thérapeutiques in Silico, Université Paris Diderot,
Paris, France
SARA E. NICHOLS  Department of Chemistry and Biochemistry, Center for Theoretical
Biological Physics, University of California, San Diego, La Jolla, CA, USA
BRITTA NISIUS  Departments of Chemical Biology and Medicinal Chemistry,
University of Bonn, Bonn, Germany
RUTH NUSSINOV  Center for Cancer Research Nanobiology Program, National Cancer
Institute, Frederick, MD, USA; Department of Human Genetics and Molecular
Medicine, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel
xvi Contributors

CHRIS OOSTENBRINK  Institute of Molecular Modeling and Simulation,

University of Natural Resources and Life Sciences, Vienna, Austria
ILZA PAJEVA  Institute of Biophysics and Biomedical Engineering,
Bulgarian Academy of Sciences, Sofia, Bulgaria
TANIA PENCHEVA  Institute of Biophysics and Biomedical Engineering,
Bulgarian Academy of Sciences, Sofia, Bulgaria
ANTON POLYANSKY  Laboratory of Computational Biophysics,
Department of Structural and Computational Biology, Max Perutz Laboratories,
Vienna, Austria
CHANTAL PRÉVOST  CNRS UPR, Université Paris Diderot, Paris, France
ENRICO PURISIMA  Biotechnology Research Institute, National Research Council,
Ottawa, ON, Canada
WALTER ROCCHIA  Department of Drug Discovery and Development,
Istituto Italiano di Tecnologia, Genova, Italy
ADRIEN SALADIN  Molécules Thérapeutiques in Silico, Université Paris Diderot,
Paris, France
ANDREJ SALI  Departments of Bioengineering & Therapeutic Sciences
and Pharmaceutical Chemistry and California Institute for Quantitative
Biosciences, University of California, San Francisco, San Francisco, CA, USA
CELIA SCHIFFER  Department of Biochemistry and Molecular Pharmacology,
University of Massachusetts Medical School, Worcester, MA, USA
SEBASTIAN SCHNEIDER  Physik-Department, Technische Universit€ a t M€
unchen,
Garching, Germany
YI SHANG  Department of Chemistry, State University of New York,
Stony Brook, NY, USA
MICHAEL R. SHIRTS  Department of Chemical Engineering, University of Virginia,
Charlottesville, VA, USA
EMMA SIERECKI  Department of Pharmacology, University of California,
San Diego, La Jolla, CA, USA
MILE ŠIKIĆ  Department of Electronic Systems and Information Processing,
University of Zagreb, Zagreb, Croatia
CARLOS SIMMERLING  Department of Chemistry, State University of New York,
Stony Brook, NY, USA
WILLIAM SINKO  Department of Chemistry and Biochemistry, Center for Theoretical
Biological Physics, University of California, San Diego, La Jolla, CA, USA
TRAIAN SULEA  Biotechnology Research Institute, National Research Council,
Ottawa, ON, Canada
SANDOR VAJDA  Departments of Biomedical Engineering and Chemistry,
Biomolecular Engineering Research Center, Boston University, Boston, MA, USA
AALT D. J. VAN DIJK  Wageningen UR, Plant Research International,
Wageningen, The Netherlands
SALVADOR VENTURA ZAMORA  Department de Bioquı́mica i Biologia Molecular
and Institut de Biotecnologia i de Biomedicina, Universitat Autónoma de Barcelona,
Barcelona, Spain
BRUNO O. VILLOUTREIX  Molécules Thérapeutiques in Silico, Université Paris Diderot,
Paris, France
 Contributors xvii

KRISTIAN VLAHOVIČEK  Department of Molecular Biology, Division of Biology,

Bioinformatics Group, University of Zagreb, Zagreb, Croatia
MARTIN VOGT  Departments of Chemical Biology and Medicinal Chemistry,
University of Bonn, Bonn, Germany
YI WANG  Department of Chemistry and Biochemistry, Center for Theoretical
Biological Physics, University of California, San Diego, La Jolla, CA, USA
ANNE MAI WASSERMANN  Departments of Chemical Biology and Medicinal Chemistry,
University of Bonn, Bonn, Germany
JEFF WERESZCZYNSKI  Department of Chemistry and Biochemistry,
Center for Theoretical Biological Physics, University of California, San Diego,
La Jolla, CA, USA
ANGELA WILKINS  Department of Molecular and Human Genetics, Baylor College
of Medicine, Houston, TX, USA; W. M. Keck Center for Interdisciplinary Bioscience
Training, Houston, TX, USA
CHUNG F. WONG  Department of Chemistry and Biochemistry,
University of Missouri-St. Louis, St. Louis, MO, USA
MARTIN ZACHARIAS  Physik-Department, Technische Universit€ a t M€
unchen,
Garching, Germany
BOJAN ZAGROVIC  Laboratory of Computational Biophysics, Department of Structural
and Computational Biology, Max Perutz Laboratories, Vienna, Austria
RUBEN ZUBAC  Laboratory of Computational Biophysics, Department of Structural
and Computational Biology, Max Perutz Laboratories, Vienna, Austria
 Part I

Drug Binding Site Prediction, Design, and Descriptors

 Chapter 1

A Molecular Dynamics Ensemble-Based Approach

for the Mapping of Druggable Binding Sites
Anthony Ivetac and J. Andrew McCammon

Abstract
An expanding repertoire of “allosteric” drugs is revealing that structure-based drug design (SBDD) is not
restricted to the “active site” of the target protein. Such compounds have been shown to bind distant
regions of the protein topography, potentially providing higher levels of target specificity, reduced toxicity
and access to new regions of chemical space. Unfortunately, the location of such allosteric pockets is not
obvious in the absence of a bound crystal structure and the ability to predict their presence would be useful
in the discovery of novel therapies. Here, we describe a method for the prediction of “druggable” binding
sites that takes protein flexibility into account through the use of molecular dynamics (MD) simulation.
By using a dynamic representation of the target, we are able to sample multiple protein conformations that
may expose new drug-binding surfaces. We perform a fragment-based mapping analysis of individual
structures in the MD ensemble using the FTMAP algorithm and then rank the most prolific probe-
binding protein residues to determine potential “hot-spots” for further examination. This approach has
recently been applied to a pair of human G-protein-coupled receptors (GPCRs), resulting in the detection
of five potential allosteric sites.

Key words: Allosteric, Molecular dynamics simulation, Docking, Binding site, Drug design

1. Introduction

Structure-based drug design (SBDD) efforts are typically initiated

when a high-resolution crystal structure of the target protein
complexed with a small molecule is available. The co-crystallized
ligand is usually some form of the endogenous substrate/agonist
or a synthetic drug compound with affinity for the same binding
site. This region of the protein surface is referred to as the “active”
or “orthosteric” site and is highly conserved among closely related
proteins. Relatively recently however, it has emerged that there
are other “druggable” sites on the protein surface, which may be
bound by therapeutic small molecules and which are spatially
distinct from known active sites (1, 2). Such pockets are known

Riccardo Baron (ed.), Computational Drug Discovery and Design, Methods in Molecular Biology, vol. 819,
DOI 10.1007/978-1-61779-465-0_1, # Springer Science+Business Media, LLC 2012

3
4 A. Ivetac and J.A. McCammon

as “allosteric” sites, the binding of which can modulate function

through a variety of proposed mechanisms that perturb protein
dynamics (3). Some well-known FDA-approved allosteric drugs
include the protein kinase inhibitor Gleevec, the calcium-sensing
receptor modulator Cinacalcet, and the HIV-1 reverse transcrip-
tase inhibitor Etravirine. Allosteric drugs are attractive for numer-
ous reasons, perhaps the most powerful of which is their potential
for enhanced target specificity. The ability to better discriminate
between binding sites belonging to related targets is crucial in
reducing “off-target” activity related to certain harmful side
effects and is thought to be possible because allosteric sites are
less well conserved than orthosteric sites (4). It has also been
observed that hitherto identified allosteric compounds are struc-
turally more diverse than their orthosteric counterparts, suggest-
ing larger regions of chemical space are available for their design
and optimization (2).
Despite progress in the screening and identification of allosteric
drugs, the structural biology of their binding sites is still poorly
understood and there has consequently been a lack of SBDD
for such compounds. Considering advances in high-resolution
structure determination, the ability to computationally predict
potential allosteric sites from an unbound protein structure
would clearly facilitate the discovery of novel therapeutic com-
pounds and elucidate the binding of existing drugs.
A number of algorithms have been reported for the detection
of druggable binding pockets, given an atomic protein structure as
input (5, 6). These vary in complexity, ranging from a simple
shape-based representation of the protein surface, to the addition
of energy-based calculations, and to molecular dynamics (MD)
simulations performed in the presence of small molecules. In this
work, we elected to use the FTMAP algorithm (7), whereby a
panel of probe molecules is docked to the surface of a static
protein structure in order to expose potential high-affinity sites
for drug molecules. FTMAP combines extensive probe sampling
with an energy-based scoring function and has performed
very well in the reproduction of experimentally determined
protein–ligand complexes (7–9).
Perhaps one of the best recognized weaknesses in current small
molecule docking programmes is the static representation of the
target protein (10, 11), giving rise to the term “rigid-protein flexi-
ble-ligand.” While this compromise has been convenient for often
time-consuming docking calculations, it is a poor reflection of the
highly dynamic process of molecular recognition and our under-
standing that proteins exist in an ensemble of conformational sub-
states (12, 13). Furthermore, it has been noted previously that many
novel allosteric sites were not obvious from the unbound form of the
protein (1), suggesting that such pockets may have a transient
character which may therefore elude predictions using experimental
 1 A Molecular Dynamics Ensemble‐Based Approach for the Mapping. . . 5

structures alone. A number of techniques have been proposed for

the introduction of flexibility into a static protein model for the
enhancement of molecular docking, varying from simple sidechain
modifications to full backbone mobility (14, 15). Here, we employ
all-atom MD simulation (16) of the target protein in explicit solvent
in order to sample new conformations that may reveal druggable
cavities. MD simulation offers full protein flexibility in a realistic
solvent environment, such that dramatic rearrangements, which can
alter the topography, are possible. MD simulation has become a
popular method in the investigation of protein dynamics and has
successfully been integrated into virtual screening efforts to opti-
mize lead discovery (17).
In this work, we present a method for the discovery of poten-
tial novel drug binding sites, whereby the computational mapping
tool FTMAP is coupled with an MD-based ensemble of target
protein conformations. This approach has recently been used to
map a series of five potential allosteric sites on the surface of the
human b1 and b2 adrenergic receptors (bARs) (18) and was
inspired by the work of Landon et al. (19), who used a similar
technique with the influenza neuraminidase target. To illustrate
the method, we use the human b2AR and the retroviral HIV-1
reverse transcriptase (RT) as membrane-bound and water-soluble
examples of targets, describing how the ensembles are generated
and how the mapping results are combined.

2. Methods

In the following section, we describe the four main procedures

involved in our flexible-target mapping protocol, which have been
illustrated in Fig. 1. Acknowledging there is significant scope for
variation in the specific algorithms used to complete each step, we
describe one strategy and suggest alternatives in Notes 4.

2.1. Ensemble The first step involves sampling the target protein’s conformational
Generation landscape to obtain novel structures that are distinct from the initial,
experimental structure, and may expose novel druggable sites.
While many methods are available for biomolecular conformational
sampling (see Note 1), we have opted to use the widespread MD
simulation technique, which has been described elsewhere (20).
An MD simulation charts the time evolution of a protein structure
from its experimental starting conformation, essentially producing a
trajectory, or “movie” of protein motion with thousands of frames,
or “snapshots” that can be extracted for analysis. This is the
most time-consuming step in our protocol; however, the resulting
trajectory can have many applications in addition to the one
6 A. Ivetac and J.A. McCammon

Fig. 1. Overview of the four main stages of the flexible mapping procedure, with HIV-1 RT as an example. Input to the
procedure is a single experimental structure of the target and output is a ranked list of residues which may form
druggable binding sites.

described here and there may already exist MD data for the target of
interest. In our work, we used the popular Gromacs MD simulation
package (21) together with the Gromos-96 biomolecular force-
field (22). To mitigate the well-known issue of incomplete sampling
of larger proteins, we have used a multi-copy approach (23),
whereby a series of simulations with different initial velocities are
carried out in preference to a single longer trajectory. For the RT
system we performed a series of four 30 ns simulations, and for the
b2AR system we performed a series of four 60 ns simulations
(for details on the MD setup protocol, please see ref. (18, 24)).
All simulations were performed in atomic detail and in the presence
of explicit water molecules, with the b2AR system including a phos-
pholipid bilayer. Both proteins were simulated in the absence of co-
crystallized ligands.

2.2. Conformer Perhaps one of the biggest challenges in the use of large structural
Selection ensembles for protein–ligand docking is the selection of a subset
of the MD frames for analysis, given the intractability of using
 1 A Molecular Dynamics Ensemble‐Based Approach for the Mapping. . . 7

every single conformer. There is a wide variety of selection criteria

that can be used to categorize each frame, the choice depending
on the goal of the subsequent analysis (see Note 2). In our
protocol, we sought to dramatically reduce the size of each ensem-
ble and select a representative set of conformers that capture
diverse protein topographies. Inspired by techniques to eliminate
redundancy in large structural datasets used in virtual screening
(as discussed in (17)), we used the RMSD-based clustering
method provided by the “g_cluster” tool in the Gromacs package
(using the “gromos” method; see Note 3). By adjusting the cutoff
value for membership of each cluster, we were able to divide each
ensemble into approximately 20 clusters, the top 15 of which were
used in the subsequent mapping step. For each cluster, we took
the centroid member as the representative structure for mapping.
To illustrate the diversity of the MD-generated structures, the
15 representative conformers from the b2AR system are shown
in Fig. 2.

Fig. 2. Conformational diversity of the MD ensemble. Fifteen representative structures of the b2AR, after RMSD-based
clustering.
8 A. Ivetac and J.A. McCammon

2.3. Site Mapping Given the reduced MD ensemble of 15 conformers, the next step is
to perform a search of druggable pockets on the surface of each.
While a number of algorithms are available for binding site predic-
tion (see Note 4), we elected to use the FTMAP algorithm (7),
inspired by encouraging correlations with experimentally solved
protein–ligand complex structures and previous work from our
group which used its predecessor, CS-Map (19). The FTMAP
software is provided as a web-based service (http://ftmap.bu.edu),
whereby a typical protein can be mapped overnight, by simply
uploading the protein coordinates. The results of the mapping are
made available on the web server and include a PDB file which
contains the input protein structure, along with a series of probe
molecules which represent favorable binding sites for that probe
type. The probe molecules are locally divided into “consensus sites”
(assigned in the output PDB file by a unique chain identifier), which
can be considered clusters where multiple probe types bind well and
which may be indicative of a druggable site. Figure 3 shows example
FTMAP output structures, with a range of consensus sites distri-
buted over the protein surface, each containing varying types of
probe molecules. Another useful output file from the FTMAP server
contains the number of non-bonded interactions between protein
residues and probe molecules. We use this data to rank the protein
residues and determine which are the most popular probe interac-
tion sites over the entire ensemble.

Fig. 3. Examples of FTMAP output for individual conformations of the HIV-1 RT (a) and
b2AR (b). Bound probe molecules belonging to consensus sites are shown in black stick
representation.
 1 A Molecular Dynamics Ensemble‐Based Approach for the Mapping. . . 9

2.4. Hot-Spot With FTMAP analysis performed on each member of the ensemble,
Identification the next step is to combine the results and define local “hot-spots”
of interest, for further investigation. To achieve this, we simply rank
the protein residues by the average number of non-bonded interac-
tions they make with probe molecules across the entire ensemble.
Thus, residues that bind probe molecules in multiple different
protein conformations will be scored highly and those binding
only occasionally will score poorly. While residues binding probe
molecules infrequently in the dynamics of the protein may still be of
interest, we have decided to prioritize the most common sites
(see Note 5). In our previous work (18), we arbitrarily decided to
focus on the top 40 probe-interacting residues of b2AR, which
included a mixture of residues known to bind orthosteric ligands,
in addition to residues in new regions of the protein surface. By
analyzing the distribution of the residues, we were able to define a
series of five potential allosteric sites, which we then examined in the
context of existing experimental and structural data to support
a potential allosteric role. In Fig. 4, we show the top 40 probe-
interacting residues for both our example proteins, illustrating the
existence of clusters that may constitute new binding sites. For both
systems, we see that the known drug binding site is well identified, in
addition to a range of new, potentially druggable locations that are
not known to be currently targeted by drugs. Work to identify small
molecules binding at some of these sites is currently in progress.

Fig. 4. Hot-spot identification for the HIV-1 RT (a) and b2AR (b) systems. The top 40
probe-interacting protein residues are shown in black stick representation. For HIV-1 RT
we indicate the known binding site for the NNRTI class of allosteric inhibitors, while for
b2AR we indicate the known binding site for drugs targeting the orthosteric site. For
each protein, clusters of residues are found in novel regions of the protein surface,
which may be able to modulate activity of the protein and may therefore be amenable to
drug design.
10 A. Ivetac and J.A. McCammon

3. Conclusions

We have presented a method for the identification of potential

druggable sites on a protein of interest, which takes structural
flexibility into account through MD simulation. We have shown
in previous work that such flexibility exposes fragment binding
sites which were not visible in the experimental structure alone
and may thus lead to the discovery of new therapeutic com-
pounds. Given a series of sites detected by this method and
supporting evidence to fortify their candidacy as drug targets,
we suggest that virtual screening could next be used to identify
small molecules that bind at those sites. Compounds identified
with affinity for the sites could then be experimentally validated
using an appropriate assaying technique. In addition, a fragment-
based approach could be adopted, whereby bound probe mole-
cules could be “grown” or “linked” to form completely novel
high-affinity compounds.

4. Notes

1. A number of alternative techniques are available for the

computational modeling of protein dynamics and generation
of a diverse structural ensemble. In this work we have used
traditional all-atom MD simulation; however, we could have
equally used a Monte Carlo approach for conformational
sampling. Alternatively, a number of adaptations of classical
MD simulation have recently been proposed, which aim to
address sampling deficiencies and promise to generate more
diverse ensembles—these include accelerated MD (25), con-
formational flooding (26), and replica exchange (27).
2. The selection of representative protein conformers from the
MD ensemble is another area with scope for many different
methods and which could have substantial impact on the
results. Here, we have clustered MD snapshots by global
structural similarity, in order to extract a small set of diverse
topographies; however, other criteria could equally be used,
depending on the target. For example, snapshots could be
selected based on some measure of conformational energy or
based on certain known conformational changes that may be
important to protein function.
3. We clustered trajectory frames according to the RMSD of the
Ca atoms of the core protein structure, so as to categorize
the conformers by global structural diversity and not bias the
 1 A Molecular Dynamics Ensemble‐Based Approach for the Mapping. . . 11

segregation to any local region. If there is a particular area of

interest targeted for druggability (e.g., a specific portion of
the protein surface), the subset of residues comprising this
region may be used in the clustering step instead.
4. There are also a number of alternative algorithms to FTMAP
for the mapping of druggable sites on each protein conformer.
Many suggestions can be found in (6). It may be advantageous
to use a range of algorithms and define consensus sites that are
identified across different prediction methods.
5. The hot-spot identification step is another area where different
approaches can be taken. Here, we have suggested the ranking
of probe-interacting residues by their mean performance
across the whole ensemble. However, there may be sites of
interest which are exposed relatively rarely in the dynamics
of the protein and which may therefore only be discovered
in one or a few representative conformers. We therefore rec-
ommend that the results from individual FTMAP runs are
examined for such “cryptic” sites.

Acknowledgments

This work has been supported in part by the National Science

Foundation (NSF), the National Institutes of Health (NIH),
the Howard Hughes Medical Institute (HHMI), the National
Biomedical Computation Resource (NBCR), the Center for
Theoretical Biological Physics (CTBP), San Diego Supercomputer
Center (SDSC), and the NSF Supercomputer Centers.

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 Chapter 2

Analysis of Protein Binding Sites by Computational

Solvent Mapping
David R. Hall, Dima Kozakov, and Sandor Vajda

Abstract
Computational solvent mapping globally samples the surface of target proteins using molecular probes—small
molecules or functional groups—to identify potentially favorable binding positions. The method is based on
X-ray and NMR screening studies showing that the binding sites of proteins also bind a large variety of
fragment-sized molecules. We have developed the multistage mapping algorithm FTMap (available as a server
at http://ftmap.bu.edu/) based on the fast Fourier transform (FFT) correlation approach. Identifying regions
of low free energy rather than individual low energy conformations, FTMap reproduces the available experi-
mental mapping results. Applications to a variety of proteins show that the probes always cluster in important
subsites of the binding site, and the amino acid residues that interact with many probes also bind the specific
ligands of the protein. The “consensus” sites at which a number of different probes cluster are likely to be
“druggable” sites, capable of binding drug-size ligands with high affinity. Due to its sensitivity to conforma-
tional changes, the method can also be used for comparing the binding sites in different structures of a protein.

Key words: Protein structure, Protein–ligand interactions, Binding site, Binding hot spots,
Fragment-based ligand design, Druggability, Binding site comparison, Docking

1. Introduction

The binding sites of proteins generally include smaller regions called

hot spots that are major contributors to the binding free energy, and
hence are crucial to the binding of any ligand at that particular
site (1). In drug design applications such hot spots can be identified
by screening for the binding of fragment-sized organic molecules
(2–4). Since the binding of the small compounds is very weak, it
is usually detected by Nuclear Magnetic Resonance (SAR by NMR
(3, 4)) or by X-ray crystallography (2, 5–8) methods. Results
confirm that the hot spots of proteins bind a variety of small mole-
cules, and that the fraction of the “probe” molecules binding to a
particular site predicts the potential importance of the site and can
be considered a measure of druggability (3, 4).

Riccardo Baron (ed.), Computational Drug Discovery and Design, Methods in Molecular Biology, vol. 819,
DOI 10.1007/978-1-61779-465-0_2, # Springer Science+Business Media, LLC 2012

13
14 D.R. Hall et al.

Fig. 1. Schematic figure of computational solvent mapping using two probes. Each circle and hexagon represents one of
the two probes. (a) Each probe is sampled around the surface of the protein to (b) find the minima where a probe clusters.
(c) The consensus site where two probe clusters overlap, but occupy slightly different positions.

Solvent mapping has been developed as a computational

analogue of the NMR and X-ray based screening experiments (9).
The method places molecular probes—small organic molecules
containing various functional groups—on a dense grid defined
around the protein, finds favorable positions using empirical free
energy functions, further refines the selected poses by free energy
minimization, clusters the low energy conformations, and ranks the
clusters on the basis of the average free energy (10). To determine
the hot spots, we find consensus sites, i.e., regions on the protein
where clusters of different probes overlap, and rank these sites in
terms of the number of overlapping probe clusters (10). This prin-
ciple is illustrated by the schematic figure (Fig. 1) for the case of
mapping a protein with only two probes (represented as circles
and hexagons, respectively), each forming a few clusters on the
protein surface. While the clusters overlap in the main consensus
site, the distributions of different probes may slightly differ, result-
ing in the arrangement shown in Fig. 1c. Thus, in principle the
mapping can identify both the “hot spots” of the binding site
and the functional groups that tend to bind at specific locations
within it. The consensus site, binding the largest number of probe
clusters, is considered the main hot spot (10, 11). The number
of probe clusters at a particular consensus site (CS) correlates with
the importance of that site for binding (12). The main hot spot
and other hot spots within a 7 Å radius predict a site that can
potentially bind drug-size ligands. These results can be used for
 2 Analysis of Protein Binding Sites by Computational Solvent Mapping 15

the prediction of binding sites, and helped to better understand the

principles that govern the weakly specific binding of small molecules
to functional sites of proteins (13–17). We have developed the
multistage mapping algorithm FTMap (10), based on the fast Four-
ier transform (FFT) correlation approach. FTMap performs all steps
of the mapping algorithm, and is available as a server at http://
ftmap.bu.edu/.

2. Software
Requirements
This method requires a molecular viewer for preparation of crystal
structures for mapping and analysis of results. This chapter
assumes that PyMol (http://pymol.org), an Open Source molec-
ular viewer available on Windows, Mac OS X, and Linux, will be
used. Additionally, an Internet connection and web browser are
required to use the various servers throughout the method.

3. Methods

3.1. Finding a Protein Computational solvent mapping techniques rely on the user to
Structure provide the 3D structure of the protein. The vast majority of
published structures of proteins can be found in the Protein
Data Bank (PDB) in the PDB format. The simplest way to find a
structure is by searching the PDB website (http://www.pdb.org)
for the name of the protein. The PDB also provides an “Advanced
Search,” where a sequence can be searched against the PDB using
BLAST (Fig. 2). The search by name relies on authors titling their
structure, paper, or chains in the protein with the same name a

Fig. 2. Advanced search interface for searching the Protein Data Bank (PDB) by sequence.
16 D.R. Hall et al.

Fig. 3. Refining a query in the PDB by presence of ligands and experimental method.

user uses in their search. Thus, it can often be advantageous to use

the sequence-based search.
For many proteins, there will be more than one structure in the
PDB. In general, the FTMap server produces better results from a
high-resolution unbound crystal structure. Having ligands in the
binding site often influences the shape of the site, sometimes dis-
turbing the ability to detect hot spots. An initial search on the PDB
website can be refined by whether the structure has ligands along
with the experimental method (Fig. 3). Additionally, the query
results can be sorted by resolution. Note though that the PDB
classifies many structures as having ligands even if they are unbound.
If a structure has an innate metal ion, or if cryoprotectants such as
glycerol are seen, the structure will be labeled as having a ligand,
despite not having a ligand in the binding site of interest.

3.2. Server The FTMap server is available for free use by academics at http://
Submission ftmap.bu.edu. After creating an account, you can submit jobs as
shown in Fig. 4. If you are using a structure from the pdb, you can
specify the pdb id and the chains. Note that HETATM records
within the pdb file are automatically stripped out. There are no
parameters for the majority of HETATMs from the PDB on the
server. The server does contain parameters for many common
metals though, such as iron, magnesium, and zinc. If you want to
include these, you should specify them as a chain by the letter “H”
for HETATM, followed by the residue name, and then by the chain
id. In Fig. 4, HZNA stands for the zinc from chain A of the protein.
If using an NMR protein, the model can be specified (see Note 1).
If a protein has been prepared as a pdb file for mapping, as in
preparing a single domain of a multidomain protein (see Note 2),
 2 Analysis of Protein Binding Sites by Computational Solvent Mapping 17

Fig. 4. FTMap job submission interface.

Fig. 5. FTMap job download interface.

this file can be uploaded by clicking on Upload PDB in the

interface. The chains can be specified as described above.
If you created a masking file (see Note 3), it may be uploaded
under Advanced Options.
Lastly, to look for binding sites in a protein–protein interaction
site, a special PPI mode has been incorporated into the FTMap server.
After submitting a protein through the server, you should
wait for an e-mail informing you of job completion. Depending
on the load on the server, a job can take from 2 h to a full day.

3.3. Analysis After a job completes, three files will be available for download, a
of Results pdb file containing the mapping, and two text files with counts of
nonbonded and hydrogen-bonded interactions to each residue on
the protein (Fig. 5).
18 D.R. Hall et al.

3.3.1. Analysis of Mapping The pdb containing the mapping is specially formatted to be split
into multiple objects when loaded into PyMol. Additionally, it is
recommended to place the following code into a pymol startup
file. This code should be placed file named pymolrc.py in your
home directory on Windows (C:\Users\USERNAME\) or a file
named .pymolrc in your home directory on Mac OS X (/Users/
USERNAME/) or Linux (/home/USERNAME/). These func-
tions allow you to easily color clusters by rank, disable and enable
clusters, and rename the objects loaded in from an FTMap job.
This last task is especially important if loading multiple FTMap
jobs into a single PyMol Session as the object names may
overwrite each other.

from pymol import cmd, util

def colorClusters():
util.cbac('*.000.*')
util.cbap('*.001.*')
util.cbay('*.002.*')
util.cbas('*.003.*')
util.cbaw('*.004.*')
util.cbab('*.005.*')
util.cbao('*.006.*')
util.cbag('*.007.*')
util.cbam('*.008.*')
util.cbak('*.009.*')

def disableClusters(rank='all'):
if (rank == 'all'):
cmd.disable('*.*.*')
else:
select = "*.%03d.*" % int(rank)
cmd.disable(select)

def enableClusters(rank='all'):
if (rank == 'all'):
cmd.enable('*.*.*')
else:
select = "*.%03d.*" % int(rank)
cmd.enable(select)

def renameFTMap(protname):
stored.clusters=[]
cmd.iterate('crosscluster* and index 1',
'stored.clusters.append(model)')

for cluster in stored.clusters:

namepieces = cluster.split('.')
namepieces[0] = protname #set first element to protname
if (namepieces[-1] == "pdb"):
namepieces.pop()
name = '.'.join(namepieces)
cmd.set_name(cluster, name)

cmd.group(protname+'_clusters', protname+'.*')

cmd.set_name('protein', protname)

cmd.extend('cc', colorClusters)
cmd.extend('colorClusters', colorClusters)
cmd.extend('dc', disableClusters)
cmd.extend('disableClusters', disableClusters)
cmd.extend('ec', enableClusters)
cmd.extend('enableClusters', enableClusters)
cmd.extend('rf', renameFTMap)
cmd.extend('renameFTMap', renameFTMap)
 2 Analysis of Protein Binding Sites by Computational Solvent Mapping 19

When the mapping is opened in PyMol, several objects are

created. The protein submitted for mapping is labeled “protein.”
The individual crossclusters from mapping are labeled “crosscluster.
rank.population.pdb.” Each crosscluster represents a location where
multiple different probe types clustered with a 4 Å radius. These
locations are the hot spots for binding. In looking for a druggable
pocket, there should be a large population crosscluster (population
greater than 10) with several nearby crossclusters of lower popula-
tion. An example in Fig. 6a is the mapping of PDB 1w50, an apo
structure of b-secretase. The largest crosscluster, with population
19, is seen in a pocket surrounded by a variety of other crossclusters.
Drug-like molecules have been developed for b-secretase, such as
the one shown in Fig. 6b, a submicromolar inhibitor (18) that uses
the hot spots defined by mapping.
If in analyzing the mapping, the majority of the results are
going into an area between two structural domains rather than a
well-defined pocket; the protein should be separated into the
individual structural domains to be mapped independently (see
Note 2). If the consensus site is in the location of a tightly bound
coenzyme, but other druggable sites are desired, a masking file
should be created to eliminate results in the region around the
coenzyme (see Note 3).

3.3.2. Analysis of Contacts While visual examination of the mapping provides a large amount of
information that can be used for structural design of a molecule,
analysis of the provided lists of hydrogen-bonded and nonbonded
contacts made by probes in mapping can provide additional infor-
mation on specific residues to target. These files have four columns,
with the first three columns identifying the residue index, chain, and
residue type. The fourth column contains the number of hydrogen-
bond or nonbonded contacts, the top 2,000 results for each of the
probes in mapping formed with a particular residue. The file can be
sorted on this column using UNIX tools, as shown in Fig. 7, on Mac
OS X or Linux, or may be imported into a spreadsheet program such
as Microsoft Excel to be sorted. In Fig. 7, the results for the
mapping of PDB 1w50, an apo b-secretase, are shown. The top
two residues for hydrogen bonds are ASP 228 and ASP 32. These
residues were found to form hydrogen bonds to a large number of
fragments by Astex Therapeutics (19). The top two residues for
nonbonded contacts are Phe108 and Leu30, which are used
by the bulk of the submicromolar inhibitor shown in Fig. 6b.
The top hydrogen-bond and nonbonded contacts can provide
information of use in structure-based drug design.
20 D.R. Hall et al.

Fig. 6. Mapping of apo b-secretase (1w50) showing a pocket that (a) contains a large crosscluster with smaller cluster
neighbors which (b) agree well with the binding of a submicromolar inhibitor (2ohu).
 2 Analysis of Protein Binding Sites by Computational Solvent Mapping 21

Fig. 7. Analysis of the top hydrogen-bonded and nonbonded contacts on Mac OS X or Linux.

4. Notes

1. Many structures in the PDB have multiple copies of a protein

in a structure. Frequently crystals will have multiple copies of
a protein in an asymmetric unit, resulting in multiple chains
with the same sequence. If using a structure solved by NMR, a
number of models will be reported. In either case, there are
multiple different structures of the same protein. All these
structures submitted to the server, and the structure with
the largest consensus site population, that is the sum of the
populations of crossclusters in the binding site, should be
chosen for analysis after mapping.
2. The FTMap algorithm works best on single domains of
proteins. If a protein has multiple domains, each domain
should be mapped and analyzed independently. The PDB
website provides access to three different methods for
determination of protein domains, SCOP, CATH, and
PFAM, on the “Derived Data” tab for a structure. This data
relies on outside groups to update the data, so it frequently is
not available for the newest PDB structures, but both CATH
and PFAM can be searched by sequence to assign domains by
similarity to previously evaluated PDB structures.
Figure 8 shows the derived data for PDB 1efv. Each
method assigned two domains to chain A of the structure
and a single domain to chain B. If you are interested in
mapping chain B, then you can proceed with the mapping,
but if you are interested in chain A, the structure should be
split into separate domains. The PDB does not provide infor-
mation on where the breaks between these domains occur.
This information must be obtained from the domain assign-
ment servers. CATH and PFAM have pages for each PDB on
their servers, showing the boundaries in the sequence
between the domains as shown in Fig. 9b, c. SCOP provides
this information in their “SCOP parseable file” named dir.des.
scop.txt. This file can be searched using your favorite text
editor, or using grep on UNIX-like systems as shown in
Fig. 9a. While the three domain assignments disagree on the
exact domain boundary, they agree to within a couple
22 D.R. Hall et al.

Fig. 8. Derived data for PDB 1efv, showing that each method assigns two domains to
chain A and a single domain to chain B.

Fig. 9. Mapping of domains to sequences from (a) SCOP, (b) CATH, (c) PFAM.
 2 Analysis of Protein Binding Sites by Computational Solvent Mapping 23

Fig. 10. Preparation of a protein domain for mapping in PyMol by (a) loading of the PDB, (b) showing the sequence,
(c) selection of the domain, and (d) saving of the selection object.

residues. FTMap will not be sensitive to which exact assign-

ment you use give or take a couple residues.
To submit the domains of chain A separately to FTMap,
PDB files of the individual domains must be prepared. The
simplest method for this is using PyMol. Once PyMol has
been launched, a specific protein from the PDB can be loaded
via Plugin->PDB Loader Service (Fig. 10a). To see the
sequence of this protein, the user should click on the S in
the lower right hand corner of the viewer (Fig. 10b). Portions
of the sequence can then be “selected” by clicking on the
sequence above the protein. In Fig. 10c, residues 20–204 of
1efv have been selected, creating a selection object called
“sele.” This is shown on the protein as a large number of
dots, which can be seen to cover one structural domain of
the protein. Finally, the structure of the selected sequence can
24 D.R. Hall et al.

Fig. 10. (continued)

be saved by going to File->Save Molecule. . . and then select-

ing “sele” in the dialog (Fig. 10d). This can be repeated for
each structural domain.
3. Many proteins have strong binding sites that bind coenzymes,
but developers of molecules would rather their molecule bind
elsewhere. This is the case, for example, with kinase inhibitors
that bind outside the ATP-binding site. FTMap is able to
mask a region of a protein from mapping. That is, it will
prevent probes from going into that region of the protein.
FTMap uses a masking file in the PDB format of the
 2 Analysis of Protein Binding Sites by Computational Solvent Mapping 25

Fig. 11. Creation of a mask in the ATP-binding region of a protein by (a) selection of the ATP analogue and (b) expansion
of the selection into the site.
26 D.R. Hall et al.

coordinates of residues on the protein where you do not want

the probes to bind. These files can be prepared using PyMol.
First, load your protein via the PDB Loader Service as shown
in Fig. 10a. In Fig. 11, we develop a mask for the ATP-
binding site of PDB 3A99. Right clicking on the ATP ana-
logue in the site brings up a menu where the analogue can be
selected by choosing residue->select (Fig. 11a). Once the
selection has been created, the selection can be expanded to
the atoms near the analogue by right clicking on the selection
and choosing actions->around->atoms within 8A (Fig. 11b).
This selection can then be saved by File->Save Molecule. . . as
shown in Fig. 10d.

Acknowledgment

This work has been supported by grant GM064700 from the

National Institutes of Health.

References
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spot of binding energy in a hormone-receptor A, Vitkup D, Petsko GA, Ringe D. (2006)
interface. Science, 267, 383–386. Multiple solvent crystal structures: probing
2. Mattos, C., and Ringe, D. (1996). Locating binding sites, plasticity and hydration. J Mol
and characterizing binding sites on proteins. Biol. 357: 1471–1482.
Nat. Biotechnol., 14, 595–599. 9. Dennis S, Kortvelyesi T, Vajda S. (2002)
3. Hajduk, P. J., Huth, J. R., and Tse, C. (2005) Computational mapping identifies the bind-
Predicting protein druggability. Drug Discov ing sites of organic solvents on proteins. Proc.
Today 10, 1675–1682. Natl. Acad. Sci. USA., 99: 4290–4295, 2002.
4. Hajduk, P.J., Huth, J. R., and Fesik, S. W. 10. Brenke R, Kozakov D, Chuang G-Y, Beglov
(2005). Druggability indices for protein tar- D, Hall D, Landon MR, Mattos C, Vajda S.
gets derived from NMR-based screening data. (2009) Fragment-based identification of
J. Med. Chem. 48: 2518–2525. druggable “hot spots” of proteins using
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Jeffery, C. J., Mattos, C., Petsko, G. A, formatics, 25: 621–627.
Ringe, D. (1996) An experimental approach 11. Silberstein M, Dennis S, Brown III L, Kort-
to mapping the binding surfaces of crystalline velyesi T, Clodfelter K, Vajda S. (2003) Iden-
proteins J. Phys. Chem. 100: 2605–2611, tification of substrate binding sites in enzymes
1996. by computational solvent mapping, J. Molec.
6. English AC, Done SH, Caves LS, Groom CR, Biol. 332: 1095–1113.
Hubbard RE. (1999) Locating interaction 12. Landon MR, Lancia DR Jr, Yu J, Thiel SC,
sites on proteins: the crystal structure of ther- Vajda S. (2007) Identification of hot spots
molysin soaked in 2% to 100% isopropanol. within druggable binding sites of proteins by
Proteins 37: 628¯640. computational solvent mapping. J. Med.
7. English AC, Groom CR, Hubbard RE. Chem., 50: 1231–1240.
(2001) Experimental and computational 13. Vajda S, Guarnieri F. (2006) Characterization
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protein. Protein Eng. 14: 47¯59. imental and computational methods. Current
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14. Landon MR, Lieberman RL, Hoang QQ, Ju 17. Chuang, G-Y., Kozakov, D., Brenke, R.,
S, Caaveiro JM, Orwig SD, Kozakov D, Beglov, D., Guarnieri, F., and Vajda, S.
Brenke R, Chuang G-Y, Beglov D, Vajda S, (2009) Binding hot spots and amantadine
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Exploring the Variety of Random
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The Queen versus

Billy and
Other Stories
THE QUEEN VERSUS
BILLY AND
OTHER STORIES

By LLOYD OSBOURNE

Charles Scribner’s Sons

New York . . . . 1900
 Copyright, 1900, by
Charles Scribner’s Sons

THE DEVINNE PRESS.

 Contents
Page
The Queen versus Billy 3
The Beautiful Man of Pingalap 31
The Dust of Defeat 65
The Happiest Day of his Life 109
Father Zosimus 127
Frenchy’s Last Job 171
The Devil’s White Man 213
The Phantom City 237
Amatua’s Sailor 287
THE QUEEN VERSUS BILLY
 THE QUEEN VERSUS BILLY

I T was the Sandfly, Captain Toombs, that brought the news to

Sydney and intercepted her Majesty’s third-class cruiser Stingaree,
as she lay in Man-of-War Cove, with her boats hoisted in and a deck-
load of coal as high as her bulwarks, on the eve of a long trip into
the western Pacific. It was the same old story—another white man
sent to his last account in the inhospitable Solomons, where if the
climate does not kill you the black man soon will: “Thomas Hysslop
Biggar, commonly known as ‘Captain Tom’; aged forty-six; British
subject; occupation, trader in coprah; place of residence, Sunflower
Bay, island of Guadalcanar; murdered by the natives in September,
1888, between the 7th and the 24th, and his station looted and
burned.” There was trouble in store for Sunflower Bay; they had
killed Collins in 1884, and Casseroles the Frenchman in 1887, and
had drawn upon themselves an ominous attention by firing into the
Meg Merrilies in the course of the same year. Murder was becoming
too frequent in Sunflower Bay, and Captain Casement, while policing
those sweltering seas, was asked to “conduct an inquiry into the
alleged murder of T. H. Biggar, and take what punitive measures he
judged to be necessary.”
It was not everybody who would have liked such a task; in dealing
with savages the innocent are too often lumped with the guilty, and
while you are scattering death and canister among the evil-doers,
you are often mangling their wives and children in a way horrible to
think of. Captain Casement had seen such things in the course of his
eventful service, and though no stickler where his duty was
concerned, he was neither a brute nor a coward. He was a simple
gentleman of character, parts, and conscience, with refined tastes,
and a horror of shedding innocent blood. Under his command were
five officers: Facey, acting first lieutenant, Burder, acting second,
Assistant Paymaster Pickthorn, Engineer Sennett, Dr. Roche, ten
marines, and a crew of eighty-eight men.
After a roundabout cruise through the pleasant groups of Fiji,
Tongataboo, and Samoa, with little to occupy him save official
dinners, tennis parties, and an occasional dance ashore, Captain
Casement headed his ship for the wild western islands and pricked
out a course for Sunflower Bay. One hot morning, when the damp,
moist air made everything sticky to the touch, and the whole ship
sweated like a palm-house from stem to stern, the Stingaree ran
past the towering cliffs and roaring breakers of Guadalcanar, and let
go her anchor off the blow-hole in Sunflower Bay. It was a
melancholy spot to look at, though beautiful in a gloomy and savage
fashion, and the only signs of man’s occupancy were the blackened
ruin of the trader’s house, a small mountain of coal half covered with
creepers, and a flagstaff surmounted by a skull. There was no visible
beach, for the mangroves ran to the water’s edge, save where it had
been partially cleared away by the man whose murder they had
come to avenge; nor did the closest scrutiny with the glass betray
any tell-tale smoke or the least sign of habitation. Captain Casement
surveyed the place with his keen, practised eyes, and the longer he
looked the less he liked it. The desolation jarred upon his nerves,
and his heart fell a little as the blow-hole burst hoarsely under the
ship’s quarter, and the everlasting breakers on the outer reef droned
their note of menace and alarm.
“Goodness gracious!” he said, in his abrupt, impatient fashion, as he
stood beside Facey on the bridge and superintended the laying of
the kedge. “I don’t half like the look of it, Mr. Facey; it’s a damned
nasty-looking place.”
The first lieutenant nodded. He was a burly, inarticulate man, to
whom speech was always a serious matter.
“And see here, Facey,” went on the captain. “Guns don’t matter
much; none of the devils shoot fit to speak of; but their poisoned
arrows are the very deuce—you know that was the way Goodenough
was killed—and you must keep your weather eye lifting.”
“Am I to go, sir?” asked the lieutenant.
“Yes,” said Casement. “You must take Pickthorn and twenty-five men
in the first cutter. Send Burder in the second, with twenty more, to
cover your landing. And for God’s sake, Facey, keep cool, and neither
get flustered nor over-friendly! Don’t shoot unless you have to; and
always remember they are the most treacherous savages in the
world. Be gentle and firm, and do everything with as little fuss and
as great a show of confidence as you can.”
“All right, sir,” said Facey.
Half an hour later, Facey, with twenty-five well-armed men, had
vanished into the mangroves, while Burder and his crew lay forty
yards off the shore in the second cutter, the officer devouring “Under
Two Flags,” and the men smoking and yarning in the bottom of the
boat. On the Stingaree two light guns were cast loose and made
ready to open fire at a moment’s notice, and a lookout man was
stationed in the maintop. The doctor busied himself in dismal
preparation, while the captain paced the bridge with quick and
anxious steps, fretting for the safety of his party ashore.
Hour after hour passed and brought never a sound from the
melancholy woods. The fierce sun mounted to the zenith and sank
again into the western sky. Casement was beside himself with
suspense; a cup of tea served him for lunch, and he smoked one
cigar after another. A deep foreboding brooded over the ship; the
men sat or walked uneasily about the waist; the maintop was
clustered with anxious blue-jackets; and old Quinn, the gunner, a
half-crazy zealot whose religious convictions were of the extremest
order, pattered off prayers beside the shotted guns. Towards five
o’clock, when things were looking desperate and all began to fear
the very worst, a sudden shout roused the ship, and the shore party,
noisy and triumphant, were seen streaming down to the beach. A
few moments later the two boats pulled slowly off to the ship,
Facey’s company the richer by a black man, whose costume
consisted of little more than the ropes he was bound with. A
thundering cheer hailed them as they swept under the stern and
drew up at the starboard gangway, and Facey was soon reporting
himself on the bridge.
“I can’t tell you what a relief it is to see you,” said the captain. “I
wouldn’t pass another such day for a thousand pounds!”
Facey was dog-tired, and his tattered clothes and scratched face
gave evidence of a toilsome march. But he was in a boisterous good
humour. He had acquitted himself with marked success, and was
thankful to have brought back his party and himself safe and sound.
“Well, how did you make out?” asked the captain.
“We landed at the trader’s house,” began Facey, “followed a path
that led inland, and reached some Kanaka huts. Not a soul in ’em;
clean gone, every man jack. Followed along a well beaten path
which led us into the next bay, bearing north-northeast half-east,
keeping the liveliest lookout all the time. Three miles along we ran
into another village, chock-a-block with niggers. It looked a nasty
go; lots of guns and spears, and everybody pretty skittish, kind of
they would and they wouldn’t! I recollected your orders and went
slow; you know what I mean, sir—worked off the presents, and
smoked my pipe leisurely. By and by they came round, tricky as the
devil, on to make friends or to eat us alive, whichever seemed the
more promising. I let out what I wanted, and bit by bit found out
that all the Sunflower Bay crowd were there, even to old Jibberik,
the chief—him Toombs said was the biggest scoundrel of the lot. He
looked pretty sick and knew mighty well what we were after. I talked
broadsides to that old man, and put it to him that he had better give
up the chaps who had killed the trader than waltz back to the ship
and be shot instanter himself—for somebody had to go, I said; and
just as soon as I got the old codger alongside of me I gave him to
understand that he was my bird, and kept my cocked pistol pointed
at his belly. After no end of a fuss, and lots of frothing and loud talk,
with things looking precious ugly now and again, we ended by
coming out on top. Then they dragged along a young nigger named
Billy, a returned labour-boy from the Queensland plantations, they
said, and handed him over to me as the murderer. I thought it was
more than likely they’d give us some cheap nigger they had no use
for, or some worn-out old customer, as they did in Pentecost to
Dewar of the Royalist; but I think this Billy was all right. A lot of
niggers—Billy’s own push, I suppose—looked as black as fits and
wouldn’t come round for a long time. Then I lashed the prisoner’s
hands and tied him to one of our men, and talked pretty straight to
Jib. I made him promise he’d bring his people back at once, and be
down on the beach, himself and two others, to-morrow morning to
give evidence against Billy.”
“You’ve done well, Mr. Facey,” said Casement, as his lieutenant drew
to a close, “and I tell you the story sha’n’t lose when I report it to
the admiral. You had better go now and get your clothes off,” he
added.
Facey jumped to his feet. “I am sure I am awfully obliged to you,
sir,” he said.
“Ugh, that’s all right,” said Casement, in his testy way. “What have
you done with the prisoner?”
“Turned him over to the sergeant for safe-keeping, sir,” returned the
officer.
“Leg-irons?” asked Casement.
“Leg-irons, handcuffs, and a dog-chain,” returned Facey, with a grin.
“He’s cost too much to take any chances of his getting off.”
The first thing next morning, old Jibberik was brought aboard with
his two companions. He was a disgusting old gorilla of a man, with a
hairy chest and a cold, leering eye—a mere scarecrow of humanity,
of a type incredibly cruel and debased. He had worked up enough
courage overnight to beg for everything within sight, and he
fingered the clothes and accoutrements of the seamen like a greedy
child. His two friends were not a whit behind him, either in manners
or appearance. They clicked and chattered like monkeys, and
showed extraordinary fearlessness in that armed ship amid the
swarming whites; the only man they seemed to dread was old
Jibberik himself; and they wilted under his piercing glance like
flowers in the sun, whenever his baleful attention fell their way.
Four bells was the time set for the court martial; at nine o’clock
Casement sent for Facey and told him he must prepare to defend
the prisoner.
“Burder will prosecute for the Queen,” he said. “Pickthorn will act as
clerk. Sennett, Roche, and I will compose the court.”
The first lieutenant was overcome. “I don’t think I can, sir,” he said
feebly. “I never did such a thing in my life; I wouldn’t know where to
begin, or to leave off, for that matter.”
“You can leave off when we hang your prisoner,” Casement returned,
with his bull-doggish air. “Of course, it’s all a damned farce,” he went
on. “Somebody’s got to act for the nigger; it’s printed that way in the
book.”
“I’ll move for an adjournment,” said Facey.
“I’ll be hanged if you will,” said the captain. “It’s a beastly business,
and we have got to put it through.”
Facey groaned.
“Well, do you think I like it?” said Casement.
The lieutenant saluted and walked away to find his prisoner.
Billy was clanking his chains in a canvas hutch alongside the sick-
bay, where a man lay dying. He looked up as Facey approached, and
his face brightened as he recognised his captor. He was a good-
looking young negro, and the symmetry of his limbs, and his air of
intelligence and capacity, stood out in pleasant contrast with the rest
of his comrades in Sunflower Bay.
“Billy,” said Facey, “they are going to make judge and jury for you by
and by; and I am to talky-talky for you.”
“All same Queensland,” returned Billy. “May the Lord have mercy on
your sinful soul!”
Facey was stupefied. “Where in thunder did you learn that?” he
demanded.
“Oh, me savvy too much,” said Billy.
“Now, see here,” said the lieutenant. “You didn’t kill that trader?”
“Yes, I kill him,” said Billy, cheerfully.
“You did?” cried the other.
“White fellow no good; I kill him,” said the prisoner.
“If you tell that to the captain he’ll shoot you,” said Facey. If the
prisoner was to be defended he was going to give him all the help
he could.
The black boy looked distressed and nodded a forlorn assent.
“You’ll be a big fool to say that,” said Facey.
“White fellow no good; I kill him,” repeated Billy.
“You unmitigated idiot, you’ll do for yourself,” cried the lieutenant,
angrily. “What’s the good of my talking for you if you can’t stand up
for yourself?”
Billy began to whimper; the other’s loud voice and threatening
demeanour seemed to overwhelm him.
Facey was struck with contrition. “Now shut up that snivelling,” he
said, more kindly. “Tell me the truth, Bill. Isn’t this some
humbuggery of old Jib’s—a regular plant, to shield somebody else at
the cost of your hide?”
Billy rolled his eyes, and wiped away the tears with a grimy paw.
“White fellow no good; I kill—”
“You be damned!” cried his legal adviser.
At ten o’clock the court martial was assembled on the quarter-deck.
The captain, with his brawny shoulders thrown forward, and his
hands deep in his trouser pockets, had all the air of a man in the
throes of indigestion. On either side of him were Sennett and Roche;
and in front, beside a table covered with a flag, was Pickthorn, with
a clerkly outfit and a Bible. Billy stood in chains beside a couple of
marines, looking extremely depressed. The old gorillas, their filthy
kilts bulging with what they had begged or pilfered, were in charge
of the sergeant, who had all he could do to prevent their spitting on
the deck.
Facey was the first one sworn. He deposed as to the capture and
identity of the prisoner. Then Billy was led up to the table and told to
plead.
“Kiss the book and say whether you murdered the trader or not,”
said the captain.
“White fellow no good; I kill him,” quavered the prisoner.
“Pleads guilty,” said Casement to the clerk.
“What did you do it for?” demanded the court.
Billy reiterated his stock phrase.
“Take him away,” said the captain.
Jibberik was the next witness. He kissed the book as though it were
his long-lost brother, and looked almost unabashed enough to beg it
of Pickthorn. I shall not weary the reader with his laboured English,
that lingua Franca of the isles which in the Western Pacific is known
as Beach da Mar. He told a pretty plain story: Billy and the trader
had always been on bad terms. One night, crazy with palm-toddy,
Billy had sneaked down to Captain Tom’s house and shot him
through the body as he was reading a book at supper. As to the
subsequent burning and looting of the station the old savage was
none so clear, sheltering himself in the unintelligibility of which he
was a master. His two companions followed suit, and drew the noose
a little tighter round Billy’s throat.
Then rose Burder for the Queen. He was a cheeky youngster, with
pink cheeks, a glib tongue, and no end of assurance.
“I don’t propose to waste the time of the honourable court,” he
began; “but if ever there was a flat-footed, self-confessed murderer,
I would say it is the dusky gentleman in the dock. The blood of
Biggar cries aloud for vengeance, and it would be a shame if it cried
in vain,” he said. He would point to that dreary ruin of which the
defunct had been the manly ornament, radiating civilisation round
him like a candle in the dark, and then to that black monster, who
had felled him down. This kind of thing had got to stop in the
Solomon Islands; the natives were losing all respect for whites, and
he put it to the court whether they would not jeopardise the life of
the new trader if they acquitted the murderer of the old. Now that
they had got their hand in, he would go even further, and hang up
with Billy the three witnesses for the prosecution, old Jib and the
other brace of jossers, who had villain and cutthroat stamped—
“Stick to the prisoner,” cried the court.
“I bow to correction, sir,” went on Burder. “I say again, this is no
time for half-measures; and I say that Sunflower Bay will be a better
place to live in without Mr. Billy. I leave it to the honourable court,
with every confidence, to vindicate justice in these islands by
condemning the prisoner to the extreme penalty of the law. The
case for the Queen is closed, gentlemen.”
“I believe you appear for the defence, Mr. Facey?” said Casement, as
the Queen’s prosecutor took his seat.
“I do, sir,” returned the first lieutenant, nervously.
“I should like to say, first of all,” he began, “that I will not cross-
examine these dirty old savages who have given evidence against
my client. I quite agree with everything my honourable friend has
said regarding them, and I cannot think that the court will attach
undue importance to any evidence they may have given. We’ve been
told that the Kanakas are losing all respect for whites, and that if we
don’t take some strong measures there will be the deuce to pay in
these islands. Perhaps there will be; but is that the British justice
we’re so proud of, or is it fair play, gentlemen, to the unfortunate
wretch who is trembling before you? From what I’ve seen of the
whites in this group, I can say emphatically that I’m in a line with
the Kanakas. Now, as to this Billy: What is there against him but his
own confession? and that, I beg leave to point out, ought not to be
taken as conclusive. As like as not he is the scapegoat for the whole
bay, and has been coached up to tell this story under the screw. Just
look one moment at old Jib there, and see how his friends wither
when his eyes fall their way. For all we know to the contrary, his
gibberish and click-click may be to the tune of ‘Billy, you son of a
gun, I’ll cut you into forty pieces, or flay you alive if you don’t stick
to what I’ve told you.’ After all, what have we learned from Billy?
Nothing more than this: ‘White fellow no good; I kill him.’ Is that
what anybody would call a full confession? Does it give any clew or
any details as to the motive or the carrying out of this murder? It
may be, indeed, that Billy is a monomaniac with a confirmed
delusion that he has killed Biggar; the court may smile, but I think I
am right in stating that such things have occurred and have even led
to miscarriages of justice in the past. I tell you, gentlemen, I believe
it was the whole blooming bay that killed Biggar, and that Billy was
just as guilty or just as innocent as the rest. And there is one thing I
feel mortal sure about: that if we take the prisoner outside the
heads we will soon get the gag off his mouth, and learn a good deal
more about this ugly business. Under old Jib’s search-light he’s got
to keep a close lip; but take him out to sea, and I answer for it he
won’t be so reticent. In conclusion, gentlemen, I say again that the
evidence in this case is inconclusive; that the honourable gentleman
who has appeared for the Queen has failed to make out a convincing
case against my client; that Billy’s confession in itself is not a
sufficient proof that he committed the crime charged against him;
and that we cannot take the life of a human being on such flimsy
and unsupported evidence.”
A dead silence fell upon the court when Facey drew his case to a
close and resumed his seat. Nothing could be heard but the
scratching of Pickthorn’s pen and the reverberating growl of the
blow-hole as it fretted and fumed within for the screaming blast
which was soon to follow. Casement rammed his hands deeper into
his pockets, gnawed his tawny mustache, and protruded his chin. At
last, with a start, he awoke from his reverie, and barked out:
“Mr. Sennett, as the youngest member, it is for you to speak first.”
“I think he’s guilty, sir,” said Sennett.
Casement turned his quick glance on Roche.
“Same here,” said the doctor.
“The finding of the court,” said the captain after another pause, “is
that the prisoner Billy is guilty of the murder of T. H.—what’s his
name?—Biggar, at Sunflower Bay, on the blank day of September,
1888, and is condemned to be shot as an example to the island.
Sentence to be deferred until I get the ship back from New Ireland,
where I’ve to look into that Carbutt business and the outrage at
MacCarthy’s Inlet, on the chance of the prisoner making a further
confession and implicating others in his crime. The court is
dismissed.”
“Beg pardon, sir,” said Pickthorn, looking up from his writing as the
others rose to their feet. “What am I to call the case?—the Queen
versus Billy what?”
“Billy nothing,” said the captain, savagely. “Call him William Pickthorn
if you think it sounds better.”
The verdict of the court was explained to Jibberik, and the old rogue
and his pair of friends were landed in the cove, the boat returning to
find the ship with anchor weighed and the loosened sails flapping on
the yards. In a few minutes she was steaming out to sea, and every
one grew confident that Billy’s tongue would soon wag as he saw
Sunflower Bay dwindle behind him. But the dogged savage stuck to
his tale; he had but one reply to all inquiries, to all probing and
pumping for further particulars of the murder. On his side the
conversation began and ended with: “White fellow no good; I kill
him.” On other topics he could be drawn out at will, and proved
himself a most tractable, sweet-tempered, and far from unintelligent
fellow. The men got to like him immensely, keeping him in perpetual
tobacco and providing him with more grog than was quite good for
him. In the fo’castle it was rank heresy to call him a murderer or to
express any doubts regarding his innocence. He became at once the
pet and the mystery of the ship, and his canvas cell the rallying-point
for all the little gaieties on board. He played cards well, was an apt
pupil on the accordion, and at checkers he was the master of the
ship! And he not only beat you, but he beat you handsomely,
shaking hands before and after the event, like a prizefighter in the
ring.
Casement felt very uneasy about the boy; he grew more and more
uncomfortable at heart, and it was the talk of the ship that the
problem of Billy was weighing on the “old man” like a hundredweight
of bricks. The whole business preyed upon him unceasingly and he
dreaded each passing day that brought the execution ever nearer.
Billy kept him sleepless in the steaming nights; Billy faced him like a
spectre at his solitary board; Billy’s face blurred the pages of the
books and magazines he had laid up for these dreary days in the
Solomons. Casement visited his prisoner twice a day, against the
better judgment that bade him keep away and try to forget him. He
never said much after his first two ineffectual attempts to wrestle
with Billy’s stereotyped phrase and to extort further information; but,
chewing a cigar, he would stare the black creature out of
countenance for ten minutes at a time, with a look of the strongest
annoyance and disfavor, as though his patience could not much
longer withstand the strain.
The officers were not a whit behind their captain. Billy’s artless ways
and boundless good humour had won the whole ward-room to his
side; and his grim determination to die, at once bewildered and
exasperated every soul on board. The strange spectacle offered of a
hundred men at work to persuade their prisoner to recall his
damning confession, and on pins and needles to save him from a
fate he himself seemed not to fear. The captain as good as told
Facey that if the boy would assert his innocence he would scarcely
venture to shoot him; and this intelligence Facey handed on to his
client, and, incidentally, to the whole ship’s company. Never was a
criminal so beset. Every man on board tried in his turn to shake
Billy’s obstinacy, and to paint, in no uncertain colours, the dreadful
fate the future held in store for him. One and all they retired
discomfited, some with curses, others on the verge of tears. They
swore at him for a fool; they cajoled him as they would a child; they
acted out his last end with all fidelity to detail, even to a firing
platoon saying “Bang, bang!” in dreadful unison, while a couple of
seamen made Billy roll the deck in agony. The black boy would
shudder and wipe his frightened eyes; but his fortitude was
unshaken.
“White fellow no good; I kill him.”
Then old Quinn got after him—wild-eyed, tangle-haired old Quinn,
the gunner, who was half cracked on religion. He prayed and
blubbered beside the wretched boy, overwhelming him with red-hot
appeals and perfervid oratory. Billy became an instant convert, and
got to love old Quinn as a dog his master. There was no more card-
playing in Billy’s cell, no more rum or tobacco; even checkers fell
under the iron ban of old Quinn, to whom every enjoyment was
hateful. Billy learned hymns instead, and would beguile the weary
sentry on the watch with his tuneful rendering of “Go Bury thy
Sorrow,” or “Nearer, my God, to Thee.” He was possessed, too, of a
Bible that Quinn gave him, from which the old gunner would read, in
his strident, overbearing voice, the sweet gospel of charity and good
will. But if old Quinn accomplished much, he ran, as they all ran at
last, into that stone wall of words which Billy raised against the
world. Contrition for the murder which had doomed him to die was
what Billy would not show or profess in any way to feel. Rant though
old Quinn might, and beseech on bended knees, with his eyes
burning and his great frame shaking with agitation, he could extort
from his convert no other answer than the one which all knew so
well. Billy’s eyes would snap and his mouth harden.
“White fellow no good; I kill him.”
As the days passed, and the ship made her way from bay to bay,
from island to island, in the course of her policing cruise among
those lawless whites and more than savage blacks, the captain grew
desperate with the problem of Billy. They all said that Casement
looked ten years older, and that something would soon happen to
the “old man” if Billy did not soon skip out; and the “old man”
showed all the desire in the world to bring about so desirable a
consummation. Billy was accorded every liberty; his chains had long
been things of the past, and no sentinel now guarded him in his cell
or watched him periodically in his sleep. Billy was free to go where
he would; and it was the fervent hope of all that he would lose no
time in making his way ashore. But though Casement stopped at
half a hundred villages, and laid the ship as close ashore as he dared
risk her, still, for the life of him, Billy would not budge. Then they
thought him afraid of sharks, which are plentiful in those seas, and
kept the dinghy at the gangway, in defiance of every regulation, in
the hope that the prisoner would deign to use it. But Billy showed no
more desire to quit the ship than Casement himself, or old Quinn. He
did the honours of the man-of-war to visiting chiefs, and seemed to
be proud of his assured position on board. Go ashore? Escape? Not
for worlds!
Then the captain determined upon new measures. He passed a hint
to Facey, and Facey passed it to the mess, and the mess to the blue-
jackets, that they were making things too comfortable for their
prisoner. For a while Billy’s easy life came to an abrupt conclusion.
His best friends began to kick and cuff him without mercy. He was
rope’s-ended by the bo’sun’s mate, and the cook threw boiling water
over his naked skin. The boy’s heart almost broke at this, and he
went about dejected and unhappy for the first time since he had
come aboard. But no harsh usage, no foul words, could drive him to
desert the ship. He stuck to it like a barnacle, for all the captain spun
out the cruise to an unconscionable length and stopped at all sorts
of places that offered a favorable landing for the prisoner. But if Billy
grew sad and moody under the stress of whippings and bad words,
it was as nothing to the change in Casement himself, who turned
daily greyer and more haggard as he pricked a course back to
Sunflower Bay. Of course, he maintained a decent reserve all along,
and betrayed, in words at least, not a sign of his consuming anxiety
to rid himself of Billy. But at last even his iron front broke down. It
was on the bridge, to Facey, when the ship had just dropped anchor
in Port McGuire, not forty miles from Sunflower Bay.
“Mr. Facey,” he said, “send Mr. Burder ashore with an armed party;
tell him just to show himself a bit and come off again.”
“Yes, sir,” said Facey.
“I am thinking they might take that fellow Billy to translate for
them,” he went on, shamefacedly.
The first lieutenant turned to go.
“Hold on,” said the captain, suddenly lowering his voice and drawing
his subordinate close to him. “Just you pass it on to Burder that I
wouldn’t skin him alive—you know what I mean—if—well, suppose
that black fellow cut his lucky altogether—”
Facey smiled.
“Of course,” rasped out the captain, “I can’t tolerate any dereliction
of duty; but if the young devil made a break for it—”
“Ay, ay, sir,” returned the first lieutenant, and darted down the brass
steps three at a time. He called Burder aside and gave his
instructions to that discreet youngster, who was sharp to see the
point without the need for awkward explanations. A broad grin ran
round the boat when Billy was made to descend and take his place
beside Burder in the stern; and so palpable and open was the whole
business that some aboard even shook the negro by the hand and
bade him God-speed.
A couple of hours later Burder embarked again and headed for the
ship in a tearing hurry. A chuckle ran along the decks as not a sign
of Billy could be made out, and the nearing boat soon put the last
doubt at rest. There was no black boy among the blue-jackets.
Burder skipped up the steps and saluted the captain on the bridge.
“I have to report the escape of Billy, sir,” he said, with inimitable
gravity and assurance. “I scarcely know how it came to happen, sir,
but he managed to bolt as he was walking between Miller and
Cracroft.”
“This is a very serious matter,” said the captain, with ill-concealed
cheerfulness. “I don’t know but what it is my duty to reprimand you
very severely for your carelessness. However, if he’s gone, he’s gone,
I suppose. I hope you took measures to recapture him?”
“Yes, sir,” returned Burder. “Looked for him high and low, sir.”
“Poor Billy!” said the captain, with a smile that spoke volumes. “We’ll
say no more about it, Mr. Burder; it may be all for the best; but
remember, sir, it mustn’t happen again.”
“No, sir,” said Burder.
“How did you manage it, old man?” was the eager question that met
the youngster as he took shelter in the ward-room and ordered “a
beer.” All his messmates were round him, save Facey, who was
officer of the deck and could not do more than hang in the doorway.
“I tell you it wasn’t easy,” said the boy. “We promenaded all round
the place, and I tried like fun to shake him off. I sent him errands
and hid behind trees, and talked of how we were going to shoot him
to-morrow—but it was all no blooming good! I was at my wits’ end
at last, and had almost made up my mind to tie him to a tree and
run for it, when I got a bright idea. I pretended I had dropped my
canteen under a banyan a mile behind the town, a kind of cemetery
banyan, full of dead men’s bones—a rummy place, I can tell you.
And when we got down near the boat, I took the nigger on one side
and bade him go and fetch it. ‘And don’t you come back without it,
Billy,’ said I. ‘I’ll be dismissed the service if I can’t account for that
canteen!’ Then he asked how long I was going to stay, and I said a
week; and he went off like a lamb, while we squared away for the
ship. Didn’t you see the jossers pull!”
It had been the merest pretence that had taken the war-ship into
Port McGuire, and now that her merciful errand had been so
successfully accomplished, and Billy reluctantly torn at last from
those who had to kill him, Captain Casement lost no time in ordering
the ship to sea. But as the winch tugged at the anchor, and the
great hull crept up inch by inch to the tautened chain, a sudden yell
roused the captain on the bridge and struck him as cruelly as one of
those poisoned arrows he feared so much.
“Billy, on the starboard bow!”
Sure enough, a black poll protruded above the rippling bosom of the
bay, and two frantic arms were seen driving a familiar dark
countenance on a course towards the vessel. It was Billy indeed, his
honest face marked with anguish and despair as he fought his way
to regain his prison.
Casement groaned. And for this he had been holding the cruiser two
long weeks in those God-forsaken islands, and had invented one
excuse upon another to delay his return to Sunflower Bay! Billy had
been given a hundred chances to escape, and now, like a bad penny,
here he was again, ready to precipitate the catastrophe which could
no longer be postponed.
A great laugh went up when Billy presented himself on deck,
exhausted, dripping like a spaniel, and sorely hurt in spirit. He began
at once to blurt out the story of the canteen, and made a bee-line
for Burder; but that intrepid youngster could afford to listen to no
explanations, and in self-defence had to order Billy into the hands of
the marines, who led him away protesting.
Casement’s patience was now quite at an end. He headed the ship
for Sunflower Bay, and spared no coal to bring her there in short
order. Three hours after they had passed out of the heads of Port
McGuire the Stingaree was at anchor off the blow-hole.
Facey was drinking a whisky-and-soda, and preparing himself, as
best he could, for the ordeal he knew to be before him, when the
captain’s servant entered the ward-room and requested his presence
in the cabin.
“Mr. Facey,” said the captain, “take the doctor and the pay and forty
men well armed from the ship, and when you’ve assembled the
village take that Billy and shoot him.”
“Yes, sir,” said the lieutenant, turning very pale.
“Faugh,” rasped Casement, “it makes me sick. Damn the boy, why
couldn’t he cut? Well, be off with you, and kill him as decently as
you know how.”
Billy did not at first realize how seriously he was involved in the
plans of the shore party that was making ready. He dropped into one
of the boats light-heartedly enough, and took his place cheerfully
between two marines with loaded rifles. But the mournful hush of all
about him, the eyes that turned and would not meet his own, the
tenderness and sorrow which was expressed in every movement, in
every furtive look, of his whilom comrades, all stirred and shook him
with consternation. No one laughed at his little antics. He tickled the
man next him, and nudged him, his friend Tommy, who could whistle
like a blackbird and do amazing tricks with cards; but instead of an
answering grin, Tommy’s eyes filled with tears and he stared straight
in front of him. Billy was whimpering before they were half ashore,
and some understanding of the fate in store for him began to
struggle through his thick head.
There was no need to assemble the village. It was there to meet
them, old Jibberik and all, silent, funereal, and expectant. The men
were marched up to the charred remains of the trader’s house and
formed up on three sides of a square, leaving the fourth open to the
sea. To this space Billy was led by Facey and old Quinn, the gunner.
The negro looked about him like a frightened child and clung to the
old man.
“Will you give the prisoner a minute to make his peace with God?”
asked old Quinn.
Facey nodded.
Quinn plunged down on his knees, Billy beside him. For a brief space
the gunner pattered prayers thick and fast, like a man with no time
to lose.
“Billy,” he said at last, “as you stand on the brink of that river we all
must cross, as the few seconds run out that you have still to live and
breathe and make your final and everlasting peace with the God you
have so grievously offended, let me implore you to show some
sorrow, some contrition, for the awful act that has brought you to
this! Billy, tell God you are sorry that you killed Biggar.”
For a moment Billy made no answer. At last, in a husky voice, he
said:
“You mean Cap’n Tom, who live here before?”
“Him you hurled into eternity with all his sins hot on him. Yes,
Captain Tom, the trader.”
“No!” cried Billy, with a strangled cry. “Me no sorry. White fellow no
good; I kill him.”
“Quinn,” cried Facey, “your time’s up.” The first lieutenant’s face was
livid, and his hands trembled as he bound Billy’s eyes with a silk
handkerchief.
“Stand right there, Billy,” said the officer, turning the prisoner round
to face the firing party, that was already drawn up.
“Good-bye, Missy Facey and gennelmen all,” whimpered the boy.
“Good-bye, Billy,” returned the other. “Now, men,” he added, as he
ran his eye along the faltering faces, “no damned squeamishness; if
you want to help the nigger, you’ll shoot straight. For God’s sake
don’t mangle him.
“Fire!”
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