Point Mutations In Proteins

PIM1, is a serine/threonine proto-oncogene kinase, involved in many biological functions, including cell survival, proliferation, and differentiation, thus play a key role in oncogenesis. It plays a crucial role in the onset and progression of various hematopoietic and non-hematopoietic malignancies, including acute myeloid leukemia and prostate cancer. Mutations in PIM1, especially in its kinase domain, can induce abnormal structural changes and thus alter functionalities that can lead to disease progression and other complexities. Herein, we have performed an extensive analysis of the PIM1 mutations at sequence and structure level while utilizing state-of-the-art computational approaches. Based on the impact on PIM1, numerous pathogenic and destabilizing mutations were identified and subsequently analyzed in detail. Finally, two amino acid substitutions (W109C and F147C) in the kinase domain of PIM1 were selected to explore their impact on the PIM1 structure in a time evolution ma...

The rigidity and flexibility of homologous psychrophilic (P), mesophilic (M) and thermophilic (T) proteins have been investigated at the global and local levels in terms of 'packing factor' and 'atomic fluctuations' obtained from B-factors. For comparison of atomic fluctuations, correction of errors by considering errors in B-factors from all sources in a consolidated manner and conversion of the fluctuations to the same temperature have been suggested and validated. Results indicate no differences in the global values like average packing factor among the three classes of protein homologs but at local levels there are differences. Comparison of homologous proteins triplets show that the average atomic fluctuations at a given temperature obey the order P > M >T. Packing factors and the atomic fluctuations are anti-correlated suggesting that altering the rigidity of the active site might be a potential strategy to make tailor made psychrophilic or thermophilic proteins from their mesophilic homologs.

PIM1, is a serine/threonine proto-oncogene kinase, involved in many biological functions, including cell survival, proliferation, and differentiation, thus play a key role in oncogenesis. It plays a crucial role in the onset and progression of various hematopoietic and nonhematopoietic malignancies, including acute myeloid leukemia and prostate cancer. Mutations in PIM1, especially in its kinase domain, can induce abnormal structural changes and thus alter functionalities that can lead to disease progression and other complexities. Herein, we have performed an extensive analysis of the PIM1 mutations at sequence and structure level while utilizing state-of-the-art computational approaches. Based on the impact on PIM1, numerous pathogenic and destabilizing mutations were identified and subsequently analyzed in detail. Finally, two amino acid substitutions (W109C and F147C) in the kinase domain of PIM1 were selected to explore their impact on the PIM1 structure in a time evolution manner using all-atom molecular dynamics (MD) simulations for 200 ns. MD results indicate significant conformational altercations in the structure of PIM1, especially upon F147C mutation. This study provides a significant insight into the PIM1 dysfunction upon single amino acid substitutions, which can be utilized to get insights into the molecular basis of PIM1-associated disease progression.

PIM1, is a serine/threonine proto-oncogene kinase, involved in many biological functions, including cell survival, proliferation, and differentiation, thus play a key role in oncogenesis. It plays a crucial role in the onset and progression of various hematopoietic and non-hematopoietic malignancies, including acute myeloid leukemia and prostate cancer. Mutations in PIM1, especially in its kinase domain, can induce abnormal structural changes and thus alter functionalities that can lead to disease progression and other complexities. Herein, we have performed an extensive analysis of the PIM1 mutations at sequence and structure level while utilizing state-of-the-art computational approaches. Based on the impact on PIM1, numerous pathogenic and destabilizing mutations were identified and subsequently analyzed in detail. Finally, two amino acid substitutions (W109C and F147C) in the kinase domain of PIM1 were selected to explore their impact on the PIM1 structure in a time evolution ma...

Knowledge of how frequently different types of residues are found near each other in protein structures has been widely used in threading and in simulating protein folding. In this paper we show that the residueresidue pair frequencies can be reproduced by a simple, physical model. The central component is the nonpolar in-charge out character. This character was captured by obtaining for each type of residue the relative density at a given distance from the protein's center of geometry. These densities were conveniently fitted to exponential or linear functions of the radial distance and used to generate atomic positions. To account for chain connectivity, distances between residue pairs were constrained by independent Gaussian functions, which have increasing means and deviations for increasing sequence separations. Interactions between nonpolar residues and between charged residues were found to extend up to a distance of ∼7.5 Å and the interaction potentials extracted appear to be an intrinsic property. This radial-distance based model, constructed and tested on a set of 243 nonhomologous proteins, has a clear physical basis and may hold important clues for structure prediction.

The energetics of ␣-helix formation are fairly well understood and the helix content of a given amino acid sequence can be calculated with reasonable accuracy from helix-coil transition theories that assign to the different residues specific effects on helix stability. In internal helical positions, alanine is regarded as the most stabilizing residue, whereas glycine, after proline, is the more destabilizing. The difference in stabilization afforded by alanine and glycine has been explained by invoking various physical reasons, including the hydrophobic effect and the entropy of folding. Herein, the contribution of these two effects and that of hydrophilic area burial is evaluated by analyzing Ala and Gly mutants implemented in three helices of apoflavodoxin. These data, combined with available data for similar mutations in other proteins (22 Ala/Gly mutations in ␣-helices have been considered), allow estimation of the difference in backbone entropy between alanine and glycine and evaluation of its contribution and that of apolar and polar area burial to the helical stabilization typically associated to Gly3 Ala substitutions. Alanine consistently stabilizes the helical conformation relative to glycine because it buries more apolar area upon folding and because its backbone entropy is lower. However, the relative contribution of polar area burial (which is shown to be destabilizing) and of backbone entropy critically depends on the approximation used to model the structure of the denatured state. In this respect, the excised-peptide model of the unfolded state, proposed by Creamer and coworkers (1995), predicts a major contribution of polar area burial, which is in good agreement with recent quantitations of the relative enthalpic contribution of Ala and Gly residues to ␣-helix formation.

The molecular mechanism of ethanol governed unfolding of an enzymatic protein, Chymotrypsin Inhibitor 2 (CI2) in water-ethanol mixed solutions has been studied by using combined Molecular Dynamics simulations and ONIOM study. The residue specific solvation of the unfolded protein and the interactions between the individual amino-acid residues of the protein with ethanol as well as water have been investigated. The results are compared with that obtained from the folded state of the protein. Further, emphasis has been given to explore the residue's preferential site of attraction towards the nature of the solvents. The heterogeneous structuring of water and ethanol around the hydrophobic and hydrophilic surfaces of the protein is found to correlate well with their available surface areas to the solvents. Both hydrophobic as well as hydrophilic interactions are found to have important contribution in rupturing protein's secondary structural segments. Further, residue-water as well

We previously suggested that proteins gain more stability from the burial and hydrogen bonding of polar groups than from the burial of nonpolar groups (Pace, C. N. (2001) Biochemistry 40, 310-313). To study this further, we prepared eight Thr-to-Val mutants of RNase Sa, four in which the Thr side chain is hydrogen-bonded and four in which it is not. We measured the stability of these mutants by analyzing their thermal denaturation curves. The four hydrogen-bonded Thr side chains contribute 1.3 ؎ 0.9 kcal/mol to the stability; those that are not still contribute 0.4 ؎ 0.9 kcal/mol to the stability. For 40 Thr-to-Val mutants of 11 proteins, the average decrease in stability is 1.0 ؎ 1.0 kcal/mol when the Thr side chain is hydrogen-bonded and 0.0 ؎ 0.5 kcal/mol when it is not. This is clear evidence that hydrogen bonds contribute favorably to protein stability. In addition, we prepared four Val-to-Thr mutants of RNase Sa, measured their stability, and determined their crystal structures. In all cases, the mutants are less stable than the wild-type protein, with the decreases in stability ranging from 0.5 to 4.4 kcal/mol. For 41 Val-to-Thr mutants of 11 proteins, the average decrease in stability is 1.8 ؎ 1.3 kcal/mol and is unfavorable for 40 of 41 mutants. This shows that placing an-OH group at a site designed for a-CH 3 group is very unfavorable. So,-OH groups can contribute favorably to protein stability, even if they are not hydrogen-bonded, if the site was selected for an-OH group, but they will make an unfavorable contribution to stability, even if they are hydrogen-bonded, when they are placed at a site selected for a-CH 3 group. The contribution that polar groups make to protein stability depends strongly on their environment.

Molecular dynamics simulations have been used to compute the difference in the unfolding free energy between wild-type barnase and the mutant in which Ile-96 is replaced by alanine. The simulations yield results (-3.42 and -5.21 kcal/mol) that compare favorably with experimental values (-3.3 and -4.0 kcal/mol). The major contributions to the free energy difference arise from bonding terms involving degrees of freedom of the mutated side chain and from nonbonded interactions of that side chain with its environment in the folded protein. By comparison with simulations of an extended peptide in the absence of solvent, used as a reference state, hydration effects are shown to play a minor role in the overall free energy balance for the Ile----Ala transformation. The implications of these results for our understanding of the hydrophobic effect and its contribution to protein stability are discussed.

The roles of electrostatic interactions in protein folding stability have been a matter of debate, largely due to the complexity in the theoretical treatment of these interactions. We have developed computational methods for calculating electrostatic effects on protein folding stability. To rigorously test and further refine these methods, here we carried out experimental studies into electrostatic effects on the folding stability of the human 12-kD FK506 binding protein (FKBP12). This protein has a close homologue, FKBP12.6, with amino acid substitutions in only 18 of their 107 residues. Of the 18 substitutions, 8 involve charged residues. Upon mutating FKBP12 residues at these 8 positions individually into the counterparts in FKBP12.6, the unfolding free energy (ΔG u) of FKBP12 changed by −0.3 to 0.7 kcal/mol. Accumulating stabilizing substitutions resulted in a mutant with a 0.9 kcal/mol increase in stability. Additional charge mutations were grafted from a thermophilic homologue, MtFKBP17, which aligns to FKBP12 with 31% sequence identity over 89 positions. Eleven such charge mutations were studied, with ΔΔG u varying from −2.9 to 0.1 kcal/mol. The predicted electrostatic effects by our computational methods with refinements herein had a root-mean-square deviation of 0.9 kcal/mol from the experimental ΔΔG u values on 16 single mutations of FKBP12. The difference in ΔΔG u between mutations grafted from FKBP12.6 and those from MtFKBP17 suggests that more distant homologues are less able to provide guidance for enhancing folding stability.

A cold-active a-amylase was purified from culture supernatants of the antarctic psychrophile Alteromonas haloplanctis A23 grown at 4 "C. In order to contribute to the understanding of the molecular basis of cold adaptations, crystallographic studies of this cold-adapted enzyme have been initiated because a threedimensional structure of a mesophilic counterpart, pig pancreatic a-amylase, already exists. a-Amylase from A. haloplanctis, which shares 53% sequence identity with pig pancreatic a-amylase, has been crystallized and data to 1.85 A have been collected. The space group is found to be C222, with a = 71.40 A, b = 138.88 A, and c = 115.66 A. Until now, a three-dimensional structure of a psychrophilic enzyme is lacking.

The rigidity and flexibility of homologous psychrophilic (P), mesophilic (M) and thermophilic (T) proteins have been investigated at the global and local levels in terms of ‘packing factor’ and ‘atomic fluctuations’ obtained from B-factors. For comparison of atomic fluctuations, correction of errors by considering errors in B-factors from all sources in a consolidated manner and conversion of the fluctuations to the same temperature have been suggested and validated. Results indicate no differences in the global values like average packing factor among the three classes of protein homologs but at local levels there are differences. Comparison of homologous proteins triplets show that the average atomic fluctuations at a given temperature obey the order P > M >T. Packing factors and the atomic fluctuations are anti-correlated suggesting that altering the rigidity of the active site might be a potential strategy to make tailor made psychrophilic or thermophilic proteins from the...

Understanding protein stability is critical for the application of enzymes in biotechnological processes. The structural basis for the stability of thermally adapted chitinases has not yet been examined. In this study, the amino acid sequences and X-ray structures of psychrophilic, mesophilic, and hyperthermophilic chitinases were analyzed using computational and molecular dynamics (MD) simulation methods. From the findings, the key features associated with higher stability in mesophilic and thermophilic chitinases were fewer and/or shorter loops, oligomerization, and less flexible surface regions. No consistent trends were observed between stability and amino acid composition, structural features, or electrostatic interactions. Instead, unique elements affecting stability were identified in different chitinases. Notably, hyperthermostable chitinase had a much shorter surface loop compared to psychrophilic and mesophilic homologs, implying that the extended floppy surface region in ...

Loops or unordered regions of a protein are structurally dynamic and are strongly implicated in activity, stability and proteolytic susceptibility of proteins. Diminished activity of proteins at lower temperatures is considered to be due to compromised dynamics of the protein at lower temperatures. To evolve an active mesophilic lipase (Bacillus subtilis) at low temperatures, we subjected all the loop residues (n 5 88) to site saturation mutagenesis (SSM). Based on a three-level screening protocol, we identified 14 substitutions, among 16 000 mutant population, which contributed to a substantial increase in activity at 58 8 8 8 8C. Based on the preliminary activity of recombinants at several temperatures, 5 substitutions among the 14 were found to be beneficial. A recombinant of these five mutations, named as 5CR, exhibited 7-fold higher catalytic efficiency than wild-type (WT) lipase at 108 8 8 8 8C. All the mutants, individually and in a recombinant (5CR), were characterized by substratebinding parameters, melting temperatures and secondary structure. 5CR was similar to WT in substrate preferences and showed a significant improvement in activity at both lower and higher temperatures compared with the WT. To establish the contribution of mutations on the dynamics of the protein, we performed 100-ns molecular dynamics (MD) simulations on the WT and mutant lipase at 10 and 378 8 8 8 8C. The root mean square fluctuations (RMSFs) indeed showed that the mutations enhance the protein dynamics locally in the loop region having a catalytic residue, which may help in improved activities at lower temperatures.

Tumor suppressor gene, STK11, encodes for serine-threonine kinase, which has a critical role in regulating cell growth and apoptosis. Mutations of the same lead to the inactivation of STK11, which eventually causes different types of cancer. In this study, we focused on identifying those driver mutations through analyzing structural variations of mutants, viz., D194N, E199K, L160P, and Y49D. Native and the mutants were analyzed to determine their geometrical deviations such as root-mean-square deviation, root-mean-square fluctuation, radius of gyration, potential energy, and solvent-accessible surface area using conformational sampling technique. Additionally, the global minimized structure of native and mutants was further analyzed to compute their intramolecular interactions and distribution of secondary structure. Subsequently, simulated thermal denaturation and docking studies were performed to determine their structural variations, which in turn alter the formation of active co...

E2 ubiquitin-conjugating enzymes are crucial mediators of protein ubiquitination, which strongly influence the ultimate fate of the target substrates. Recently, it has been shown that the activity of several enzymes of the ubiquitination pathway is finely tuned by phosphorylation, an ubiquitous mechanism for cellular regulation, which modulates protein conformation. In this contribution, we provide the first rationale, at the molecular level, of the regulatory mechanism mediated by casein kinase 2 (CK2) phosphorylation of E2 Cdc34-like enzymes. In particular, we identify two co-evolving signature elements in one of the larger families of E2 enzymes: an acidic insertion in b4a2 loop in the proximity of the catalytic cysteine and two conserved key serine residues within the catalytic domain, which are phosphorylated by CK2. Our investigations, using yeast Cdc34 as a model, through 2.5 ms molecular dynamics simulations and biochemical assays, define these two elements as an important phosphorylation-controlled switch that modulate opening and closing of the catalytic cleft. The mechanism relies on electrostatic repulsions between a conserved serine phosphorylated by CK2 and the acidic residues of the b4a2 loop, promoting E2 ubiquitin charging activity. Our investigation identifies a new and unexpected pivotal role for the acidic loop, providing the first evidence that this loop is crucial not only for downstream events related to ubiquitin chain assembly, but is also mandatory for the modulation of an upstream crucial step of the ubiquitin pathway: the ubiquitin charging in the E2 catalytic cleft.

We have developed a method for calculating the association energy of quaternary complexes starting from their atomic coordinates. The association energy is described as the sum of two solvation terms and an energy term to account for the loss of translational and rotational entropy. The calculated solvation energy, using atomic solvation parameters and the solvent accessible surface areas, has a correlation of 96% with experimentally determined values. We have applied this methodology to examine intermediates in viral assembly and to assess the contribution isomerization makes to the association energy of molecular complexes. In addition, we have shown that the calculated association can be used as a predictive tool for analyzing modeled molecular complexes.

The hydrophobic core packing in four-a-helical bundles appears to be crucial for stabilizing the protein structure. To examine the structural basis of hydrophobic stabilization, the crystal structures of the Leu 3 Val (L41V) and Leu 3 Ala (L41A) substitutions of the core residue Leu41 of the ROP protein have been determined. Both substitutions are destabilizing and lead to formation of cavities. The main responses to mutations are the collapse of the central part of the a-helix containing the site of mutation, shifts of internal water molecules, and in L41A, the trapping of a water molecule in a cavity engineered by the mutation. For both mutants, these effects limit the increase in cavity size to less than 10 A Ê 3 , while an increase of 37 A Ê 3 and 100 A Ê 3 is expected for L41V and L41A, respectively, in the absence of any cavity size reducing effects. The mobility of internal side-chains is increased and in L41A, it reaches values typical for exposed residues. A parameter (Án h ) is introduced as a measure of the number of van der Waals contacts lost. For ROP, barnase and T4 lysozyme mutants, there is a good correlation between Án h and the free energy of unfolding ÁÁG relative to wild-type protein. The Án h value turns out to be more suitable for analysing structural and energetic responses to mutation than other parameter, such as cavity volumes and packing densities. Possible evolutionary implications of the ÁÁG versus Án h relationship are discussed.

Among the different classes of enzymes involved in the ubiquitin pathway, E2 ubiquitin-conjugating enzymes occupy a central role in the ubiquitination cascade. Cdc34-like E2 enzymes are characterized by a 12-14 residue insertion in the proximity of the catalytic site, known as the acidic loop. Cdc34 ubiquitin-charging activity is regulated by CK2-dependent phosphorylation and the regulatory mechanism involves the acidic loop. Indeed, the phosphorylation stabilizes the loop in an open conformation that is competent for ubiquitin charging.

a b s t r a c t E2 ubiquitin-conjugating enzymes are key elements of the ubiquitin (Ub) pathway, since they influence processivity and topology of the Ub chain assembly and, as a consequence, the fate of the target substrates. E2s are multi-domain proteins, with accessory N-terminal or C-terminal domains that can contribute to the specificity for the cognate Ub-like molecules, or even the E3. In this context, the thorough structural characterization of E2 accessory domains is mandatory, in particular when they are associated to specific functions.

The degree of similarity in the three-dimensional structures of two proteins can be examined by comparing the patterns of hydrophobicity found in their amino acid sequences. Each type of amino acid residue is assigned a numerical hydrophobicity, and the correlation coefficient ru is computed between all pairs of residues in the two sequences.

L’isoforme M2 du pyruvate kinase (PKM2) est responsable de la conversion du phosphoénolpyruvate (PEP) en pyruvate. Cette étape est la dernière étape de catalyse dans le processus de glycolyse puis joue un rôle essentiel dans la fermentation lactique effectuée chez les cellules anaérobiques, notamment les cellules myocardiques. De plus, le pyruvate kinase joue un rôle important dans le métabolisme des cellules cancéreuses. Au cours de cette expérience, nous caractérisent une mutante (K422R) de la PKM2 à l’aide de dosages enzymatiques des deux formes de l’enzyme en présence de certains effecteurs étant déjà identifiés comme régulateurs allostérique de la PKM2. La mutation a été confirmée à l’aide de séquençage et alignement de séquence avec la séquence protéique de la PKM2, retrouvée sur NCBI. Nos résultats démontrent que la mutation K422R favorise l’état T tétramérique et inactive de la PKM2 à cause de formation de liens stabilisantes au sein de la structure globulaire. La PKM2, sous sa forme active, existe plutôt sous l’état R tétramérique (3). Notre étude met en évidence une façon efficace de réguler l’activité enzymatique de la PKM2 à travers un changement dans la structure cristalline du type sauvage. Cette information s’avère utile pour des études dans le traitement du cancer qui vise à inhiber la fonction de la PKM2.

The stability scale of 20 amino acid residues is derived from a database of 1023 mutation experiments on 35 proteins. The resulting scale of hydrophobic residues has an excellent correlation with the octanol-to-water transfer free energy corrected with an additional Flory-Huggins molarvolume term (correlation coefficient r ‫؍‬ 0.95, slope ‫؍‬ 1.05, and a near zero intercept). Thus, hydrophobic contribution to folding stability is characterized remarkably well by transfer experiments. However, no corresponding correlation is found for hydrophilic residues. Both the hydrophilic portion and the entire scale, however, correlate strongly with average burial accessible surface (r ‫؍‬ 0.76 and 0.97, respectively). Such a strong correlation leads to a near uniform value of the atomic solvation parameters for atoms C, S, O/N, O ؊0.5 , and N ؉0.5,1 . All are in the range of 12-28 cal mol ؊1 Å ؊2 , close to the original estimate of hydrophobic contribution of 25-30 cal mol ؊1 Å ؊2 to folding stability. Without any adjustable parameters, the new stability scale and new atomic solvation parameters yielded an accurate prediction of protein-protein binding free energy for a separate database of 21 protein-protein complexes (r ‫؍‬ 0.80 and slope ‫؍‬ 1.06, and r ‫؍‬ 0.83 and slope ‫؍‬ 0.93, respectively). Proteins 2002;49:483-492.

Previous experimental studies on thermostable lipase from Shewanella putrefaciens suggested the maximum activity at higher temperatures, but with little information on its conformational profile. In this study, the three-dimensional structure of lipase was predicted and a 60 ns molecular dynamics (MD) simulation was carried out at temperatures ranging from 300 to 400 K to better understand its thermostable nature at the molecular level. MD simulations were performed in order to predict the optimal activity of thermostable lipase. The results suggested strong conformational temperature dependence. The thermostable lipase maintained its bio-active conformation at 350 K during the 60 ns MD simulations.

Background: Protein dynamics influence protein function and stability and modulate conformational changes. Such motions depend on the underlying networks of intramolecular interactions and communicating residues within the protein structure. Here, we provide the first characterization of the dynamic fingerprint of the dimeric alkaline phosphatase (AP) from the cold-adapted Vibrio strain G15-21 (VAP), which is among the APs with the highest known k cat at low temperatures. Methods: Multiple all-atom explicit solvent molecular dynamics simulations were employed in conjunction with different metrics to analyze the dynamical patterns and the paths of intra-and intermolecular communication. Results: Interactions and coupled motions at the interface between the two VAP subunits have been characterized, along with the networks of intramolecular interactions. It turns out a low number of intermolecular interactions and coupled motions, which result differently distributed in the two monomers. The paths of long-range communication mediated from the catalytic residues to distal sites were also characterized, pointing out a different information flow in the two subunits. Conclusions: A pattern of asymmetric flexibility is evident in the two identical subunits of the VAP dimer that is intimately linked to a different distribution of intra-and intermolecular interactions. The asymmetry was also evident in pairs of cross-correlated residues during the dynamics. General significance: The results here discussed provide a structural rationale to the half-of-site mechanism previously proposed for VAP and other APs, as well as a general framework to characterize asymmetric dynamics in homomeric enzymes.

To understand water-protein interactions in solution, the electrostatic field is calculated by solving the Poisson-Boltzmann equation, and the free energy surface of water is mapped by translating and rotating an explicit water molecule around the protein. The calculation is applied to T4 lysozyme with data available on the conservation of solvent binding sites in 18 crystallographically independent molecules. The free energy maps around the ordered water sites provide information on the relationship between water positions in crystal structure and in solution. Results show that almost all conserved sites and the majority of nonconserved sites are within 1.3 Å of local free energy minima. This finding is in sharp contrast to the behavior of randomly placed water molecules in the boundary layer, which, on the average, must travel more than 3 Å to the nearest free energy minimum. Thus, the solvation sites are at least partially determined by protein-water interactions rather than by crystal packing alone. The characteristic water residence times, obtained from the free energies at the local minima, are in good agreement with nuclear magnetic resonance experiments. Only about half of the potential sites show up as ordered water in the 1.7 Å resolution X-ray structure. Crystal packing interactions can stabilize weak or mobile potential sites (in fact, some ordered water positions are not close to free energy minima) or can prevent water from occupying certain sites. Apart from a few buried water molecules that are strong binders, the free energies are not very different for conserved and nonconserved sites. We show that conservation of a water site between two crystals occurs if the positions of protein atoms, primarily contributing to the free energy at the local minimum, do not substantially change from one structure to the other. This requirement can be correlated with the nature of the side chain contacting the water molecule in the site.

A cold-active a-amylase was purified from culture supernatants of the antarctic psychrophile Alteromonas haloplanctis A23 grown at 4 "C. In order to contribute to the understanding of the molecular basis of cold adaptations, crystallographic studies of this cold-adapted enzyme have been initiated because a threedimensional structure of a mesophilic counterpart, pig pancreatic a-amylase, already exists. a-Amylase from A. haloplanctis, which shares 53% sequence identity with pig pancreatic a-amylase, has been crystallized and data to 1.85 A have been collected. The space group is found to be C222, with a = 71.40 A, b = 138.88 A, and c = 115.66 A. Until now, a three-dimensional structure of a psychrophilic enzyme is lacking.

Ras-related protein (Rab-5a) is primarily involved in the regulation of early endosome fusion during endocytosis and takes part in the budding process. During GTP hydrolysis, Rab5a was spotted in the cytoplasmic side of early endo-somes in association with the GTP. Previous study suggested that the substitution of alanine with proline at position 30 of Rab5a reduces the GTPase activity around 12-fold, while, with arginine substitution stimulates the intrinsic GTP hydrolysis by 5-fold. Most of the other substitutions at this position show a little or no effect on the GTPase activity. In this paper, structure analysis and molecular dynamics (MD) simulation studies of human Rab5a and its mutants have been extensively carried out. The effect of binding of a non-hydrolyzable GTP analog guanosine-5′-(β, γ)-imidotriphos-phate (GppNHp) with Rab5a and its mutants are described. The objective of the current study is to perform a detailed examination of structural flexibility of Rab5a and its mutants p.Ala30Pro and p.Ala30Arg using MD simulations. Our observations suggest that mutant p.Ala30Arg stabilize the protein molecule when bound to GppNHp which offers additional contacts. Despite an in silico approach, this study provides a deep insight into the impact of mutation on the structure , function, stability, and mechanism of binding of GppNHp to the Rab5a at molecular level.

Factor X (FX) is one of the major players in the blood coagulation cascade. Upon activation to FXa, it converts prothrombin to thrombin, which in turn converts fibrinogen into fibrin (blood clots). FXa deficiency causes hemostasis defects, such as intracranial bleeding, hemathrosis, and gastrointestinal blood loss. Herein, we have analyzed a pool of pathogenic mutations, located in the FXa catalytic domain and directly associated with defects in enzyme catalytic activity. Using chymotrypsinogen numbering, they correspond to D102N, T135M, V160A, G184S, and G197D. Molecular dynamics simulations were performed for 1.68 μs on the wild-type and mutated forms of FXa. Overall, our analysis shows that four of the five mutants considered, D102N, T135M, V160A, and G184S, have rigidities higher than those of the wild type, in terms of both overall protein motion and, specifically, subpocket S4 flexibility, while S1 is rather insensitive to the mutation. This acquired rigidity can clearly impact the substrate recognition of the mutants.

Ras-related protein (Rab-5a) is primarily involved in the regulation of early endosome fusion during endocytosis and takes part in the budding process. During GTP hydrolysis, Rab5a spotted in the cytoplasmic side of early endosomes in association with the GTP. Previous study suggested that substitution of alanine with proline at position 30 of Rab5a reduces the GTPase activity around 12-fold. While, with arginine substitution stimulates the intrinsic GTP hydrolysis by 5-fold. Most of the other substitutions at this position show a little or no effect on the GTPase activity. In this paper, structure analysis and molecular dynamics (MD) simulation studies of human Rab5a and its mutants have been extensively carried out. The effect of binding of a nonhydrolyzable GTP analog guanosine-5’-(β,γ)-imidotriphosphate (GppNHp) with Rab5a and its mutants are described. The objective of the current study is to perform a detailed examination of structural flexibility of Rab5a and its mutants p.Ala30Pro and p.Ala30Arg using MD simulations. Our observations suggest that mutant p.Ala30Arg stabilize the protein molecule when bound to GppNHp which offers additional contacts. Despite of an in silico approach, this
study provides a deep insight into the impact of mutation on the structure, function, stability and mechanism of binding of GppNHp to the Rab5a at molecular level.

E2 ubiquitin-conjugating enzymes are crucial mediators of protein ubiquitination, which strongly influence the ultimate fate of the target substrates. Recently, it has been shown that the activity of several enzymes of the ubiquitination pathway is finely tuned by phosphorylation, an ubiquitous mechanism for cellular regulation, which modulates protein conformation. In this contribution, we provide the first rationale, at the molecular level, of the regulatory mechanism mediated by casein kinase 2 (CK2) phosphorylation of E2 Cdc34-like enzymes. In particular, we identify two co-evolving signature elements in one of the larger families of E2 enzymes: an acidic insertion in b4a2 loop in the proximity of the catalytic cysteine and two conserved key serine residues within the catalytic domain, which are phosphorylated by CK2. Our investigations, using yeast Cdc34 as a model, through 2.5 ms molecular dynamics simulations and biochemical assays, define these two elements as an important phosphorylation-controlled switch that modulate opening and closing of the catalytic cleft. The mechanism relies on electrostatic repulsions between a conserved serine phosphorylated by CK2 and the acidic residues of the b4a2 loop, promoting E2 ubiquitin charging activity. Our investigation identifies a new and unexpected pivotal role for the acidic loop, providing the first evidence that this loop is crucial not only for downstream events related to ubiquitin chain assembly, but is also mandatory for the modulation of an upstream crucial step of the ubiquitin pathway: the ubiquitin charging in the E2 catalytic cleft.

Accurate identification of cavities is important in the study of protein structure, stability, design, and ligand binding. Identification and quantitation of cavities is a nontrivial problem because most cavities are connected to the protein exterior. We describe a computational procedure for quantitating cavity volumes and apply this to derive an estimate of the hydrophobic driving force in protein folding. A grid-based Monte Carlo procedure is used to position water molecules on the surface of a protein. A Voronoi procedure is used to identify and quantitate empty space within the solvated protein.

The cold-adapted ␣-amylase from Pseudoalteromonas haloplanktis (AHA) is a multidomain enzyme capable of reversible unfolding. Coldadapted proteins, including AHA, have been predicted to be structurally flexible and conformationally unstable as a consequence of a high lysine-to-arginine ratio. In order to examine the role of low arginine content in structural flexibility of AHA, the amino groups of lysine were guanidinated to form homoarginine (hR), and the structure-function-stability properties of the modified enzyme were analyzed by transverse urea gradient-gel electrophoresis. The extent of modification was monitored by MALDI-TOF-MS, and correlated to changes in activity and stability. Modifying lysine to hR produced a conformationally more stable and less active ␣-amylase. The k cat of the modified enzyme decreased with a concomitant increase in ⌬H # and decrease in K m . To interpret the structural basis of the kinetic and thermodynamic properties, the hR residues were modeled in the AHA X-ray structure and compared to the X-ray structure of a thermostable homolog. The experimental properties of the modified AHA were consistent with K106hR forming an intra-Domain B salt bridge to stabilize the active site and decrease the cooperativity of unfolding. Homo-Arg modification also appeared to alter Ca 2؉ and Cl ؊ binding in the active site. Our results indicate that replacing lysine with hR generates mesophilic-like characteristics in AHA, and provides support for the importance of lysine residues in promoting enzyme cold adaptation. These data were consistent with computational analyses that show that AHA possesses a compositional bias that favors decreased conformational stability and increased flexibility. Proteins 2006;64:486 -501.

Protein structure and dynamics are crucial for protein function. Thus, the study of conformational properties can be very informative for characterizing new proteins and to rationalize how residue substitutions at specific protein sites affect its dynamics, activity and thermal stability. Here, we investigate the structure and dynamics of the recently isolated cold-adapted acylaminoacyl peptidase from Sporosarcina psychrophila (SpAAP) by the integration of simulations, circular dichroism, mass spectrometry and other experimental data. Our study notes traits of cold-adaptation, such as lysine-to-arginine substitutions and a lack of disulphide bridges. Cold-adapted enzymes are generally characterized by a higher number of glycine residues with respect to their warm-adapted counterparts. Conversely, the SpAAP glycine content is lower than that in the warm-adapted variants. Nevertheless, glycine residues are strategically located in proximity to the functional sites in SpAAP, such as the active site and the linker between the two domains.. In particular, G457 reduces the steric hindrance around the nucleophile elbow. Our results suggest a local weakening of the intramolecular interactions in the cold-adapted enzyme. This study offers a basis for the experimental mutagenesis of SpAAP and related enzymes. The approaches employed in this study may also provide a more general framework to characterize new protein structures in the absence of X-ray or NMR data.

To further investigate the mechanism and function of allosteric activation by chloride in some ␣-amylases, the structure of the bacterial ␣-amylase from the psychrophilic micro-organism Pseudoalteromonas haloplanktis in complex with nitrate has been solved at 2.1 Å, as well as the structure of the mutants Lys300Gln (2.5 Å) and Lys300Arg (2.25 Å). Nitrate binds strongly to ␣-amylase but is a weak activator. Mutation of the critical chloride ligand Lys300 into Gln results in a chloride-independent enzyme, whereas the mutation into Arg mimics the binding site as is found in animal ␣-amylases with, however, a lower affinity for chloride. These structures reveal that the triangular conformation of the chloride ligands and the nearly equatorial coordination allow the perfect accommodation of planar trigonal monovalent anions such as NO 3 − , explaining their unusual strong binding. It is also shown that a localized negative charge such as that of Cl − , rather than a delocalized charge as in the case of nitrate, is essential for maximal activation. The chloride-free mutant Lys300Gln indicates that chloride is not mandatory for the catalytic mechanism but strongly increases the reactivity at the active site. Disappearance of the putative catalytic water molecule in this weakly active mutant supports the view that chloride helps to polarize the hydrolytic water molecule and enhances the rate of the second step in the catalytic reaction.

The importance of electrostatics in catalysis has been emphasized in the literature for a large number of enzymes. We examined this hypothesis for the Bacillus licheniformis a-amylase by constructing site-directed mutants that were predicted to change the pK a values of the catalytic residues and thus change the pH±activity profile of the enzyme. To change the pK a of the catalytic residues in the active site, we constructed mutations that altered the hydrogen bonding network, mutations that changed the solvent accessibility, and mutations that altered the net charge of the molecule. The results show that changing the hydrogen bonding network near an active site residue or changing the solvent accessibility of an active site residue will very likely result in an enzyme with drastically reduced activity. The differences in the pH±activity profiles for these mutants were modest. pH±activity profiles of mutants which change the net charge on the molecule were significantly different from the wild-type pH±activity profile. The differences were, however, difficult to correlate with the electrostatic field changes calculated. In several cases we observed that pH±activity profiles shifted in the opposite direction compared to the shift predicted from electrostatic calculations. This strongly suggests that electrostatic effects cannot be solely responsible for the pH±activity profile of the B. licheniformis a-amylase.

The ubiquitin-conjugating enzyme Cdc34 (cell division cycle 34) plays an essential role in promoting the G 1 -S-phase transition of the eukaryotic cell cycle and is phosphorylated in vivo. In the present study, we investigated if phosphorylation regulates Cdc34 function. We mapped the in vivo phosphorylation sites on budding yeast Cdc34 (yCdc34; Ser 207 and Ser 216 ) and human Cdc34 (hCdc34 Ser 203 , Ser 222 and Ser 231 ) to serine residues in the acidic tail domain, a region that is critical for Cdc34's cell cycle function. CK2 (protein kinase CK2) phosphorylates both yCdc34 and hCdc34 on these sites in vitro. CK2-mediated phosphorylation increased yCdc34 ubiquitination activity towards the yeast Saccharomyces cerevisiae Sic1 in vitro, when assayed in the presence of its cognate SCF Cdc4 E3 ligase [where SCF is Skp1 (S-phase kinase-associated protein 1)/cullin/F-box]. Similarly, mutation of the yCdc34 phosphorylation sites to alanine, aspartate or glutamate residues altered Cdc34-SCF Cdc4 -mediated Sic1 ubiquitination activity. Similar results were obtained when yCdc34's ubiquitination activity was assayed in the absence of SCF Cdc4 , indicating that phosphorylation regulates the intrinsic catalytic activity of Cdc34. To evaluate the in vivo consequences of altered Cdc34 activity, wild-type yCdc34 and the phosphosite mutants were introduced into an S. cerevisiae cdc34 deletion strain and, following synchronization in G 1 -phase, progression through the cell cycle was monitored. Consistent with the increased ubiquitination activity in vitro, cells expressing the phosphosite mutants with higher catalytic activity exhibited accelerated cell cycle progression and Sic1 degradation. These studies demonstrate that CK2-mediated phosphorylation of Cdc34 on the acidic tail domain stimulates Cdc34-SCF Cdc4 ubiquitination activity and cell cycle progression.

The Gō-like potential at a residual level has been successfully applied to the folding of proteins in many previous works. However, taking into consideration more detailed structural information in the atomic level, the definition of contacts used in these traditional Gō-models may not be suitable for all-atom simulations. Here, in this work, we develop a rational definition of contacts considering the screening effect in the crowded intramolecular environment. In such a scheme, a large amount of screened atom pairs are excluded and the number of contacts is decreased compared to the case of the traditional definition. These contacts defined by such a new definition are compatible with the all-atom representation of protein structures. To verify the rationality of the new definition of contacts, the folding of proteins CI2 and SH3 is simulated by all-atom molecular dynamics simulations. A high folding cooperativity and good correlation of the simulated ⌽-values with those obtained experimentally, especially for CI2, are found. This suggests that the all-atom Gō-model is improved compared to the traditional Gō-model. Based on the comparison of the ⌽-values, the roles of side chains in the folding are discussed, and it is concluded that the side-chain structures are more important for local contacts in determining the transition state structures. Moreover, the relations between side chain and backbone orderings are also discussed.

Mutational experiments show how changes in the hydrophobic cores of proteins affect their stabilities. Here, we estimate these effects computationally, using four-body likelihood potentials obtained by simplicial neighborhood analysis of protein packing (SNAPP). In this procedure, the volume of a known protein structure is tiled with tetrahedra having the center of mass of one amino acid side-chain at each vertex. Log-likelihoods are computed for the 8855 possible tetrahedra with equivalent compositions from structural databases and amino acid frequencies. The sum of these four-body potentials for tetrahedra present in a given protein yields the SNAPP score. Mutations change this sum by changing the compositions of tetrahedra containing the mutated residue and their related potentials. Linear correlation coef®cients between experimental mutational stability changes, Á(ÁG unfold ), and those based on SNAPP scoring range from 0.70 to 0.94 for hydrophobic core mutations in ®ve different proteins. Accurate predictions for the effects of hydrophobic core mutations can therefore be obtained by virtual mutagenesis, based on changes to the total SNAPP likelihood potential. Signi®cantly, slopes of the relation between Á(ÁG unfold ) and ÁSNAPP for different proteins are statistically distinct, and we show that these protein-speci®c effects can be estimated using the average SNAPP score per residue, which is readily derived from the analysis itself. This result enhances the predictive value of statistical potentials and supports previous suggestions that`c omparable'' mutations in different proteins may lead to different Á(ÁG unfold ) values because of differences in their¯exibility and/or conformational entropy.

Mutational experiments show how changes in the hydrophobic cores of proteins affect their stabilities. Here, we estimate these effects computationally, using four-body likelihood potentials obtained by simplicial neighborhood analysis of protein packing (SNAPP). In this procedure, the volume of a known protein structure is tiled with tetrahedra having the center of mass of one amino acid side-chain at each vertex. Log-likelihoods are computed for the 8855 possible tetrahedra with equivalent compositions from structural databases and amino acid frequencies. The sum of these four-body potentials for tetrahedra present in a given protein yields the SNAPP score. Mutations change this sum by changing the compositions of tetrahedra containing the mutated residue and their related potentials. Linear correlation coef®cients between experimental mutational stability changes, Á(ÁG unfold ), and those based on SNAPP scoring range from 0.70 to 0.94 for hydrophobic core mutations in ®ve different proteins. Accurate predictions for the effects of hydrophobic core mutations can therefore be obtained by virtual mutagenesis, based on changes to the total SNAPP likelihood potential. Signi®cantly, slopes of the relation between Á(ÁG unfold ) and ÁSNAPP for different proteins are statistically distinct, and we show that these protein-speci®c effects can be estimated using the average SNAPP score per residue, which is readily derived from the analysis itself. This result enhances the predictive value of statistical potentials and supports previous suggestions that`c omparable'' mutations in different proteins may lead to different Á(ÁG unfold ) values because of differences in their¯exibility and/or conformational entropy.

To shed light on the molecular features related to cold-adaptation in serine-proteases, we have carried out molecular dynamics simulations of homologous mesophilic and psychrophilic trypsins, with particular attention to evaluation of intramolecular interactions and flexibility.

The cold-active, chloride-dependent R-amylase from Pseudoalteromonas haloplanktis (AHA) is one of the best characterized psychrophilic enzymes, and shares high sequence and structural similarity with its mesophilic porcine counterpart (PPA). An atomic detail comparative analysis was carried out by performing more than 60 ns of multiple-replica explicit-solvent molecular dynamics simulations on the two enzymes in order to characterize the differences in ensemble properties and dynamics in solution between the two homologues. We find in both enzymes high flexibility clusters in the surroundings of the substrate-binding groove, primarily involving the long loops that protrude from the main domain's barrel structure. These loops are longer in PPA and extend further away from the core of the barrel, where the active site is located: essential fluctuations in PPA mainly affect the highly solvent-accessible portions of these loops, whereas AHA is characterized by greater flexibility in the immediate surroundings of the active site. Furthermore, detailed analysis of activesite dynamics has revealed that elements previously identified through X-ray crystallography as involved in substrate binding in both enzymes undergo concerted motions that may be linked to catalysis.

The stability scale of 20 amino acid residues is derived from a database of 1023 mutation experiments on 35 proteins. The resulting scale of hydrophobic residues has an excellent correlation with the octanol-to-water transfer free energy corrected with an additional Flory–Huggins molar-volume term (correlation coefficient r = 0.95, slope = 1.05, and a near zero intercept). Thus, hydrophobic contribution to folding stability is characterized remarkably well by transfer experiments. However, no corresponding correlation is found for hydrophilic residues. Both the hydrophilic portion and the entire scale, however, correlate strongly with average burial accessible surface (r = 0.76 and 0.97, respectively). Such a strong correlation leads to a near uniform value of the atomic solvation parameters for atoms C, S, O/N, O−0.5, and N+0.5,1. All are in the range of 12–28 cal mol−1 Å−2, close to the original estimate of hydrophobic contribution of 25–30 cal mol−1 Å−2 to folding stability. Without any adjustable parameters, the new stability scale and new atomic solvation parameters yielded an accurate prediction of protein–protein binding free energy for a separate database of 21 protein–protein complexes (r = 0.80 and slope = 1.06, and r = 0.83 and slope = 0.93, respectively). Proteins 2002;49:483–492. © 2002 Wiley-Liss, Inc.

MD simulations and continuum electrostatics calculations have been used to study the observed differences in thermostability of cold- and warm-active uracil DNA glycosylase (UDG). The present study focuses on the role of ion pairs and how they affect the thermal stability of the two enzymes. Analysis of the MD generated structural ensembles show that cod UDG (cUDG) and human UDG (hUDG) have 11 and 12 ion pairs which are present in at least 30% of the conformations. The electrostatic contribution of the ion pairs, computed using continuum electrostatics, is slightly more favorable in cUDG at 298 K. This is primarily attributed to more optimized interactions between the ion pairs and nearby dipoles/charges in cUDG. More global salt bridges are found in hUDG and are more stabilizing when compared to cUDG, possibly playing a role in maintaining overall stability and reducing conformational entropy. Both enzymes contain one three-member ionic network, but the one found in hUDG is far more stabilizing. Our results also suggest that care should be taken when performing statistical analysis of crystal structures with respect to ion pairs, and that crystallization conditions must be carefully examined when performing such analysis. Proteins 2008. © 2007 Wiley-Liss, Inc.

E2 ubiquitin-conjugating enzymes are crucial mediators of protein ubiquitination, which strongly influence the ultimate fate of the target substrates. Recently, it has been shown that the activity of several enzymes of the ubiquitination pathway is finely tuned by phosphorylation, an ubiquitous mechanism for cellular regulation, which modulates protein conformation. In this contribution, we provide the first rationale, at the molecular level, of the regulatory mechanism mediated by casein kinase 2 (CK2) phosphorylation of E2 Cdc34-like enzymes. In particular, we identify two co-evolving signature elements in one of the larger families of E2 enzymes: an acidic insertion in b4a2 loop in the proximity of the catalytic cysteine and two conserved key serine residues within the catalytic domain, which are phosphorylated by CK2. Our investigations, using yeast Cdc34 as a model, through 2.5 ms molecular dynamics simulations and biochemical assays, define these two elements as an important phosphorylation-controlled switch that modulate opening and closing of the catalytic cleft. The mechanism relies on electrostatic repulsions between a conserved serine phosphorylated by CK2 and the acidic residues of the b4a2 loop, promoting E2 ubiquitin charging activity. Our investigation identifies a new and unexpected pivotal role for the acidic loop, providing the first evidence that this loop is crucial not only for downstream events related to ubiquitin chain assembly, but is also mandatory for the modulation of an upstream crucial step of the ubiquitin pathway: the ubiquitin charging in the E2 catalytic cleft.

The cold-active, chloride-dependent R-amylase from Pseudoalteromonas haloplanktis (AHA) is one of the best characterized psychrophilic enzymes, and shares high sequence and structural similarity with its mesophilic porcine counterpart (PPA). An atomic detail comparative analysis was carried out by performing more than 60 ns of multiple-replica explicit-solvent molecular dynamics simulations on the two enzymes in order to characterize the differences in ensemble properties and dynamics in solution between the two homologues. We find in both enzymes high flexibility clusters in the surroundings of the substrate-binding groove, primarily involving the long loops that protrude from the main domain's barrel structure. These loops are longer in PPA and extend further away from the core of the barrel, where the active site is located: essential fluctuations in PPA mainly affect the highly solvent-accessible portions of these loops, whereas AHA is characterized by greater flexibility in the immediate surroundings of the active site. Furthermore, detailed analysis of activesite dynamics has revealed that elements previously identified through X-ray crystallography as involved in substrate binding in both enzymes undergo concerted motions that may be linked to catalysis.

Networks and clusters of intramolecular interactions, as well as their ''communication'' across the three-dimensional architecture have a prominent role in determining protein stability and function. Special attention has been dedicated to their role in thermal adaptation. In the present contribution, seven previously experimentally characterized mutants of a cold-adapted a-amylase, featuring mesophilic-like behavior, have been investigated by multiple molecular dynamics simulations, essential dynamics and analyses of correlated motions and electrostatic interactions. Our data elucidate the molecular mechanisms underlying the ability of single and multiple mutations to globally modulate dynamic properties of the cold-adapted a-amylase, including both local and complex unpredictable distal effects. Our investigation also shows, in agreement with the experimental data, that the conversion of the cold-adapted enzyme in a warm-adapted variant cannot be completely achieved by the introduction of few mutations, also providing the rationale behind these effects. Moreover, pivotal residues, which are likely to mediate the effects induced by the mutations, have been identified from our analyses, as well as a group of suitable candidates for protein engineering. In fact, a subset of residues here identified (as an isoleucine, or networks of mesophilic-like salt bridges in the proximity of the catalytic site) should be considered, in experimental studies, to get a more efficient modification of the features of the cold-adapted enzyme.

Molecular dynamics simulations of representative mesophilic and psycrophilic elastases have been carried out at different temperatures to explore the molecular basis of cold adaptation inside a specific enzymatic family. The molecular dynamics trajectories have been compared and analyzed in terms of secondary structure, molecular flexibility, intramolecular and protein-solvent interactions, unravelling molecular features relevant to rationalize the efficient catalytic activity of psychrophilic elastases at low temperature. The comparative molecular dynamics investigation reveals that modulation of the number of protein-solvent interactions is not the evolutionary strategy followed by the psycrophilic elastase to enhance catalytic activity at low temperature. In addition, flexibility and solvent accessibility of the residues forming the catalytic triad and the specificity pocket are comparable in the cold-and warm-adapted enzymes. Instead, loop regions with different amino acid composition in the two enzymes, and clustered around the active site or the specificity pocket, are characterized by enhanced flexibility in the cold-adapted enzyme. Remarkably, the psycrophilic elastase is characterized by reduced flexibility, when compared to the mesophilic counterpart, in some scattered regions distant from the functional sites, in agreement with hypothesis suggesting that local rigidity in regions far from functional sites can be beneficial for the catalytic activity of psychrophilic enzymes.