Comparative void-volume analysis of psychrophilic and mesophilic enzymes: Structural bioinformatics of psychrophilic enzymes reveals sources of core flexibility

Background

Psychrophiles, cold-adapted organisms, have adapted to live at low temperatures by using a variety of mechanisms. Their enzymes are active at cold temperatures by being structurally more flexible than mesophilic enzymes. Even though, there are some indications of the possible structural mechanisms by which psychrophilic enzymes are catalytic active at cold temperatures, there is not a generalized structural property common to all psychrophilic enzymes.

Results

We examine twenty homologous enzyme pairs from psychrophiles and mesophiles to investigate flexibility as a key characteristic for cold adaptation. B-factors in protein X-ray structures are one way to measure flexibility. Comparing psychrophilic to mesophilic protein B-factors reveals that psychrophilic enzymes are more flexible in 5-turn and strand secondary structures. Enzyme cavities, identified using CASTp at various probe sizes, indicate that psychrophilic enzymes have larger average cavity sizes at probe radii of 1.4-1.5 Å, sufficient for water molecules. Furthermore, amino acid side chains lining these cavities show an increased frequency of acidic groups in psychrophilic enzymes.

Conclusions

These findings suggest that embedded water molecules may play a significant role in cavity flexibility, and therefore, overall protein flexibility. Thus, our results point to the important role enzyme flexibility plays in adaptation to cold environments.

     

Adaptability in protein structures: structural dynamics and implications in ligand design

The basic framework of understanding the mechanisms of protein functions is achieved from the knowledge of their structures which can model the molecular recognition. Recent advancement in the structural biology has revealed that in spite of the availability of the structural data, it is nontrivial to predict the mechanism of the molecular recognition which progresses via situation-dependent structural adaptation. The mutual selectivity of protein–protein and protein–ligand interactions often depends on the modulations of conformations empowered by their inherent flexibility, which in turn regulates the function. The mechanism of a protein’s function, which used to be explained by the ideas of ‘lock and key’ has evolved today as the concept of ‘induced fit’ as well as the ‘population shift’ models. It is felt that the ‘dynamics’ is an essential feature to take into account for understanding the mechanism of protein’s function. The design principles of therapeutic molecules suffer from the problems of plasticity of the receptors whose binding conformations are accurately not predictable from the prior knowledge of a template structure. On the other hand, flexibility of the receptors provides the opportunity to improve the binding affinity of a ligand by suitable substitution that will maximize the binding by modulating the receptors surface. In this paper, we discuss with example how the protein’s flexibility is correlated with its functions in various systems, revealing the importance of its understanding and for making applications. We also highlight the methodological challenges to investigate it computationally and to account for the flexible nature of the molecules in drug design.     

Fold recognition of the human immunodeficiency virus type 1 V3 loop and flexibility of its crown structure during the course of adaptation to a host

The third hypervariable (V3) region of the HIV-1 gp120 protein is responsible for many aspects of viral infectivity. The tertiary structure of the V3 loop seems to influence the coreceptor usage of the virus, which is an important determinant of HIV pathogenesis. Hence, the information about preferred conformations of the V3-loop region and its flexibility could be a crucial tool for understanding the mechanisms of progression from an initial infection to AIDS. Taking into account the uncertainty of the loop structure, we predicted the structural flexibility, diversity, and sequence fitness to the V3-loop structure for each of the sequences serially sampled during an asymptomatic period. Structural diversity correlated with sequence diversity. The predicted crown structure usage implied that structural flexibility depended on the patient and that the antigenic character of the virus might be almost uniform in a patient whose immune system is strong. Furthermore, the predicted structural ensemble suggested that toward the end of the asymptomatic period there was a change in the V3-loop structure or in the environment surrounding the V3 loop, possibly because of its proximity to the gp120 core.     

Dynameomics: large-scale assessment of native protein flexibility

Structure is only the first step in understanding the interactions and functions of proteins. In this paper, we explore the flexibility of proteins across a broad database of over 250 solvated protein molecular dynamics simulations in water for an aggregate simulation time of approximately 6 micros. These simulations are from our Dynameomics project, and these proteins represent approximately 75% of all known protein structures. We employ principal component analysis of the atomic coordinates over time to determine the primary axis and magnitude of the flexibility of each atom in a simulation. This technique gives us both a database of flexibility for many protein fold families and a compact visual representation of a particular protein's native-state conformational space, neither of which are available using experimental methods alone. These tools allow us to better understand the nature of protein motion and to describe its relationship to other structural and dynamical characteristics. In addition to reporting general properties of protein flexibility and detailing many dynamic motifs, we characterize the relationship between protein native-state flexibility and early events in thermal unfolding and show that flexibility predicts how a protein will begin to unfold. We provide evidence that fold families have conserved flexibility patterns, and family members who deviate from the conserved patterns have very low sequence identity. Finally, we examine novel aspects of highly inflexible loops that are as important to structural integrity as conventional secondary structure. These loops, which are difficult if not impossible to locate without dynamic data, may constitute new structural motifs.     

A survey of flexible protein binding mechanisms and their transition states using native topology based energy landscapes

Many cellular functions rely on interactions between protein pairs and higher oligomers. We have recently shown that binding mechanisms are robust and owing to the minimal frustration principle, just as for protein folding, are governed primarily by the protein's native topology, which is characterized by the network of non-covalent residue-residue interactions. The detailed binding mechanisms of nine dimers, a trimer, and a tetramer, each involving different degrees of flexibility and plasticity during assembly, are surveyed here using a model that is based solely on the protein topology, having a perfectly funneled energy landscape. The importance of flexibility in binding reactions is manifested by the fly-casting effect, which is diminished in magnitude when protein flexibility is removed. Many of the grosser and finer structural aspects of the various binding mechanisms (including binding of pre-folded monomers, binding of intrinsically unfolded monomers, and binding by domain-swapping) predicted by the native topology based landscape model are consistent with the mechanisms found in the laboratory. An asymmetric binding mechanism is often observed for the formation of the symmetric homodimers where one monomer is more structured at the binding transition state and serves as a template for the folding of the other monomer. Phi values were calculated to show how the structure of the binding transition state ensemble would be manifested in protein engineering studies. For most systems, the simulated Phi values are reasonably correlated with the available experimental values. This agreement suggests that the overall binding mechanism and the nature of the binding transition state ensemble can be understood from the network of interactions that stabilize the native fold. The Phi values for the formation of an antibody-antigen complex indicate a possible role for solvation of the interface in biomolecular association of large rigid proteins.     

Freedom and restraint: themes in virus capsid assembly

Viruses assemble protective capsids from several copies of one or a few structural proteins. This is accomplished through a combination of conformational flexibility and control mechanisms that restrict this flexibility. This review will discuss some of these mechanisms in light of the many recent results in this area.     

Insights into correlated motions and long-range interactions in CheY derived from molecular dynamics simulations

CheY is a response regulator protein involved in bacterial chemotaxis. Much is known about its active and inactive conformations, but little is known about the mechanisms underlying long-range interactions or correlated motions. To investigate these events, molecular dynamics simulations were performed on the unphosphorylated, inactive structure from Salmonella typhimurium and the CheY-BeF(3)(-) active mimic structure (with BeF(3)(-) removed) from Escherichia coli. Simulations utilized both sequences in each conformation to discriminate sequence- and structure-specific behavior. The previously identified conformational differences between the inactive and active conformations of the strand-4-helix-4 loop, which are present in these simulations, arise from the structural, and not the sequence, differences. The simulations identify previously unreported structure-specific flexibility features in this loop and sequence-specific flexibility features in other regions of the protein. Both structure- and sequence-specific long-range interactions are observed in the active and inactive ensembles. In the inactive ensemble, two distinct mechanisms based on Thr-87 or Ile-95 rotameric forms, are observed for the previously identified g+ and g- rotamer sampling by Tyr-106. These molecular dynamics simulations have thus identified both sequence- and structure-specific differences in flexibility, long-range interactions, and rotameric form of key residues. Potential biological consequences of differential flexibility and long-range correlated motion are discussed.     

Synapse maturation and structural plasticity at Drosophila neuromuscular junctions

The Drosophila larval neuromuscular junction has recently emerged as a powerful model system to characterize the cellular and molecular events involved in the formation and flexibility of synapses. The combination of molecular, genetic, electrophysiological and anatomical approaches has revealed, for example, the functional significance of the discs-large gene product (a novel synapse-organizing protein) in the nervous system. This protein is involved in the clustering of at least one ion channel and in the structural modification of glutamatergic synapses during target muscle growth. The manipulation of the genes encoding ion channels, components of second-messenger cascades, and cell adhesion molecules is beginning to tease apart the mechanisms underlying structural synaptic plasticity.     

On the relation between residue flexibility and residue interactions in proteins

B-factor from X-ray crystal structure can well measure protein structural flexibility, which plays an important role in different biological processes, such as catalysis, binding and molecular recognition. Understanding the essence of flexibility can be helpful for the further study of the protein function. In this study, we attempted to correlate the flexibility of a residue to its interactions with other residues by representing the protein structure as a residue contact network. Here, several well established network topological parameters were employed to feature such interactions. A prediction model was constructed for B-factor of a residue by using support vector regression (SVR). Pearson correlation coefficient (CC) was used as the performance measure. CC values were 0.63 and 0.62 for single amino acid and for the whole sequence, respectively. Our results revealed well correlations between B-factors and network topological parameters. This suggests that the protein structural flexibility could be well characterized by the inter-amino acid interactions in a protein.     

Conformational Effects on the Circular Dichroism of Human Carbonic Anhydrase II: A Multilevel Computational Study

Circular Dichroism (CD) spectroscopy is a powerful method for investigating conformational changes in proteins and therefore has numerous applications in structural and molecular biology. Here a computational investigation of the CD spectrum of the Human Carbonic Anhydrase II (HCAII), with main focus on the near-UV CD spectra of the wild-type enzyme and it seven tryptophan mutant forms, is presented and compared to experimental studies. Multilevel computational methods (Molecular Dynamics, Semiempirical Quantum Mechanics, Time-Dependent Density Functional Theory) were applied in order to gain insight into the mechanisms of interaction between the aromatic chromophores within the protein environment and understand how the conformational flexibility of the protein influences these mechanisms. The analysis suggests that combining CD semi empirical calculations, crystal structures and molecular dynamics (MD) could help in achieving a better agreement between the computed and experimental protein spectra and provide some unique insight into the dynamic nature of the mechanisms of chromophore interactions.     

Conservation of Flexible Residue Clusters among Structural and Functional Enzyme Homologues

Conformational flexibility between structural ensembles is an essential component of enzyme function. Although the broad dynamical landscape of proteins is known to promote a number of functional events on multiple time scales, it is yet unknown whether structural and functional enzyme homologues rely on the same concerted residue motions to perform their catalytic function. It is hypothesized that networks of contiguous and flexible residue motions occurring on the biologically relevant millisecond time scale evolved to promote and/or preserve optimal enzyme catalysis. In this study, we use a combination of NMR relaxation dispersion, model-free analysis, and ligand titration experiments to successfully capture and compare the role of conformational flexibility between two structural homologues of the pancreatic ribonuclease family: RNase A and eosinophil cationic protein (or RNase 3). In addition to conserving the same catalytic residues and structural fold, both homologues show similar yet functionally distinct clusters of millisecond dynamics, suggesting that conformational flexibility can be conserved among analogous protein folds displaying low sequence identity. Our work shows that the reduced conformational flexibility of eosinophil cationic protein can be dynamically and functionally reproduced in the RNase A scaffold upon creation of a chimeric hybrid between the two proteins. These results support the hypothesis that conformational flexibility is partly required for catalytic function in homologous enzyme folds, further highlighting the importance of dynamic residue sectors in the structural organization of proteins.     

Structural basis of cellulosome efficiency explored by small angle X-ray scattering

Cellulose, the main structural component of plant cell walls, is the most abundant carbohydrate polymer in nature. To break down plant cell walls, anaerobic microorganisms have evolved a large extracellular enzyme complex termed cellulosome. This megadalton catalytic machinery organizes an enzymatic assembly, tenaciously bound to a scaffolding protein via specialized intermodular "cohesin-dockerin" interactions that serve to enhance synergistic activity among the different catalytic subunits. Here, we report the solution structure properties of cellulosome-like assemblies analyzed by small angle x-ray scattering and molecular dynamics. The atomic models, generated by our strategy for the free chimeric scaffoldin and for binary and ternary complexes, reveal the existence of various conformations due to intrinsic structural flexibility with no, or only coincidental, inter-cohesin interactions. These results provide primary evidence concerning the mechanisms by which these protein assemblies attain their remarkable synergy. The data suggest that the motional freedom of the scaffoldin allows precise positioning of the complexed enzymes according to the topography of the substrate, whereas short-scale motions permitted by residual flexibility of the enzyme linkers allow "fine-tuning" of individual catalytic domains.     

Evolution of structurally disordered proteins promotes neostructuralization

Protein structure is generally more conserved than sequence, but for regions that can adopt different structures in different environments, does this hold true? Understanding how structurally disordered regions evolve altered secondary structure element propensities as well as conformational flexibility among paralogs are fundamental questions for our understanding of protein structural evolution. We have investigated the evolutionary dynamics of structural disorder in protein families containing both orthologs and paralogs using phylogenetic tree reconstruction, protein structure disorder prediction, and secondary structure prediction in order to shed light upon these questions. Our results indicate that the extent and location of structurally disordered regions are not universally conserved. As structurally disordered regions often have high conformational flexibility, this is likely to have an effect on how protein structure evolves as spatially altered conformational flexibility can also change the secondary structure propensities for homologous regions in a protein family.     

Conserved tertiary couplings stabilize elements in the PDZ fold,leading to characteristic patterns of domain conformational flexibility

Single‐domain allostery has been postulated to occur through intramolecular pathways of signaling within a protein structure. We had previously investigated these pathways by introducing a local thermal perturbation and analyzed the anisotropic propagation of structural changes throughout the protein. Here, we develop an improved approach, the Rotamerically Induced Perturbation (RIP), that identifies strong couplings between residues by analyzing the pathways of heat‐flow resulting from thermal excitation of rotameric rotations at individual residues. To explore the nature of these couplings, we calculate the complete coupling maps of 5 different PDZ domains. Although the PDZ domain is a well conserved structural fold that serves as a scaffold in many protein–protein complexes, different PDZ domains display unique patterns of conformational flexibility in response to ligand binding: some show a significant shift in a set of α‐helices, while others do not. Analysis of the coupling maps suggests a simple relationship between the computed couplings and observed conformational flexibility. In domains where the α‐helices are rigid, we find couplings of the α‐helices to the body of the protein, whereas in domains having ligand‐responsive α‐helices, no couplings are found. This leads to a model where the α‐helices are intrinsically dynamic but can be damped if sidechains interact at key tertiary contacts. These tertiary contacts correlate to high covariation contacts as identified by the statistical coupling analysis method. As these dynamic modules are exploited by various allosteric mechanisms, these tertiary contacts have been conserved by evolution.     

Relation between flexibility and positively selected HIV‐1 protease mutants against inhibitors

The antiretroviral chemotherapy helps to reduce the mortality of HIVs infected patients. However, RNA dependant virus replication has a high mutation rate. Human immunodeficiency virus Type 1 protease plays an essential role in viral replication cycle. This protein is an important target for therapy with viral protein inhibitors. There are few works using normal mode analysis to investigate this problem from the structural changes viewpoint. The investigation of protein flexibility may be important for the study of processes associated with conformational changes and state transitions. The normal mode analysis allowed us to investigate structural changes in the protease (such as flexibility) in a straightforward way and try to associate these changes with the increase of fitness for each positively selected HIV‐1 mutant protease of patients treated with several protease inhibitors (saquinavir, indinavir, ritonavir, nelfinavir, lopinavir, fosamprenavir, atazanavir, darunavir, and tripanavir) in combination or separately. These positively selected mutations introduce significant flexibility in important regions such as the active site cavity and flaps. These mutations were also able to cause changes in accessible solvent area. This study showed that the majority of HIV‐1 protease mutants can be grouped into two main classes of protein flexibility behavior. We presented a new approach to study structural changes caused by positively selected mutations in a pathogen protein, for instance the HIV‐1 protease and their relationship with their resistance mechanism against known inhibitors. The method can be applied to any pharmaceutically relevant pathogen proteins and could be very useful to understand the effects of positively selected mutations in the context of structural changes. Proteins 2012; © 2012 Wiley Periodicals, Inc.     

Statistical analysis of structural determinants for protein–DNA‐binding specificity

DNA‐binding proteins play critical roles in biological processes including gene expression, DNA packaging and DNA repair. They bind to DNA target sequences with different degrees of binding specificity, ranging from highly specific (HS) to nonspecific (NS). Alterations of DNA‐binding specificity, due to either genetic variation or somatic mutations, can lead to various diseases. In this study, a comparative analysis of protein–DNA complex structures was carried out to investigate the structural features that contribute to binding specificity. Protein–DNA complexes were grouped into three general classes based on degrees of binding specificity: HS, multispecific (MS), and NS. Our results show a clear trend of structural features among the three classes, including amino acid binding propensities, simple and complex hydrogen bonds, major/minor groove and base contacts, and DNA shape. We found that aspartate is enriched in HS DNA binding proteins and predominately binds to a cytosine through a single hydrogen bond or two consecutive cytosines through bidentate hydrogen bonds. Aromatic residues, histidine and tyrosine, are highly enriched in the HS and MS groups and may contribute to specific binding through different mechanisms. To further investigate the role of protein flexibility in specific protein–DNA recognition, we analyzed the conformational changes between the bound and unbound states of DNA‐binding proteins and structural variations. The results indicate that HS and MS DNA‐binding domains have larger conformational changes upon DNA‐binding and larger degree of flexibility in both bound and unbound states. Proteins 2016; 84:1147–1161. © 2016 Wiley Periodicals, Inc.     

Predicting Evolutionary Site Variability from Structure in Viral Proteins: Buriedness,Packing, Flexibility,and Design

Several recent works have shown that protein structure can predict site-specific evolutionary sequence variation. In particular, sites that are buried and/or have many contacts with other sites in a structure have been shown to evolve more slowly, on average, than surface sites with few contacts. Here, we present a comprehensive study of the extent to which numerous structural properties can predict sequence variation. The quantities we considered include buriedness (as measured by relative solvent accessibility), packing density (as measured by contact number), structural flexibility (as measured by B factors, root-mean-square fluctuations, and variation in dihedral angles), and variability in designed structures. We obtained structural flexibility measures both from molecular dynamics simulations performed on nine non-homologous viral protein structures and from variation in homologous variants of those proteins, where they were available. We obtained measures of variability in designed structures from flexible-backbone design in the Rosetta software. We found that most of the structural properties correlate with site variation in the majority of structures, though the correlations are generally weak (correlation coefficients of 0.1–0.4). Moreover, we found that buriedness and packing density were better predictors of evolutionary variation than structural flexibility. Finally, variability in designed structures was a weaker predictor of evolutionary variability than buriedness or packing density, but it was comparable in its predictive power to the best structural flexibility measures. We conclude that simple measures of buriedness and packing density are better predictors of evolutionary variation than the more complicated predictors obtained from dynamic simulations, ensembles of homologous structures, or computational protein design.     

Local flexibility facilitates oxidization of buried methionine residues

In proteins, all amino acid residues are susceptible to oxidation by various reactive oxygen species (ROS), with methionine and cysteine residues being particularly sensitive to oxidation. Methionine oxidation is known to lead to destabilization and inactivation of proteins, and oxidatively modified proteins can accumulate during aging, oxidative stress, and in various age-related diseases. Although the efficiency of a given methionine oxidation can depend on its solvent accessibility (evaluated from a protein structure as the accessible surface area of the corresponding methionine residue), many experimental results on oxidation rate and oxidation sites cannot be unequivocally explained by the methionine solvent accessible surface area alone. In order to explore other possible mechanisms, we analyzed a set of seventy-one oxidized methionines contained in thirty-one proteins by various bioinformatics tools. In which, 41% of the methionines are exposed, 15% are buried but with various degree of flexibility, and the rest 44% are buried and structured. Buried but highly flexible methionines can be oxidized. Buried and less flexible methionines can acquire additional local structural flexibility from flanking regions to facilitate the oxidation. Oxidation of buried and structured methionine can also be promoted by the oxidation of neighboring methionine that is more exposed and/or flexible. Our data are consistent with the hypothesis that protein structural flexibility represents another important factor favoring the oxidation process.     

Multiple flexible structure alignment using partial order graphs

MOTIVATION: Existing comparisons of protein structures are not able to describe structural divergence and flexibility in the structures being compared because they focus on identifying a common invariant core and ignore parts of the structures outside this core. Understanding the structural divergence and flexibility is critical for studying the evolution of functions and specificities of proteins. RESULTS: A new method of multiple protein structure alignment, POSA (Partial Order Structure Alignment), was developed using a partial order graph representation of multiple alignments. POSA has two unique features: (1) identifies and classifies regions that are conserved only in a subset of input structures and (2) allows internal rearrangements in protein structures. POSA outperforms other programs in the cases where structural flexibilities exist and provides new insights by visualizing the mosaic nature of multiple structural alignments. POSA is an ideal tool for studying the variation of protein structures within diverse structural families. AVAILABILITY: POSA is freely available for academic users on a Web server at http://fatcat.burnham.org/POSA