Peptide segments in protein-protein interfaces

An important component of functional genomics involves the understanding of protein association. The interfaces resulting from protein-protein interactions - (i) specific, as represented by the homodimeric quaternary structures and the complexes formed by two independently occurring protein components, and (ii) non-specific, as observed in the crystal lattice of monomeric proteins - have been analysed on the basis of the length and the number of peptide segments. In 1000 A2 of the interface area, contributed by a polypeptide chain, there would be 3.4 segments in homodimers, 5.6 in complexes and 6.3 in crystal contacts. Concomitantly, the segments are the longest (with 8.7 interface residues) in homodimers. Core segments (likely to contribute more towards binding) are more in number in homodimers (1.7) than in crystal contacts (0.5), and this number can be used as one of the parameters to distinguish between the two types of interfaces. Dominant segments involved in specific interactions, along with their secondary structural features, are enumerated.     

Side-chain conformational entropy in protein folding.

An important, but often neglected, contribution to the thermodynamics of protein folding is the loss of entropy that results from restricting the number of accessible side-chain conformers in the native structure. Conformational entropy changes can be found by comparing the number of accessible rotamers in the unfolded and folded states, or by estimating fusion entropies. Comparison of several sets of results using different techniques shows that the mean conformational free energy change (T delta S) is 1 kcal.mol-1 per side chain or 0.5 kcal.mol-1 per bond. Changes in vibrational entropy appear to be negligible compared to the entropy change resulting from the loss of accessible rotamers. Side-chain entropies can help rationalize alpha-helix propensities, predict protein/inhibitor complex structures, and account for the distribution of side chains on the protein surface or interior.     

The relationship between the flexibility of proteins and their conformational states on forming protein-protein complexes with an application to protein-protein docking

We investigate the extent to which the conformational fluctuations of proteins in solution reflect the conformational changes that they undergo when they form binary protein-protein complexes. To do this, we study a set of 41 proteins that form such complexes and whose three-dimensional structures are known, both bound in the complex and unbound. We carry out molecular dynamics simulations of each protein, starting from the unbound structure, and analyze the resulting conformational fluctuations in trajectories of 5 ns in length, comparing with the structure in the complex. It is found that fluctuations take some parts of the molecules into regions of conformational space close to the bound state (or give information about it), but at no point in the simulation does each protein as whole sample the complete bound state. Subsequent use of conformations from a clustered MD ensemble in rigid-body docking is nevertheless partially successful when compared to docking the unbound conformations, as long as the unbound conformations are themselves included with the MD conformations and the whole globally rescored. For one key example where sub-domain motion is present, a ribonuclease inhibitor, principal components analysis of the MD was applied and was also able to produce conformations for docking that gave enhanced results compared to the unbound. The most significant finding is that core interface residues show a tendency to be less mobile (by size of fluctuation or entropy) than the rest of the surface even when the other binding partner is absent, and conversely the peripheral interface residues are more mobile. This surprising result, consistent across up to 40 of the 41 proteins, suggests different roles for these regions in protein recognition and binding, and suggests ways that docking algorithms could be improved by treating these regions differently in the docking process.     

Side-chain conformational changes upon Protein-Protein Association

Conformational changes upon protein-protein association are the key element of the binding mechanism. The study presents a systematic large-scale analysis of such conformational changes in the side chains. The results indicate that short and long side chains have different propensities for the conformational changes. Long side chains with three or more dihedral angles are often subject to large conformational transition. Shorter residues with one or two dihedral angles typically undergo local conformational changes not leading to a conformational transition. A relationship between the local readjustments and the equilibrium fluctuations of a side chain around its unbound conformation is suggested. Most of the side chains undergo larger changes in the dihedral angle most distant from the backbone. The frequencies of the core-to-surface interface transitions of six nonpolar residues and Tyr are larger than the frequencies of the opposite surface-to-core transitions. The binding increases both polar and nonpolar interface areas. However, the increase of the nonpolar area is larger for all considered classes of protein complexes, suggesting that the protein association perturbs the unbound interfaces to increase the hydrophobic contribution to the binding free energy. To test modeling approaches to side-chain flexibility in protein docking, conformational changes in the X-ray set were compared with those in the docking decoy sets. The results lead to a better understanding of the conformational changes in proteins and suggest directions for efficient conformational sampling in docking protocols.     

Structural segments and residue propensities in protein-RNA interfaces: comparison with protein-protein and protein-DNA complexes

The interface of a protein molecule that is involved in binding another protein, DNA or RNA has been characterized in terms of the number of unique secondary structural segments (SSSs), made up of stretches of helix, strand and non-regular (NR) regions. On average 10-11 segments define the protein interface in protein-protein (PP) and protein-DNA (PD) complexes, while the number is higher (14) for protein-RNA (PR) complexes. While the length of helical segments in PP interaction increases with the interface area, this is not the case in PD and PR complexes. The propensities of residues to occur in the three types of secondary structural elements (SSEs) in the interface relative to the corresponding elements in the protein tertiary structures have been calculated. Arg, Lys, Asn, Tyr, His and Gln are preferred residues in PR complexes; in addition, Ser and Thr are also favoured in PD interfaces.     

Statistical analysis and prediction of protein-protein interfaces

Predicting protein-protein interfaces from a three-dimensional structure is a key task of computational structural proteomics. In contrast to geometrically distinct small molecule binding sites, protein-protein interface are notoriously difficult to predict. We generated a large nonredundant data set of 1494 true protein-protein interfaces using biological symmetry annotation where necessary. The data set was carefully analyzed and a Support Vector Machine was trained on a combination of a new robust evolutionary conservation signal with the local surface properties to predict protein-protein interfaces. Fivefold cross validation verifies the high sensitivity and selectivity of the model. As much as 97% of the predicted patches had an overlap with the true interface patch while only 22% of the surface residues were included in an average predicted patch. The model allowed the identification of potential new interfaces and the correction of mislabeled oligomeric states.     

蛋白质-蛋白质界面热点残基预测及其在线工具

蛋白质-蛋白质结合热点是界面中对结合自由能有着显著贡献的一小簇残基。捕捉和揭示这类热点残基可以加深对蛋白质间相互作用机制的理解,为蛋白质工程和药物设计提供指导。但实验技术费时费力且代价昂贵。计算工具可用于辅助和补充实验上的尝试。该文较详细、系统地介绍了蛋白质界面热点的特性、计算预测的策略与技术,并应用实例进一步说明这些方法学的特征;还介绍了界面热点的数据库和一些主要的在线预测工具,旨在为设计、挑选和应用这类工具解决特定问题的研究人员提供指南。     

Side-chain conformational entropy in protein unfolded states

The largest force disfavoring the folding of a protein is the loss of conformational entropy. A large contribution to this entropy loss is due to the side-chains, which are restricted, although not immobilized, in the folded protein. In order to accurately estimate the loss of side-chain conformational entropy that occurs upon folding it is necessary to have accurate estimates of the amount of entropy possessed by side-chains in the ensemble of unfolded states. A new scale of side-chain conformational entropies is presented here. This scale was derived from Monte Carlo computer simulations of small peptide models. It is demonstrated that the entropies are independent of host peptide length. This new scale has the advantage over previous scales of being more precise with low standard errors. Better estimates are obtained for long (e.g., Arg and Lys) and rare (e.g., Trp and Met) side-chains. Excellent agreement with previous side-chain entropy scales is achieved, indicating that further advancements in accuracy are likely to be small at best. Strikingly, longer side-chains are found to possess a smaller fraction of the theoretical maximum entropy available than short side-chains. This indicates that rotations about torsions after chi(2) are significantly affected by side-chain interactions with the polypeptide backbone. This finding invalidates previous assumptions about side-chain-backbone interactions. Proteins 2000;40:443-450.     

Structural motifs at protein-protein interfaces: protein cores versus two-state and three-state model complexes.

The general similarity in the forces governing protein folding and protein-protein associations has led us to examine the similarity in the architectural motifs between the interfaces and the monomers. We have carried out extensive, all-against-all structural comparisons between the single-chain protein structural dataset and the interface dataset, derived both from all protein-protein complexes in the structural database and from interfaces generated via an automated crystal symmetry operation. We show that despite the absence of chain connections, the global features of the architectural motifs, present in monomers, recur in the interfaces, a reflection of the limited set of the folding patterns. However, although similarity has been observed, the details of the architectural motifs vary. In particular, the extent of the similarity correlates with the consideration of how the interface has been formed. Interfaces derived from two-state model complexes, where the chains fold cooperatively, display a considerable similarity to architectures in protein cores, as judged by the quality of their geometric superposition. On the other hand, the three-state model interfaces, representing binding of already folded molecules, manifest a larger variability and resemble the monomer architecture only in general outline. The origin of the difference between the monomers and the three-state model interfaces can be understood in terms of the different nature of the folding and the binding that are involved. Whereas in the former all degrees of freedom are available to the backbone to maximize favorable interactions, in rigid body, three-state model binding, only six degrees of freedom are allowed. Hence, residue or atom pair-wise potentials derived from protein-protein associations are expected to be less accurate, substantially increasing the number of computationally acceptable alternate binding modes (Finkelstein et al., 1995).     

The kinetics of protein-protein recognition

We examine a simple kinetic model for association that incorporates the basic features of protein-protein recognition within the rigid body approximation, that is, when no large conformation change occurs. Association starts with random collision at the rate k_coll predicted by the Einstein-Smoluchowski equation. This creates an encounter pair that can evolve into a stable complex if and only if the two molecules are correctly oriented and positioned, which has a probability p_r. In the absence of long-range interactions, the bimolecular rate of association is p_r k_coll. Long-range electrostatic interactions affect both k_coll and p_r. The collision rate is multiplied by q_t, a factor larger than 1 when the molecules carry net charges of opposite sign as coulombic attraction makes collisions more frequent, and less than 1 in the opposite case. The probability p_r is multiplied by a factor q_r that represents the steering effect of electric dipoles, which preorient the molecules before they collide. The model is applied to experimental data obtained by Schreiber and Fersht (Nat. Struct. Biol. 3:427–431, 1996) on the kinetics of barnase-barstar association. When long-range electrostatic interactions are fully screened or mutated away, q_tq_r ≈1, and the observed rate of productive collision is p_r k_coll ≈10⁵ M⁻¹ · s⁻¹. Under these conditions, p_r ≈1.5 · 10⁻⁵ is determined by geometric constraints corresponding to a loss of rotational freedom. Its value is compatible with computer docking simulations and implies a rotational entropy loss ΔS_rot ≈ 22 e.u. in the transition state. At low ionic strength, long-range electrostatic interactions accelerate barnase-barstar association by a factor q_tq_rof up to 10⁵ as favorable charge-charge and charge-dipole interactions work together to make it much faster than free diffusion would allow. Proteins 28:153–161, 1997. © 1997 Wiley-Liss Inc.     

Determining the solution conformational entropy of O-linked oligosaccharides at quasi-physiological conditions: size-exclusion chromatography and molecular dynamics

The physiological functions of oligosaccharides are influenced by a number of structural parameters such as anomeric configuration, glycosidic linkage, and degree of polymerization. These parameters affect the conformation of the oligosaccharides which, in turn, is responsible for characteristics such as aptameric and enzymatic binding, chiral recognition, and the structural targeting of bacterial and parasitic recognition events. Here, we measure the solution conformational entropy (DeltaS) of two series of oligosaccharides, linear malto- and cellooligosaccharides, using size-exclusion chromatography (SEC). For each series, we have determined DeltaS as a function of degree of polymerization (DP). The choice of oligosaccharides studied also allowed us to compare the influence of anomeric configuration on DeltaS, and to do so as a function of DP. Studies were conducted in water at physiological temperature and pH in order to resemble conditions within the human body. Experimental results were augmented with results from molecular dynamics computer modeling simulations in aqueous solvent. A comparison between experimental and computational data showed how the techniques can complement each other. An example of the latter is the considerable enthalpic contribution to the chromatographic separation of alpha- and gamma-cyclodextrin, which may have gone unnoticed if not for the large discrepancy between the results obtained by the separate techniques.     

Hydrophobic folding units at protein-protein interfaces: implications to protein folding and to protein-protein association.

A hydrophobic folding unit cutting algorithm, originally developed for dissecting single-chain proteins, has been applied to a dataset of dissimilar two-chain protein-protein interfaces. Rather than consider each individual chain separately, the two-chain complex has been treated as a single chain. The two-chain parsing results presented in this work show hydrophobicity to be a critical attribute of two-state versus three-state protein-protein complexes. The hydrophobic folding units at the interfaces of two-state complexes suggest that the cooperative nature of the two-chain protein folding is the outcome of the hydrophobic effect, similar to its being the driving force in a single-chain folding. In analogy to the protein-folding process, the two-chain, two-state model complex may correspond to the formation of compact, hydrophobic nuclei. On the other hand, the three-state model complex involves binding of already folded monomers, similar to the association of the hydrophobic folding units within a single chain. The similarity between folding entities in protein cores and in two-state protein-protein interfaces, despite the absence of some chain connectivities in the latter, indicates that chain linkage does not necessarily affect the native conformation. This further substantiates the notion that tertiary, non-local interactions play a critical role in protein folding. These compact, hydrophobic, two-chain folding units, derived from structurally dissimilar protein-protein interfaces, provide a rich set of data useful in investigations of the role played by chain connectivity and by tertiary interactions in studies of binding and of folding. Since they are composed of non-contiguous pieces of protein backbones, they may also aid in defining folding nuclei.     

On the role of electrostatic interactions in the design of protein-protein interfaces

Here, the methods of continuum electrostatics are used to investigate the contribution of electrostatic interactions to the binding of four protein-protein complexes; barnase-barstar, human growth hormone and its receptor, subtype N9 influenza virus neuraminidase and the NC41 antibody, the Ras binding domain (RBD) of kinase cRaf and a Ras homologue Rap1A. In two of the four complexes electrostatics are found to strongly oppose binding (hormone-receptor and neuraminidase-antibody complexes), in one case the net effect is close to zero (barnase-barstar) and in one case electrostatics provides a significant driving force favoring binding (RBD-Rap1A). In order to help understand the wide range of electrostatic contributions that were calculated, the electrostatic free energy was partitioned into contributions of individual charged and polar residues, salt bridges and networks involving salt bridges and hydrogen bonds. Although there is no one structural feature that accounts for the differences between the four interfaces, the extent to which the desolvation of buried charges is compensated by the formation of hydrogen bonds and ion pairs appears to be an important factor. Structural features that are correlated with contribution of an individual residue to stability are also discussed. These include partial burial of a charged group in the free monomer, the formation of networks involving charged and polar amino acids, and the formation of partially exposed ion-pairs. The total electrostatic contribution to binding is found to be inversely correlated with buried total and non-polar surface area. This suggests that different interfaces can be designed to exploit electrostatic and hydrophobic forces in very different ways.     

Analysing six types of protein-protein interfaces

Non-covalent residue side-chain interactions occur in many different types of proteins and facilitate many biological functions. Are these differences manifested in the sequence compositions and/or the residue-residue contact preferences of the interfaces? Previous studies analysed small data sets and gave contradictory answers. Here, we introduced a new data-mining method that yielded the largest high-resolution data set of interactions analysed. We introduced an information theory-based analysis method. On the basis of sequence features, we were able to differentiate six types of protein interfaces, each corresponding to a different functional or structural association between residues. Particularly, we found significant differences in amino acid composition and residue-residue preferences between interactions of residues within the same structural domain and between different domains, between permanent and transient interfaces, and between interactions associating homo-oligomers and hetero-oligomers. The differences between the six types were so substantial that, using amino acid composition alone, we could predict statistically to which of the six types of interfaces a pool of 1000 residues belongs at 63-100% accuracy. All interfaces differed significantly from the background of all residues in SWISS-PROT, from the group of surface residues, and from internal residues that were not involved in non-trivial interactions. Overall, our results suggest that the interface type could be predicted from sequence and that interface-type specific mean-field potentials may be adequate for certain applications.     

Flexibility and conformational entropy in protein-protein binding

To better understand the interplay between protein-protein binding and protein dynamics, we analyzed molecular dynamics simulations of 17 protein-protein complexes and their unbound components. Complex formation does not restrict the conformational freedom of the partner proteins as a whole, but, rather, it leads to a redistribution of dynamics. We calculate the change in conformational entropy for seven complexes with quasiharmonic analysis. We see significant loss, but also increased or unchanged conformational entropy. Where comparison is possible, the results are consistent with experimental data. However, stringent error estimates based on multiple independent simulations reveal large uncertainties that are usually overlooked. We observe substantial gains of pseudo entropy in individual partner proteins, and we observe that all complexes retain residual stabilizing intermolecular motions. Consequently, protein flexibility has an important influence on the thermodynamics of binding and may disfavor as well as favor association. These results support a recently proposed unified model for flexible protein-protein association.     

Hydration of protein-protein interfaces

We present an analysis of the water molecules immobilized at the protein-protein interfaces of 115 homodimeric proteins and 46 protein-protein complexes, and compare them with 173 large crystal packing interfaces representing nonspecific interactions. With an average of 15 waters per 1000 A2 of interface area, the crystal packing interfaces are more hydrated than the specific interfaces of homodimers and complexes, which have 10-11 waters per 1000 A2, reflecting the more hydrophilic composition of crystal packing interfaces. Very different patterns of hydration are observed: Water molecules may form a ring around interfaces that remain "dry," or they may permeate "wet" interfaces. A majority of the specific interfaces are dry and most of the crystal packing interfaces are wet, but counterexamples exist in both categories. Water molecules at interfaces form hydrogen bonds with protein groups, with a preference for the main-chain carbonyl and the charged side-chains of Glu, Asp, and Arg. These interactions are essentially the same in specific and nonspecific interfaces, and very similar to those observed elsewhere on the protein surface. Water-mediated polar interactions are as abundant at the interfaces as direct protein-protein hydrogen bonds, and they may contribute to the stability of the assembly.     

A dissection of specific and non-specific protein-protein interfaces

We compare the geometric and physical-chemical properties of interfaces involved in specific and non-specific protein-protein interactions in crystal structures reported in the Protein Data Bank. Specific interactions are illustrated by 70 protein-protein complexes and by subunit contacts in 122 homodimeric proteins; non-specific interactions are illustrated by 188 pairs of monomeric proteins making crystal-packing contacts selected to bury more than 800 A2 of protein surface. A majority of these pairs have 2-fold symmetry and form "crystal dimers" that cannot be distinguished from real dimers on the basis of the interface size or symmetry. The chemical and amino acid compositions of the large crystal-packing interfaces resemble the protein solvent-accessible surface. These interfaces are less hydrophobic than in homodimers and contain much fewer fully buried atoms. We develop a residue propensity score and a hydrophobic interaction score to assess preferences seen in the chemical and amino acid compositions of the different types of interfaces, and we derive indexes to evaluate the atomic packing, which we find to be less compact at non-specific than at specific interfaces. We test the capacity of these parameters to identify homodimeric proteins in crystal structures, and show that a simple combination of the non-polar interface area and the fraction of buried interface atoms assigns the quaternary structure of 88% of the homodimers and 77% of the monomers in our data set correctly. These success rates increase to 93-95% when the residue propensity score of the interfaces is taken into consideration.     

Side-chain entropy and packing in proteins.

What role does side-chain packing play in protein stability and structure? To address this question, we compare a lattice model with side chains (SCM) to a linear lattice model without side chains (LCM). Self-avoiding configurations are enumerated in 2 and 3 dimensions exhaustively for short chains and by Monte Carlo sampling for chains up to 50 main-chain monomers long. This comparison shows that (1) side-chain degrees of freedom increase the entropy of open conformations, but side-chain steric exclusion decreases the entropy of compact conformations, thus producing a substantial entropy that opposes folding; (2) there is a side-chain “freezing” or ordering, i.e., a sharp decrease in entropy, near maximum compactness; and (3) the different types of contacts among side chains (s) and main-chain elements (m) have different frequencies, and the frequencies have different dependencies on compactness. mm contacts contribute significantly only at high densities, suggesting that main-chain hydrogen bonding in proteins may be promoted by compactness. The distributions of mm, ms, and ss contacts in compact SCM configurations are similar to the distributions in protein structures in the Brookhaven Protein Data Bank. We propose that packing in proteins is more like the packing of nuts and bolts in a jar than like the pairwise matching of jigsaw puzzle pieces.     

Enhanced sampling of protein conformational states for dynamic cross-docking within the protein-protein docking server SwarmDock

The formation of specific protein-protein interactions is often a key to a protein's function. During complex formation, each protein component will undergo a change in the conformational state, for some these changes are relatively small and reside primarily at the sidechain level; however, others may display notable backbone adjustments. One of the classic problems in the protein-docking field is to be able to a priori predict the extent of such conformational changes. In this work, we investigated three protocols to find the most suitable input structure conformations for cross-docking, including a robust sampling approach in normal mode space. Counterintuitively, knowledge of the theoretically best combination of normal modes for unbound-bound transitions does not always lead to the best results. We used a novel spatial partitioning library, Aether Engine (see Supplementary Materials ), to efficiently search the conformational states of 56 receptor/ligand pairs, including a recent CAPRI target, in a systematic manner and selected diverse conformations as input to our automated docking server, SwarmDock, a server that allows moderate conformational adjustments during the docking process. In essence, here we present a dynamic cross-docking protocol, which when benchmarked against the simpler approach of just docking the unbound components shows a 10% uplift in the quality of the top docking pose.