A "solvated rotamer" approach to modeling water-mediated hydrogen bonds at protein-protein interfaces

Water-mediated hydrogen bonds play critical roles at protein-protein and protein-nucleic acid interfaces, and the interactions formed by discrete water molecules cannot be captured using continuum solvent models. We describe a simple model for the energetics of water-mediated hydrogen bonds, and show that, together with knowledge of the positions of buried water molecules observed in X-ray crystal structures, the model improves the prediction of free-energy changes upon mutation at protein-protein interfaces, and the recovery of native amino acid sequences in protein interface design calculations. We then describe a "solvated rotamer" approach to efficiently predict the positions of water molecules, at protein-protein interfaces and in monomeric proteins, that is compatible with widely used rotamer-based side-chain packing and protein design algorithms. Finally, we examine the extent to which the predicted water molecules can be used to improve prediction of amino acid identities and protein-protein interface stability, and discuss avenues for overcoming current limitations of the approach.     

Wet and dry interfaces: the role of solvent in protein-protein and protein-DNA recognition

Water molecules are found in abundance in protein-protein and protein-DNA interfaces. Although interface solvent molecules exchange quickly with the bulk solvent, structural and biochemical data suggest that water-mediated interactions are as important as direct hydrogen bonds in the stability and specificity of recognition.     

Interaction geometry involving planar groups in protein-protein interfaces

The geometry of interactions of planar residues is nonrandom in protein tertiary structures and gives rise to conventional, as well as nonconventional (X--H...pi, X--H...O, where X = C, N, or O) hydrogen bonds. Whether a similar geometry is maintained when the interaction is across the protein-protein interface is addressed here. The relative geometries of interactions involving planar residues, and the percentage of contacts giving rise to different types of hydrogen bonds are quite similar in protein structures and the biological interfaces formed by protein chains in homodimers and protein-protein heterocomplexes--thus pointing to the similarity of chemical interactions that occurs during protein folding and binding. However, the percentage is considerably smaller in the nonspecific and nonphysiological interfaces that are formed in crystal lattices of monomeric proteins. The C--H...O interaction linking the aromatic and the peptide groups is quite common in protein structures as well as the three types of interfaces. However, as the interfaces formed by crystal contacts are depleted in aromatic residues, the weaker hydrogen bond interactions would contribute less toward their stability.     

Protein-RNA contacts at crystal packing surfaces

The crystal packing surfaces comprising protein-RNA interactions were analyzed for 50 RNA-protein crystal structures in the Protein Data Bank database. Protein-RNA crystal contacts, which represent nonspecific protein-RNA interfaces, were investigated for their amino acid propensities, hydrogen bond patterns, and backbone and side chain interactions. When compared to biologically relevant interactions, the protein-RNA crystal contacts exhibit similarities as well as differences with respect to the principles of protein-RNA interactions. Similar to what was observed at cognate protein-RNA interfaces, positively charged amino acids have high propensities at noncognate protein-RNA interfaces and preferentially form hydrogen bonds with RNA phosphate groups. In contrast, nonpolar residues are less frequently associated with noncognate interactions. These results highlight the important roles of both electrostatic and hydrogen bonding interactions, facilitated by positively charged amino acids, in mediating both specific and nonspecific protein-RNA interactions.     

Solvated docking: introducing water into the modelling of biomolecular complexes

MOTIVATION: Interfacial water, which plays an important role in mediating biomolecular interactions, has been neglected in the modelling of biomolecular complexes. METHODS: We present a solvated docking approach that explicitly accounts for the presence of water in protein-protein complexes. Our solvated docking protocol is based on the concept of the first encounter complex in which a water layer is present in-between the molecules. It mimics the pathway from this initial complex towards the final assembly in which most waters have been expelled from the interface. Docking is performed from solvated biomolecules and waters are removed in a biased Monte Carlo procedure based on water-mediated contact propensities obtained from an analysis of high-resolution crystal structures. RESULTS: We demonstrate the feasibility of this approach for protein-protein complexes representing both 'wet' and 'dry' interfaces. Solvated docking leads to improvements both in quality and scoring. Water molecules are recovered that closely match the ones in the crystal structures. AVAILABILTY: Solvated docking will be made available in the future release of HADDOCK version 2.0 (http://www.nmr.chem.uu.nl/haddock).     

Protein–protein interaction at crystal contacts

Packing contacts are crystal artifacts, yet they make use of the same forces that govern specific recognition in protein-protein complexes and oligomeric proteins. They provide examples of a nonspecific protein-protein interaction which can be compared to biologically relevant ones. We evaluate the number and size of pairwise interfaces in 152 crystal forms where the asymmetric unit contains a monomeric protein. In those crystal forms that have no element of 2-fold symmetry, we find that molecules form 8 to 10 pairwise interfaces. The total area of the surface buried on each molecule is large, up to 4400 Ų. Pairwise interfaces bury 200–1200 Ų, like interfaces generated at random in a computer simulation, and less than interfaces in protease-inhibitor or antigen-antibody complexes, which bury 1500 Ų or more. Thus, specific contacts occurring in such complexes extend over a larger surface than nonspecific ones. In crystal forms with 2-fold symmetry, pairwise interfaces are fewer and larger on average than in the absence of 2-fold symmetry. Some bury 1500–2500 Ų, like interfaces in oligomeric proteins, and create “crystal oligomers” which may have formed in the solution before crystallizing. © 1995 Wiley-Liss, Inc.     

An orientation-dependent hydrogen bonding potential improves prediction of specificity and structure for proteins and protein-protein complexes

Hydrogen bonding is a key contributor to the specificity of intramolecular and intermolecular interactions in biological systems. Here, we develop an orientation-dependent hydrogen bonding potential based on the geometric characteristics of hydrogen bonds in high-resolution protein crystal structures, and evaluate it using four tests related to the prediction and design of protein structures and protein-protein complexes. The new potential is superior to the widely used Coulomb model of hydrogen bonding in prediction of the sequences of proteins and protein-protein interfaces from their structures, and improves discrimination of correctly docked protein-protein complexes from large sets of alternative structures.     

Crystal and molecular structures of trichloro-cobalt(II) complexes of epiquinine, epiquinidine, and epidihydrocinchonine

Cinchona alkaloids are very well known antimalarials but the mechanism of their biological action still remains to be elucidated. The structural studies of active erythro and inactive threo alkaloid complexes are an important step to this aim. In this paper results of crystal structure analysis of three cobalt complexes of threo alkaloids are presented: (epiquininium)trichlorocobalt(II) (EpiQnCoCl3), (epiquinidinium)trichlorocobalt(II) (EpiQdCoCl3) and (epidihydrocinchoninium)trichlorocobalt(II) (EpiCnCoCl3). The complexes are zwitterions in which trichlorocobalt substituents are coordinated to quinoline nitrogen atoms and quinuclidine nitrogen atoms are protonated. EpiQnCoCl3 adopts uncommon conformation with quinoline moiety oriented in the opposite direction in comparison to the analogous uncomplexed alkaloid. The packing in the crystal structures is determined mainly by the hydrogen bonds, in which the chlorine atoms of substituents and solvent molecules contribute. Atoms participating in hydrogen bonds in EpiQnCoCl3 and EpiQdCoCl3 form large rings, while in EpiCnCoCl3 only chains are present. Solvent molecules are very important for the packing mode. In contrast to most erythro alkaloids, the hydroxyl oxygen atom in the title complexes forms weak or not well defined hydrogen bonds. The contribution of very weak intramolecular interactions N1--H1...O12 cannot be excluded. Such "trace" interactions can be considered a relic of the unprotonated status of an epi alkaloid.     

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.     

Calculations on crystal packing of a flexible molecule, Leu-enkephalin

Crystal packing calculations have been carried out on a substantial number of conformations of Leu-enkephalin; namely, those obtained both from crystal structures and from energy minimizations on isolated molecules, and with and without waters of crystallization. The known crystal structures represent the most energetically stable packings found. The conformations of the enkephalin molecules in the crystal are not the most stable for an isolated molecule; i.e. intermolecular interactions force the isolated molecule to change conformation in order to achieve a small packing volume and an optimal packing energy in the crystal. It is found that the packing energy of an enkephalin molecule is a reasonably smooth function of its molecular volume in the unit cell, if structures with intermolecular hydrogen bonding are excluded, and is substantially independent of other details of the molecular conformation or of the crystal packing. Hydrogen bonding provides additional stabilization of the crystal structure, and would likely permit crystallization of the system if it is sufficiently dense. Solvent molecules further stabilize the structure when they can also provide intermolecular hydrogen bonds.     

Stereochemical metrics of lectin-carbohydrate interactions: comparison with protein-protein interfaces

A global census of stereochemical metrics including interface size, hydropathy, amino acid propensities, packing and hydrogen bonding was carried out on 32 x-ray-elucidated structures of lectin-carbohydrate complexes covering eight different lectin families. It is shown that the interactions at primary binding subsites are more efficient than at other subsites. Another salient behavior found for primary subsites was a marked negative correlation between the interface size and the polar surface content. It is noteworthy that this demographic rule is delineated by lectins with unrelated phylogenetic origin, indicating that independent interface architectures have evolved through common optimization paths. The structural properties of lectin-carbohydrate interfaces were compared with those characterizing a set of 32 protein homodimers. Overall, the analysis shows that the stereochemical bases of lectin-carbohydrate and protein-protein interfaces differ drastically from each other. In comparison with protein-protein complexes, lectin-carbohydrate interfaces have superior packing efficiency, better hydrogen bonding stereochemistry, and higher interaction cooperativity. A similar conclusion holds in the comparison with protein-protein heterocomplexes. We propose that the energetic consequence of this better interaction geometry is a larger decrease in free energy per unit of area buried, feature that enables lectins and carbohydrates to form stable complexes with relatively small interface areas. These observations lend support to the emerging notion that systems differing from each other in their stereochemical metrics may rely on different energetic bases.     

Hydrogen bond connectivity patterns and hydrophobic interactions in crystal structures of small, acyclic peptides.

Crystal structures of all available unblocked linear peptides with two to five residues were retrieved from the Cambridge Structural Database and their intermolecular contacts and packing modes studied using molecular graphics. This survey reveals that interactions between hydrophobic portions of the molecules are critically important in determining the overall features of their crystal packing patterns. Distinct hydrophobic columns or layers are observed in almost all crystal structures. Analyses of the relationships between these interactions and crystal growth properties of small peptides are given. It is suggested that needle growth is promoted by hydrophobic packing, usually along a short crystallographic axis (4.6-6.0 angstroms). Also contributing to these morphologic characteristics are entropic factors associated with hydrophobic aggregation as well as tightly bound water molecules on hydrophobic faces. The paper also provides a comprehensive overview of hydrogen bond patterns in acyclic peptide crystals. It is demonstrated that one of their primary roles is to provide a scaffolding within which hydrophobic groups can aggregate. Even though there is a high density of hydrogen bonds in the crystals, often with complex patterns and networks, certain motifs are found to recur in a number of structures indicating specific hydrogen bond preferences. Water, for example, is an integral part of the hydrogen bond networks in these crystals, usually acting as the primary donor for main-chain carboxylate groups in peptide hydrates.     

Similar chemistry, but different bond preferences in inter versus intra-protein interactions

Proteins fold into a well-defined structure as a result of the collapse of the polypeptide chain, while transient protein-complex formation mainly is a result of binding of two folded individual monomers. Therefore, a protein-protein interface does not resemble the core of monomeric proteins, but has a more polar nature. Here, we address the question of whether the physico-chemical characteristics of intraprotein versus interprotein bonds differ, or whether interfaces are different from folded monomers only in the preference for certain types of interactions. To address this question we assembled a high resolution, nonredundant, protein-protein interaction database consisting of 1374 homodimer and 572 heterodimer complexes, and compared the physico-chemical properties of these interactions between protein interfaces and monomers. We performed extensive statistical analysis of geometrical properties of interatomic interactions of different types: hydrogen bonds, electrostatic interactions, and aromatic interactions. Our study clearly shows that there is no significant difference in the chemistry, geometry, or packing density of individual interactions between interfaces and monomeric structures. However, the distribution of different bonds differs. For example, side-chain-side-chain interactions constitute over 62% of all interprotein interactions, while they make up only 36% of the bonds stabilizing a protein structure. As on average, properties of backbone interactions are different from those of side chains, a quantitative difference is observed. Our findings clearly show that the same knowledge-based potential can be used for protein-binding sites as for protein structures. However, one has to keep in mind the different architecture of the interfaces and their unique bond preference.     

Computational design of a new hydrogen bond network and at least a 300-fold specificity switch at a protein-protein interface

The redesign of protein-protein interactions is a stringent test of our understanding of molecular recognition and specificity. Previously we engineered a modest specificity switch into the colicin E7 DNase-Im7 immunity protein complex by identifying mutations that are disruptive in the native complex, but can be compensated by mutations on the interacting partner. Here we extend the approach by systematically sampling alternate rigid body orientations to optimize the interactions in a binding mode specific manner. Using this protocol we designed a de novo hydrogen bond network at the DNase-immunity protein interface and confirmed the design with X-ray crystallographic analysis. Subsequent design of the second shell of interactions guided by insights from the crystal structure on tightly bound water molecules, conformational strain, and packing defects yielded new binding partners that exhibited specificities of at least 300-fold between the cognate and the non-cognate complexes. This multi-step approach should be applicable to the design of polar protein-protein interactions and contribute to the re-engineering of regulatory networks mediated by protein-protein interactions.     

Probing the role of interfacial waters in protein–DNA recognition using a hybrid implicit/explicit solvation model

When proteins bind to their DNA target sites, ordered water molecules are often present at the protein–DNA interface bridging protein and DNA through hydrogen bonds. What is the role of these ordered interfacial waters? Are they important determinants of the specificity of DNA sequence recognition, or do they act in binding in a primarily nonspecific manner, by improving packing of the interface, shielding unfavorable electrostatic interactions, and solvating unsatisfied polar groups that are inaccessible to bulk solvent? When modeling details of structure and binding preferences, can fully implicit solvent models be fruitfully applied to protein–DNA interfaces, or must the individualistic properties of these interfacial waters be accounted for? To address these questions, we have developed a hybrid implicit/explicit solvation model that specifically accounts for the locations and orientations of small numbers of DNA‐bound water molecules, while treating the majority of the solvent implicitly. Comparing the performance of this model with that of its fully implicit counterpart, we find that explicit treatment of interfacial waters results in a modest but significant improvement in protein side‐chain placement and DNA sequence recovery. Base‐by‐base comparison of the performance of the two models highlights DNA sequence positions whose recognition may be dependent on interfacial water. Our study offers large‐scale statistical evidence for the role of ordered water for protein–DNA recognition, together with detailed examination of several well‐characterized systems. In addition, our approach provides a template for modeling explicit water molecules at interfaces that should be extensible to other systems. Proteins 2013; 81:1318–1329. © 2013 Wiley Periodicals, Inc.     

On the contribution of water-mediated interactions to protein-complex stability

Protein-water interactions have long been recognized as a major determinant of chain folding, conformational stability, binding specificity and catalysis. However, the detailed effects of water on stabilizing protein-protein interactions remain elusive. A way to test experimentally the contribution of water-mediated interactions is by applying double mutant cycle analysis on pairs of residues that do not form direct interactions, but are bridged by water. Seven such interactions within the interface between TEM1 and BLIP proteins were evaluated. No significant interaction free energy was found between either of them. Water can bridge interactions, but also stabilize the structure of the monomer. To distinguish between these, we performed a bioinformatic analysis using AQUAPROT (http://bioinfo.weizmann.ac.il/aquaprot) to determine the degree of water conservation between the bound and unbound states. 29 structures of twelve complexes and 20 related monomers were analyzed. Of the 262 water molecules located within the interfaces, 145 were conserved between the unbound and bound structures. Strikingly, all 50 buried or partially buried waters in the monomer structures were conserved at the same location in the bound structures. Thus, buried waters have an important role in stabilizing the monomer fold rather than contributing to protein-protein binding, and are not replaced by residues from the incoming protein. Taking together the experimental and bioinformatics evidence suggests that exposed waters within the interface may be good sites for protein engineering, while buried or mostly buried waters should be left unchanged.     

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.     

A dissection of the protein-protein interfaces in icosahedral virus capsids

We selected 49 icosahedral virus capsids whose crystal structures are reported in the Protein Data Bank. They belong to the T=1, T=3, pseudo T=3 and other lattice types. We identified in them 779 unique interfaces between pairs of subunits, all repeated by icosahedral symmetry. We analyzed the geometric and physical chemical properties of these interfaces and compared with interfaces in protein-protein complexes and homodimeric proteins, and with crystal packing contacts. The capsids contain one to 16 subunits implicated in three to 66 unique interfaces. Each subunit loses 40-60% of its accessible surface in contacts with an average of 8.5 neighbors. Many of the interfaces are very large with a buried surface area (BSA) that can exceed 10,000 A(2), yet 39% are small with a BSA<800 A(2) comparable to crystal packing contacts. Pairwise capsid interfaces overlap, so that one-third of the residues are part of more than one interface. Those with a BSA>800 A(2) resemble homodimer interfaces in their chemical composition. Relative to the protein surface, they are non-polar, enriched in aliphatic residues and depleted of charged residues, but not of neutral polar residues. They contain one H-bond per about 200 A(2) BSA. Small capsid interfaces (BSA<800 A(2)) are only slightly more polar. They have a similar amino acid composition, but they bury fewer atoms and contain fewer H-bonds for their size. Geometric parameters that estimate the quality of the atomic packing suggest that the small capsid interfaces are loosely packed like crystal packing contacts, whereas the larger interfaces are close-packed as in protein-protein complexes and homodimers. We discuss implications of these findings on the mechanism of capsid assembly, assuming that the larger interfaces form first to yield stable oligomeric species (capsomeres), and that medium-size interfaces allow the stepwise addition of capsomeres to build larger intermediates.