Dissecting subunit interfaces in homodimeric proteins

The subunit interfaces of 122 homodimers of known three-dimensional structure are analyzed and dissected into sets of surface patches by clustering atoms at the interface; 70 interfaces are single-patch, the others have up to six patches, often contributed by different structural domains. The average interface buries 1,940 A2 of the surface of each monomer, contains one or two patches burying 600-1,600 A2, is 65% nonpolar and includes 18 hydrogen bonds. However, the range of size and of hydrophobicity is wide among the 122 interfaces. Each interface has a core made of residues with atoms buried in the dimer, surrounded by a rim of residues with atoms that remain accessible to solvent. The core, which constitutes 77% of the interface on average, has an amino acid composition that resembles the protein interior except for the presence of arginine residues, whereas the rim is more like the protein surface. These properties of the interfaces in homodimers, which are permanent assemblies, are compared to those of protein-protein complexes where the components associate after they have independently folded. On average, subunit interfaces in homodimers are twice larger than in complexes, and much less polar due to the large fraction belonging to the core, although the amino acid compositions of the cores are similar in the two types of interfaces.     

Protein–protein interactions leave evolutionary footprints: High molecular coevolution at the core of interfaces

Protein–protein interactions are essential to all aspects of life. Specific interactions result from evolutionary pressure at the interacting interfaces of partner proteins. However, evolutionary pressure is not homogeneous within the interface: for instance, each residue does not contribute equally to the binding energy of the complex. To understand functional differences between residues within the interface, we analyzed their properties in the core and rim regions. Here, we characterized protein interfaces with two evolutionary measures, conservation and coevolution, using a comprehensive dataset of 896 protein complexes. These scores can detect different selection pressures at a given position in a multiple sequence alignment. We also analyzed how the number of interactions in which a residue is involved influences those evolutionary signals. We found that the coevolutionary signal is higher in the interface core than in the interface rim region. Additionally, the difference in coevolution between core and rim regions is comparable to the known difference in conservation between those regions. Considering proteins with multiple interactions, we found that conservation and coevolution increase with the number of different interfaces in which a residue is involved, suggesting that more constraints (i.e., a residue that must satisfy a greater number of interactions) allow fewer sequence changes at those positions, resulting in higher conservation and coevolution values. These findings shed light on the evolution of protein interfaces and provide information useful for identifying protein interfaces and predicting protein–protein interactions.     

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.     

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.     

Protein-DNA interactions: structural, thermodynamic and clustering patterns of conserved residues in DNA-binding proteins

Amino acid residues, which play important roles in protein function, are often conserved. Here, we analyze thermodynamic and structural data of protein-DNA interactions to explore a relationship between free energy, sequence conservation and structural cooperativity. We observe that the most stabilizing residues or putative hotspots are those which occur as clusters of conserved residues. The higher packing density of the clusters and available experimental thermodynamic data of mutations suggest cooperativity between conserved residues in the clusters. Conserved singlets contribute to the stability of protein-DNA complexes to a lesser extent. We also analyze structural features of conserved residues and their clusters and examine their role in identifying DNA-binding sites. We show that about half of the observed conserved residue clusters are in the interface with the DNA, which could be identified from their amino acid composition; whereas the remaining clusters are at the protein-protein or protein-ligand interface, or embedded in the structural scaffolds. In protein-protein interfaces, conserved residues are highly correlated with experimental residue hotspots, contributing dominantly and often cooperatively to the stability of protein-protein complexes. Overall, the conservation patterns of the stabilizing residues in DNA-binding proteins also highlight the significance of clustering as compared to single residue conservation.     

Insights from the structural analysis of protein heterodimer interfaces

Protein heterodimer complexes are often involved in catalysis, regulation, assembly, immunity and inhibition. This involves the formation of stable interfaces between the interacting partners. Hence, it is of interest to describe heterodimer interfaces using known structural complexes. We use a non-redundant dataset of 192 heterodimer complex structures from the protein databank (PDB) to identify interface residues and describe their interfaces using amino-acids residue property preference. Analysis of the dataset shows that the heterodimer interfaces are often abundant in polar residues. The analysis also shows the presence of two classes of interfaces in heterodimer complexes. The first class of interfaces (class A) with more polar residues than core but less than surface is known. These interfaces are more hydrophobic than surfaces, where protein-protein binding is largely hydrophobic. The second class of interfaces (class B) with more polar residues than core and surface is shown. These interfaces are more polar than surfaces, where binding is mainly polar. Thus, these findings provide insights to the understanding of protein-protein interactions.     

Small-world network approach to identify key residues in protein-protein interaction

We show that protein complexes can be represented as small-world networks, exhibiting a relatively small number of highly central amino-acid residues occurring frequently at protein-protein interfaces. We further base our analysis on a set of different biological examples of protein-protein interactions with experimentally validated hot spots, and show that 83% of these predicted highly central residues, which are conserved in sequence alignments and nonexposed to the solvent in the protein complex, correspond to or are in direct contact with an experimentally annotated hot spot. The remaining 17% show a general tendency to be close to an annotated hot spot. On the other hand, although there is no available experimental information on their contribution to the binding free energy, detailed analysis of their properties shows that they are good candidates for being hot spots. Thus, highly central residues have a clear tendency to be located in regions that include hot spots. We also show that some of the central residues in the protein complex interfaces are central in the monomeric structures before dimerization and that possible information relating to hot spots of binding free energy could be obtained from the unbound structures.     

Evolvability of yeast protein-protein interaction interfaces

The functional importance of protein-protein interactions indicates that there should be strong evolutionary constraint on their interaction interfaces. However, binding interfaces are frequently affected by amino acid replacements. Change due to coevolution within interfaces can contribute to variability but is not ubiquitous. An alternative explanation for the ability of surfaces to accept replacements may be that many residues can be changed without affecting the interaction. Candidates for these types of residues are those that make interchain interaction only through the protein main chain, β-carbon, or associated hydrogen atoms. Since almost all residues have these atoms, we hypothesize that this subset of interface residues may be more easily substituted than those that make interactions through other atoms. We term such interactions "residue type independent." Investigating this hypothesis, we find that nearly a quarter of residues in protein interaction interfaces make exclusively interchain residue-type-independent contacts. These residues are less structurally constrained and less conserved than residues making residue-type-specific interactions. We propose that residue-type-independent interactions allow substitutions in binding interfaces while the specificity of binding is maintained.     

Dissecting protein-protein recognition sites

The recognition sites in 70 pairwise protein-protein complexes of known three-dimensional structure are dissected in a set of surface patches by clustering atoms at the interface. When the interface buries <2000 A2 of protein surface, the recognition sites usually form a single patch on the surface of each component protein. In contrast, larger interfaces are generally multipatch, with at least one pair of patches that are equivalent in size to a single-patch interface. Each recognition site, or patch within a site, contains a core made of buried interface atoms, surrounded by a rim of atoms that remain accessible to solvent in the complex. A simple geometric model reproduces the number and distribution of atoms within a patch. The rim is similar in composition to the rest of the protein surface, but the core has a distinctive amino acid composition, which may help in identifying potential protein recognition sites on single proteins of known structures.     

An analysis of the relationship between hydration and protein-DNA interactions.

Eleven protein-DNA crystal structures were analyzed to test the hypothesis that hydration sites predicted in the first hydration shell of DNA mark the positions where protein residues hydrogen-bond to DNA. For nine of those structures, protein atoms, which form hydrogen bonds to DNA bases, were found within 1.5 A of the predicted hydration positions in 86% of the interactions. The correspondence of the predicted hydration sites with the hydrogen-bonded protein side chains was significantly higher for bases inside the conserved DNA recognition sequences than outside those regions. In two CAP-DNA complexes, predicted base hydration sites correctly marked 71% (within 1.5 A) of protein atoms, which form hydrogen bonds to DNA bases. Phosphate hydration was compared to actual protein binding sites in one CAP-DNA complex with 78% marked contacts within 2.0 A. These data suggest that hydration sites mark the binding sites at protein-DNA interfaces.     

Uracil interference, a rapid and general method for defining protein-DNA interactions involving the 5-methyl group of thymines: the GCN4-DNA complex.

We describe a novel uracil interference method for examining protein contacts with the 5-methyl group of thymines. The protein of interest is incubated with target DNA containing randomly distributed deoxyuracil substitutions that is generated by carrying out the polymerase chain reaction in the presence of a mixture of TTP and dUTP. After separating DNA-protein complexes away from unbound DNA, the locations of deoxyuracil residues that either do or do not interfere with DNA-binding are determined by cleavage with uracil-N-glycosylase followed by piperidine. Using this uracil interference assay, we show that the methyl groups of the four core thymines, but not the two peripheral thymines, of the optimal binding site (ATG-ACTCAT) are important for high affinity binding of GCN4. Similar, but not identical, results are obtained using KMnO4 interference, another method used for studying protein-DNA interactions involving thymine residues. These observations strongly suggest that GCN4 directly contacts the 5-methyl groups of the four core thymines that lie in the major groove of the target DNA. Besides providing specific structural information about protein-DNA complexes, uracil interference should also be useful for identifying DNA-binding proteins and their target sites in eukaryotic promoter regions.     

Protein-protein interactions: hot spots and structurally conserved residues often locate in complemented pockets that pre-organized in the unbound states: implications for docking

Energetic hot spots account for a significant portion of the total binding free energy and correlate with structurally conserved interface residues. Here, we map experimentally determined hot spots and structurally conserved residues to investigate their geometrical organization. Unfilled pockets are pockets that remain unfilled after protein-protein complexation, while complemented pockets are pockets that disappear upon binding, representing tightly fit regions. We find that structurally conserved residues and energetic hot spots are strongly favored to be located in complemented pockets, and are disfavored in unfilled pockets. For the three available protein-protein complexes with complemented pockets where both members of the complex were alanine-scanned, 62% of all hot spots (DeltaDeltaG>2kcal/mol) are within these pockets, and 60% of the residues in the complemented pockets are hot spots. 93% of all red-hot residues (DeltaDeltaG>/=4kcal/mol) either protrude into or are located in complemented pockets. The occurrence of hot spots and conserved residues in complemented pockets highlights the role of local tight packing in protein associations, and rationalizes their energetic contribution and conservation. Complemented pockets and their corresponding protruding residues emerge among the most important geometric features in protein-protein interactions. By screening the solvent, this organization shields backbone hydrogen bonds and charge-charge interactions. Complemented pockets often pre-exist binding. For 18 protein-protein complexes with complemented pockets whose unbound structures are available, in 16 the pockets are identified to pre-exist in the unbound structures. The root-mean-squared deviations of the atoms lining the pockets between the bound and unbound states is as small as 0.9A, suggesting that such pockets constitute features of the populated native state that may be used in docking.     

Protein-protein interaction and quaternary structure

Protein-protein recognition plays an essential role in structure and function. Specific non-covalent interactions stabilize the structure of macromolecular assemblies, exemplified in this review by oligomeric proteins and the capsids of icosahedral viruses. They also allow proteins to form complexes that have a very wide range of stability and lifetimes and are involved in all cellular processes. We present some of the structure-based computational methods that have been developed to characterize the quaternary structure of oligomeric proteins and other molecular assemblies and analyze the properties of the interfaces between the subunits. We compare the size, the chemical and amino acid compositions and the atomic packing of the subunit interfaces of protein-protein complexes, oligomeric proteins, viral capsids and protein-nucleic acid complexes. These biologically significant interfaces are generally close-packed, whereas the non-specific interfaces between molecules in protein crystals are loosely packed, an observation that gives a structural basis to specific recognition. A distinction is made within each interface between a core that contains buried atoms and a solvent accessible rim. The core and the rim differ in their amino acid composition and their conservation in evolution, and the distinction helps correlating the structural data with the results of site-directed mutagenesis and in vitro studies of self-assembly.     

Major groove recognition by three-stranded beta-sheets: affinity determinants and conserved structural features

We present the results of a rational mutagenesis and binding-affinity study of the three-stranded beta-sheet-DNA interface in the complex formed by the amino-terminal DNA-binding domain of the Tn916 integrase protein and its cognate binding site. The relative importance of interfacial contacts present in its NMR-derived solution structure have been tested through mutagenesis, fluorescence anisotropy, and intrinsic quenching DNA-binding assays. We find that seven protein-DNA hydrogen bonds (two base-specific and five to phosphate groups) significantly contribute to the level of affinity. These interactions span the entire DNA-binding surface on the protein, but primarily originate from residues in only two strands of the sheet and loop L2. Interestingly, we show that highly populated, precisely defined intermolecular hydrogen bonds in the ensemble of conformers are invariably important for DNA-binding, implying that NMR-derived solution structures provide direct insight into the energetics of recognition. Unusual three-stranded beta-sheet-DNA interfaces have recently been discovered in three unrelated protein-DNA complexes. A comparative analysis of these structures reveals similar sheet positioning, the presence of two invariant interfacial contacts to the phosphodiester backbone, and two semi-conserved base-specific hydrogen bonds. Two of these conserved contacts significantly contribute to the affinity of the integrase-DNA complex, suggesting that the three-stranded beta-sheet DNA-binding motif exhibits conserved principles of recognition.     

Studies of protein-protein interfaces: a statistical analysis of the hydrophobic effect.

Data sets of 362 structurally nonredundant protein-protein interfaces and of 57 symmetry-related oligomeric interfaces have been used to explore whether the hydrophobic effect that guides protein folding is also the main driving force for protein-protein associations. The buried nonpolar surface area has been used to measure the hydrophobic effect. Our analysis indicates that, although the hydrophobic effect plays a dominant role in protein-protein binding, it is not as strong as that observed in the interior of protein monomers. Comparison of interiors of the monomers with those of the interfaces reveals that, in general, the hydrophobic amino acids are more frequent in the interior of the monomers than in the interior of the protein-protein interfaces. On the other hand, a higher proportion of charged and polar residues are buried at the interfaces, suggesting that hydrogen bonds and ion pairs contribute more to the stability of protein binding than to that of protein folding. Moreover, comparison of the interior of the interfaces to protein surfaces indicates that the interfaces are poorer in polar/charged than the surfaces and are richer in hydrophobic residues. The interior of the interfaces appears to constitute a compromise between the stabilization contributed by the hydrophobic effect on the one hand and avoiding patches on the protein surfaces that are too hydrophobic on the other. Such patches would be unfavorable for the unassociated monomers in solution. We conclude that, although the types of interactions are similar between protein-protein interfaces and single-chain proteins overall, the contribution of the hydrophobic effect to protein-protein associations is not as strong as to protein folding. This implies that packing patterns and interatom, or interresidue, pairwise potential functions, derived from monomers, are not ideally suited to predicting and assessing ligand associations or design. These would perform adequately only in cases where the hydrophobic effect at the binding site is substantial.     

Conserved residue clusters at protein-protein interfaces and their use in binding site identification

Background  

Biological evolution conserves protein residues that are important for structure and function. Both protein stability and function often require a certain degree of structural co-operativity between spatially neighboring residues and it has previously been shown that conserved residues occur clustered together in protein tertiary structures, enzyme active sites and protein-DNA interfaces. Residues comprising protein interfaces are often more conserved compared to those occurring elsewhere on the protein surface. We investigate the extent to which conserved residues within protein-protein interfaces are clustered together in three-dimensions.     

Residue conservation in viral capsid assembly

To evaluate the evolutionary constraints placed on viral proteins by the structure and assembly of the capsid, we calculate Shannon entropies in the aligned sequences of 45 polypeptide chains in 32 icosahedral viruses, and relate these entropies to the residue location in the three-dimensional structure of the capsids. Three categories of residues have entropies lower than the chain average implying that they are better conserved than average: residues that are buried within a subunit (the protein core), residues that contain atoms buried at an interface between subunits (the interface core), and residues that contribute to several such interfaces. The interface core is also conserved in homomeric proteins and in transient protein-protein complexes, which have only one interface whereas capsids have many. In capsids, the subunit interfaces implicate most of the polypeptide chain: on average, 66% of the capsid residues are at an interface, 34% at more than one, and 47% at the interface core. Nevertheless, we observe that the degree of residue conservation can vary widely between interfaces within a capsid and between regions within an interface. The interfaces and regions of interfaces that show a low sequence variability are likely to play major roles in the self-assembly of the capsid, with implications on its mechanism that we discuss taking adeno-associated virus as an example.     

A simple physical model for the prediction and design of protein-DNA interactions

Protein-DNA interactions are crucial for many biological processes. Attempts to model these interactions have generally taken the form of amino acid-base recognition codes or purely sequence-based profile methods, which depend on the availability of extensive sequence and structural information for specific structural families, neglect side-chain conformational variability, and lack generality beyond the structural family used to train the model. Here, we take advantage of recent advances in rotamer-based protein design and the large number of structurally characterized protein-DNA complexes to develop and parameterize a simple physical model for protein-DNA interactions. The model shows considerable promise for redesigning amino acids at protein-DNA interfaces, as design calculations recover the amino acid residue identities and conformations at these interfaces with accuracies comparable to sequence recovery in globular proteins. The model shows promise also for predicting DNA-binding specificity for fixed protein sequences: native DNA sequences are selected correctly from pools of competing DNA substrates; however, incorporation of backbone movement will likely be required to improve performance in homology modeling applications. Interestingly, optimization of zinc finger protein amino acid sequences for high-affinity binding to specific DNA sequences results in proteins with little or no predicted specificity, suggesting that naturally occurring DNA-binding proteins are optimized for specificity rather than affinity. When combined with algorithms that optimize specificity directly, the simple computational model developed here should be useful for the engineering of proteins with novel DNA-binding specificities.     

The solution structure of Lac repressor headpiece 62 complexed to a symmetrical lac operator

BACKGROUND: Lactose repressor protein (Lac) controls the expression of the lactose metabolic genes in Escherichia coli by binding to an operator sequence in the promoter of the lac operon. Binding of inducer molecules to the Lac core domain induces changes in tertiary structure that are propagated to the DNA-binding domain through the connecting hinge region, thereby reducing the affinity for the operator. Protein-protein and protein-DNA interactions involving the hinge region play a crucial role in the allosteric changes occurring upon induction, but have not, as yet, been analyzed in atomic detail. RESULTS: We have used nuclear magnetic resonance (NMR) spectroscopy and restrained molecular dynamics (rMD) to determine the structure of the Lac repressor DNA-binding domain (headpeice 62; HP62) in complex with a symmetrized lac operator. Analysis of the structures reveals specific interactions between Lac repressor and DNA that were not found in previously investigated Lac repressor-DNA complexes. Important differences with the previously reported structures of the HP56-DNA complex were found in the loop following the helix-turn-helix (HTH) motif. The protein-protein and protein-DNA interactions involving the hinge region and the deformations in the DNA structure could be delineated in atomic detail. The structures were also used for comparison with the available crystallographic data on the Lac and Pur repressor-DNA complexes. CONCLUSIONS: The structures of the HP62-DNA complex provide the basis for a better understanding of the specific recognition in the Lac repressor-operator complex. In addition, the structural features of the hinge region provide detailed insight into the protein-protein and protein-DNA interactions responsible for the high affinity of the repressor for operator DNA.