Antigen-antibody interface properties: Composition, residue interactions, and features of 53 non-redundant structures

The structures of protein antigen-antibody (Ag-Ab) interfaces contain information about how Ab recognize Ag as well as how Ag are folded to present surfaces for Ag recognition. As such, the Ab surface holds information about Ag folding that resides with the Ab-Ag interface residues and how they interact. In order to gain insight into the nature of such interactions, a data set comprised of 53 non-redundant 3D structures of Ag-Ab complexes was analyzed. We assessed the physical and biochemical features of the Ag-Ab interfaces and the degree to which favored interactions exist between amino acid residues on the corresponding interface surfaces. Amino acid compositional analysis of the interfaces confirmed the dominance of TYR in the Ab paratope-containing surface (PCS), with almost two fold greater abundance than any other residue. Additionally TYR had a much higher than expected presence in the PCS compared to the surface of the whole antibody (defined as the occurrence propensity), along with aromatics PHE, TRP, and to a lesser degree HIS and ILE. In the Ag epitope-containing surface (ECS), there were slightly increased occurrence propensities of TRP and TYR relative to the whole Ag surface, implying an increased significance over the compositionally most abundant LYS > ASN > GLU > ASP > ARG. This examination encompasses a large, diverse set of unique Ag-Ab crystal structures that help explain the biological range and specificity of Ag-Ab interactions. This analysis may also provide a measure of the significance of individual amino acid residues in phage display analysis of Ag binding.     

Evaluation of the protein interfaces that form an intermolecular four-helix bundle as studied by computer simulation

Rational design of protein surface is important for creating higher order protein structures, but it is still challenging. In this study, we designed in silico the several binding interfaces on protein surfaces that allow a de novo protein–protein interaction to be formed. We used a computer simulation technique to find appropriate amino acid arrangements for the binding interface. The protein–protein interaction can be made by forming an intermolecular four-helix bundle structure, which is often found in naturally occurring protein subunit interfaces. As a model protein, we used a helical protein, YciF. Molecular dynamics simulation showed that a new protein–protein interaction is formed depending on the number of hydrophobic and charged amino acid residues present in the binding surfaces. However, too many hydrophobic amino acid residues present in the interface negatively affected on the binding. Finally, we found an appropriate arrangement of hydrophobic and charged amino acid residues that induces a protein–protein interaction through an intermolecular four-helix bundle formation.     

An investigation of protein subunit and domain interfaces

Protein structures were collected from the Brookhaven Database of tertiary architectures that displayed oligomeric association (24 molecules) or whose polypeptide folding revealed domains (34 proteins). The subunit and domain interfaces for these proteins were respectively examined from the following aspects: percentage water-accessible surface area buried by the respective associations, surface compositions and physical characteristics of the residues involved in the subunit and domain contacts, secondary structural state of the interface amino acids, preferred polar and non-polar interactions, spatial distribution of polar and non-polar residues on the interface surface, same residue interactions in the oligomeric contacts, and overall cross-section and shape of the contact surfaces. A general, consistent picture emerged for both the domain and subunit interfaces.     

Distinct roles of overlapping and non-overlapping regions of hub protein interfaces in recognition of multiple partners

Cellular functions of an organism are maintained by protein-protein interactions. Those proteins that bind multiple partners asynchronously (date hub proteins) are important to make the interaction network coordinated. It is known that many date hub proteins bind different partners at overlapping (OV) interfaces. To understand how OV interfaces of date hub proteins can recognize multiple partners, we analyzed the difference between OV and non-overlapping (Non-OV) regions of interfaces involved in the binding of different partners. By using the structures of 16 date hub proteins with various interaction partners (ranging from 5 to 33), we compared buried surface area, compositions of amino acid residues and secondary structures, and side-chain orientations. It was found that buried interface residues are important for recognizing multiple partners, while exposed interface residues are important for determining specificity to a particular ligand. In addition, our analyses reveal that residue compositions in OV and Non-OV regions are different and that residues in OV region show diverse side-chain torsion angles to accommodate binding to multiple targets.     

Crystal structure of an antibody bound to an immunodominant peptide epitope: novel features in peptide-antibody recognition

The crystal structure of Fab of an Ab PC283 complexed with its corresponding peptide Ag, PS1 (HQLDPAFGANSTNPD), derived from the hepatitis B virus surface Ag was determined. The PS1 stretch Gln2P to Phe7P is present in the Ag binding site of the Ab, while the next three residues of the peptide are raised above the binding groove. The residues Ser11P, Thr12P, and Asn13P then loop back onto the Ag-binding site of the Ab. The last two residues, Pro14P and Asp15P, extend outside the binding site without forming any contacts with the Ab. The PC283-PS1 complex is among the few examples where the light chain complementarity-determining regions show more interactions than the heavy chain complementarity-determining regions, and a distal framework residue is involved in Ag binding. As seen from the crystal structure, most of the contacts between peptide and Ab are through the five residues, Leu3-Asp4-Pro5-Ala6-Phe7, of PS1. The paratope is predominantly hydrophobic with aromatic residues lining the binding pocket, although a salt bridge also contributes to stabilizing the Ag-Ab interaction. The molecular surface area buried upon PS1 binding is 756 A(2) for the peptide and 625 A(2) for the Fab, which is higher than what has been seen to date for Ab-peptide complexes. A comparison between PC283 structure and a homology model of its germline ancestor suggests that paratope optimization for PS1 occurs by improving both charge and shape complementarity.     

Surface, subunit interfaces and interior of oligomeric proteins

The solvent-accessible surface area (As) of 23 oligomeric proteins is calculated using atomic co-ordinates from high-resolution and well-refined crystal structures. As is correlated with the protein molecular weight, and a power law predicts its value to within 5% on average. The accessible surface of the average oligomer is similar to that of monomeric proteins in its hydropathy and amino acid composition. The distribution of the 20 amino acid types between the protein surface and its interior is also the same as in monomers. Interfaces, i.e. surfaces involved in subunit contacts, differ from the rest of the subunit surface. They are enriched in hydrophobic side-chains, yet they contain a number of charged groups, especially from Arg residues, which are the most abundant residues at interfaces except for Leu. Buried Arg residues are involved in H-bonds between subunits. We counted H-bonds at interfaces and found that several have none, others have one H-bond per 200 A2 of interface area on average (1 A = 0.1 nm). A majority of interface H-bonds involve charged donor or acceptor groups, which should make their contribution to the free energy of dissociation significant, even when they are few. The smaller interfaces cover about 700 A2 of the subunit surface. The larger ones cover 3000 to 10,000 A2, up to 40% of the subunit surface area in catalase. The lower value corresponds to an estimate of the accessible surface area loss required for stabilizing subunit association through the hydrophobic effect alone. Oligomers with small interfaces have globular subunits with accessible surface areas similar to those of monomeric proteins. We suggest that these oligomers assemble from preformed monomers with little change in conformation. In oligomers with large interfaces, isolated subunits should be unstable given their excessively large accessible surface, and assembly is expected to require major structural changes.     

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.     

Characterization of amphiphilic secondary structures in neuropeptide Y through the design, synthesis, and study of model peptides

Neuropeptide Y (NPY) has the potential to form two amphiphilic secondary structures: a polyproline II-like helix in residues 1-8, and an alpha-helix in residues 13-32. NPY dimerizes in aqueous solution and forms stable monolayers at the air-water interface, suggesting that these amphiphilic conformations are stabilized at interfaces. Furthermore, the negative molar ellipticity of monomeric NPY at 222 nm (-8500 degree cm2/dmol), suggests that hydrophobic interactions with the NH2-terminal amphiphilic structure may stabilize the alpha-helix in residues 13-32 before it binds to cell surfaces, even at physiological concentrations. In order to investigate the role of these amphiphilic structures, five NPY models with multiple substitutions in positions 13-32 have been synthesized and studied. Our data demonstrate that the surfactant properties of NPY result from its potential to form amphiphilic secondary and tertiary structures and not from specific amino acid sequences in this region. However, specific residues on the hydrophilic face of the amphiphilic alpha-helix that have been substituted in the models appear to be required to reproduce the full potency of NPY in our pharmacological assays. A possible role for the amphiphilic structures in NPY in presenting such specific determinants to cell surface receptors in the correct conformation is suggested.     

Output grating couplers on planar optical waveguides as direct immunosensors

We demonstrated the feasibility of using integrated optical output grating couplers in direct immunosensing. We monitored as functions of time, first the adsorption of an antigen (Ag) on the waveguide's surface, and subsequently, the binding of the corresponding antibody (Ab), i.e. the formation of the immuno-complex Ag-Ab. The Ag was human immunoglobulin G (h-IgG), and the Ab was rabbit anti-h-IgG. We also studied the adsorption of avidin. The refractive indices nF', thicknesses dF', and surface coverages gamma of the adsorbed adlayers and of the immuno-complex Ag-Ab, respectively, were determined.     

固有无序蛋白与蛋白质相互作用位点残基特征分析

本文对固有无序蛋白(IDPs)与其他蛋白质相互作用位点残基特征进行了研究.首先在数据库中选出满足条件的109条IDPs蛋白质链及与其他配体蛋白形成的299个IDPs-蛋白质复合物,然后提取复合物中作为相互作用位点的IDPs-蛋白质残基.这109条IDPs链中共含有50 031个氨基酸残基,其中处于作用位点的残基有4 822个.通过分析发现,20种氨基酸在形成IDPs-蛋白质相互作用位点残基时具有不同的倾向性,根据形成作用位点残基的倾向性,20种氨基酸可分成三大类:倾向型氨基酸(ILE、LEU、ARG、PHE、TYR、MET、TRP)、中间型氨基酸(GLN、GLU、THR、LYS、VAL、ASP、HIS)、非倾向型氨基酸(PRO、SER、GLY、ALA、ASN、CYS).研究结果还进一步表明,不同氨基酸在有序区域与无序区域形成IDPs-蛋白质作用位点残基的倾向性不同.其中,氨基酸TRP、LEU、ILE、CYS在有序和无序区域形成作用位点残基的差异性尤为明显,而氨基酸GLU、PHE、HIS、ALA则基本没有多大差别.对IDPs-蛋白质相互作用位点残基理化特征进行分析发现:疏水性强、侧链净电荷量较少、极性较小、溶剂可及性表面积较大、侧链体积较大、极化率较大的氨基酸比较倾向于形成作用位点残基.主成分分析结果显示,残基的极化率、侧链体积和溶剂可及表面积对作用位点残基影响最大.     

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.     

Homopolyvalent antibody-antigen interaction kinetic studies with use of a dual-polarization interferometric biosensor

We used dual-polarization interferometry (DPI) to study the interaction kinetics between a 'homopolyvalent' antigen (Ag) and a monoclonal antibody (Ab). A model system, which uses a monoclonal Ab against a homopentameric Ag, C-reactive protein (CRP), is presented with principle and experiments for the study of the interactions between an Ab and an Ag that has multiple identical epitopes. This allows evaluation of the dissociation constant (K(D)) and of the binding stoichiometry by DPI based on measurements of phase changes of Ab-Ag complexes in the transverse magnetic (TM) and transverse electric (TE) polarization modes. The average experimental value of K(D) found by the DPI technique for anti-CRP Ab was shown to be in close agreement with the value obtained by an indirect competition-enzyme-linked immunosorbent assay (ELISA). Moreover, the total number of Ab combining sites on the DPI sensor chip was calculated, and the binding stoichiometry of the surface Ag-Ab complex was obtained. This study illustrates the advantages of the DPI method in biosensing in its capacity for simultaneous evaluation of the thickness and refractive index (density, mass) of adsorbed layers. This allowed a comprehensive analysis of affinity reactions between an Ab having two binding sites and a multi-sited Ag.     

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.     

Size and structure of antigen-antibody complexes: thermodynamic parameters

The role of antigen-antibody (Ag-Ab) complexes in the immune response depends, in part, on the size of the complexes. Previously, we combined electron microscopy with classical and quasi-elastic light scattering to characterize the molecular weight distribution and the conformation of Ag-Ab complexes made from bovine serum albumin (BSA) and pairs of anti-BSA monoclonal antibodies at a single concentration and Ag:Ab molar ratio. In this report, the molecular weight distribution of Ag-Ab complexes was determined by classical light scattering at a single Ag:Ab ratio and over a range of concentrations, and binding of BSA to pairs of MAb was determined by radioimmunoassay at several Ag:Ab molar ratios. A thermodynamic model was developed for the equilibrium size distribution of Ag-Ab complexes formed between a pair of MAb, each with unique affinity and specificity, and an Ag containing a single epitope for each of the pair of MAb. The combined experimental data were used in conjunction with the model to determine the values of cyclization and polymerization constants. Successful determination of the parameters required data from both classical light scattering and electron microscopy. Cyclization constants were lower than those reported in other studies of Ag-Ab complexes; this may be attributable to our use of a protein Ag, as compared to a divalent hapten. In two out of three cases, cyclization constants increased with increasing number of Ab in the complex, in contrast to previous assumptions. The validity of the thermodynamic model was further shown by its ability, in combination with conformational and hydrodynamic model, to predict the hydrodynamic radius of the complexes over a wide range of experimental conditions.     

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.     

Substitution rates in α-helical transmembrane proteins

It has been shown previously that some membrane proteins have a conserved core of amino acid residues. This idea not only serves to orient helices during model building exercises but may also provide insight into the structural role of residues mediating helix-helix interactions. Using experimentally determined high-resolution structures of alpha-helical transmembrane proteins we show that, of the residues within the hydrophobic transmembrane spans, the residues at lipid and subunit interfaces are more evolutionarily variable than those within the lipid-inaccessible core of a polypeptide's transmembrane domain. This supports the idea that helix-helix interactions within the same polypeptide chain and those at the interface between different polypeptide chains may arise in distinct ways. To show this, we use a new method to estimate the substitution rate of an amino acid residue given an alignment and phylogenetic tree of closely related proteins. This method gives better sensitivity in the otherwise-conserved transmembrane domains than a conventional similarity analysis and is relatively insensitive to the sequences used.     

Cation–π, amino–π, π–π, and H‐bond interactions stabilize antigen–antibody interfaces

The identification of immunogenic regions on the surface of antigens, which are able to stimulate an immune response, is a major challenge for the design of new vaccines. Computational immunology aims at predicting such regions—in particular B‐cell epitopes—but is far from being reliably applicable on a large scale. To gain understanding into the factors that contribute to the antigen–antibody affinity and specificity, we perform a detailed analysis of the amino acid composition and secondary structure of antigen and antibody surfaces, and of the interactions that stabilize the complexes, in comparison with the composition and interactions observed in other heterodimeric protein interfaces. We make a distinction between linear and conformational B‐cell epitopes, according to whether they consist of successive residues along the polypeptide chain or not. The antigen–antibody interfaces were shown to differ from other protein–protein interfaces by their smaller size, their secondary structure with less helices and more loops, and the interactions that stabilize them: more H‐bond, cation–π, amino–π, and π–π interactions, and less hydrophobic packing; linear and conformational epitopes can clearly be distinguished. Often, chains of successive interactions, called cation/amino–π and π–π chains, are formed. The amino acid composition differs significantly between the interfaces: antigen–antibody interfaces are less aliphatic and more charged, polar and aromatic than other heterodimeric protein interfaces. Moreover, paratopes and epitopes—albeit to a lesser extent—have amino acid compositions that are distinct from general protein surfaces. This specificity holds promise for improving B‐cell epitope prediction. Proteins 2014; 82:1734–1746. © 2014 Wiley Periodicals, Inc.     

Dissecting the protein-RNA interface: the role of protein surface shapes and RNA secondary structures in protein-RNA recognition

Protein-RNA interactions are essential for many biological processes. However, the structural mechanisms underlying these interactions are not fully understood. Here, we analyzed the protein surface shape (dented, intermediate or protruded) and the RNA base pairing properties (paired or unpaired nucleotides) at the interfaces of 91 protein-RNA complexes derived from the Protein Data Bank. Dented protein surfaces prefer unpaired nucleotides to paired ones at the interface, and hydrogen bonds frequently occur between the protein backbone and RNA bases. In contrast, protruded protein surfaces do not show such a preference, rather, electrostatic interactions initiate the formation of hydrogen bonds between positively charged amino acids and RNA phosphate groups. Interestingly, in many protein-RNA complexes that interact via an RNA loop, an aspartic acid is favored at the interface. Moreover, in most of these complexes, nucleotide bases in the RNA loop are flipped out and form hydrogen bonds with the protein, which suggests that aspartic acid is important for RNA loop recognition through a base-flipping process. This study provides fundamental insights into the role of the shape of the protein surface and RNA secondary structures in mediating protein-RNA interactions.     

A two-stage classifier for identification of protein-protein interface residues

MOTIVATION: The ability to identify protein-protein interaction sites and to detect specific amino acid residues that contribute to the specificity and affinity of protein interactions has important implications for problems ranging from rational drug design to analysis of metabolic and signal transduction networks. RESULTS: We have developed a two-stage method consisting of a support vector machine (SVM) and a Bayesian classifier for predicting surface residues of a protein that participate in protein-protein interactions. This approach exploits the fact that interface residues tend to form clusters in the primary amino acid sequence. Our results show that the proposed two-stage classifier outperforms previously published sequence-based methods for predicting interface residues. We also present results obtained using the two-stage classifier on an independent test set of seven CAPRI (Critical Assessment of PRedicted Interactions) targets. The success of the predictions is validated by examining the predictions in the context of the three-dimensional structures of protein complexes.