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.     

Non-covalent interactions across subunit interfaces in Sm proteins

The distinguishing property of Sm protein associations is their high stability. In order to understand this property, we analyzed the interface non-covalent interactions and compared the properties of the Sm protein interfaces with those of a test set, Binding Interface Database (BID). The comparison revealed that the main differences between interfaces of Sm proteins and those of the BID set are the content of charged residues, hydrogen bonds, salt bridges, and conservation scores of interface residues. In Sm proteins, the interfaces have more hydrophobic and fewer charged residues than the surface, which is also the case for the BID test set and other proteins. However, in the interfaces, the content of charged residues in Sm proteins (26%) is substantially larger than that in the BID set (22%). Both interfaces of Sm proteins and of test set have a similar number of hydrophobic interactions per 100 Å². The interfaces of Sm proteins have substantially more hydrogen bonds than the interfaces in test set. The results show clearly that the interfaces of Sm proteins form more salt bridges compared with test set. On average, there are about 16 salt bridges per interface. The high conservation score of amino acids that are involved in non-covalent interactions in protein interfaces is an additional strong argument for their importance. The overriding conclusion from this study is that the non-covalent interactions in Sm protein interfaces considerably contribute to stability of higher order structures.     

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.     

Aromatic interactions at atom-to-atom contact and just beyond: a case study of protein interactions of NAD⁺/NADP⁺

We probed aromatic-protein interactions based on specificity of enrichment of protein residues across a contact-based cutoff. Thus, 155 protein-NAD⁺/NADP⁺ complexes were analyzed for enrichments within 10 Å of centroids of aromatic groups of the ligand when the residues were contacted and not contacted with the aromatic ligand. Specifically, neutral-adenine and cationic-nicotinamide groups of the oxidized coenzymes evoked interest to know whether the contrast of charge or the shared aromaticity will manifest in the enrichments across the cutoff. We found that when in contact, the enrichments are highly specific for nicotinamide and adenine-aromatic structures, and thus possibly complex in the basis, but when not in contact, they are generic for charge and aromaticity of the structures, and thus possibly specific in the basis. The order of enrichments over the contacted residues is Tyr > Cys > Thr > His > Asn > Ser > Met > Ile > Phe against nicotinamide-π⁺ structure and Asp > Ile > Thr > His > Arg > Tyr > Gly > Val against adenine-π structure, while the order over the non-contacted residues is Trp > Gly > His > Asn > Cys > Met > Tyr > Ser > Thr > Phe against nicotinamide-π⁺ structure and Asn > Thr > Ser > Gly > Cys > His > Val against adenine-π structure. Neutral Trp, His, Tyr, and Phe, but not cationic Arg, are thus the non-contacted residues enriched specifically against nicotinamide-π⁺ structure, while Asn, Gly, Thr, Ser, and Cys are the non-contacted residues enriched generically against both the nicotinamide-π⁺ and adenine-π aromatic structures. By analyzing the enriched groups in their geometric specificities, we found that, the enrichments against nicotinamide cation manifest the specificity expected of cation-π interaction and against nicotinamide- and adenine-aromatic groups manifest the specificity expected of dipole-π interaction. The cutoff-based method is proven valuable in probing protein-ligand interactions in the physics involved.     

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.     

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.     

Detection of Bacillus thuringiensis Cry1Ab protein based on surface plasmon resonance immunosensor

Two novel surface plasmon resonance immunosensors were fabricated for detection of the Bacillus thuringiensis Cry1Ab protein and to demonstrate their performance in analyzing Cry1Ab protein in crop samples. Sensor 2 was modified by 1,6-hexanedithiol, Au/Ag alloy nanoparticles, 3-mercaptopropionic acid, and protein A (or not [sensor 1]), with Cry1Ab monoclonal antibody. As a result, both of the immunosensors exhibited satisfactory linear responses in the Cry1Ab protein concentration ranges of 10 to 500 ng ml⁻¹ and 8 to 1000 ng ml⁻¹, and the detection limits were 5.0 and 4.8 ng ml⁻¹, respectively. The immunosensors possessed good specificity and acceptable reproducibility. In addition, crop samples could be analyzed after a simple treatment. The transgenic crops could be easily identified from the conventional ones by the two immunosensors.     

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.     

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.     

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.     

Molecular basis for the glycosphingolipid-binding specificity of α-synuclein: key role of tyrosine 39 in membrane insertion

Cell surface glycosphingolipids (GSLs) including gangliosides play a key role in the regulation of the conformation, oligomerization, and fibrillation of amyloidogenic proteins. Correspondingly, most amyloidogenic proteins possess a functional GSL-binding motif (GBM). Sequence alignments of GSL-binding proteins against the GBM of α-synuclein allowed the establishment of a consensus GBM sequence defined as K/H/R/-X(1-4)-Y/F-X(4-5)-K/H/R, where at least one of the X(1-4) residues is glycine. The GBMs of α-synuclein (34-KEGVLYVGSKTK-45) and Alzheimer's disease β-amyloid peptide (Aβ) (5-RHDSGYEVHHQK-16) consist of a structurally related loop centered on tyrosine (Y39 for α-synuclein, Y10 for Aβ). Surface pressure measurements of GSL monolayers at the air-water interface allowed us to determine the following order for α-synuclein-GSL interactions: GM3 > Gb3 > GalCer-NFA > GM1 > sulfatide > GalCer-HFA > LacCer > GM4 > GM2 > asialo-GM1 > GD3, indicating a marked preference for GSLs with one, three, or five sugar units. The insertion of α-synuclein into sphingomyelin-containing monolayers was strongly stimulated by the presence of GM3. This effect was not observed with phosphatidylcholine monolayers, suggesting that the ganglioside facilitated the insertion of α-synuclein into raft-like membrane domains. Molecular dynamics simulations suggested that the side chain of Y39 was deeply inserted between GM3 head groups. Monolayer experiments with mutant GBM peptides showed that Y39, K34, and K45 were important for GM3 binding, whereas only Y39 appeared critical for GM1 recognition. The interaction of Aβ 5-16 with GM1 involved R5, H13, H14, and K16, but not Y10. These data indicate that subtle amino acid variations in the consensus GBM of α-synuclein and Aβ conferred distinct GSL-binding properties.     

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.     

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.     

Coarse-grained models for simulations of multiprotein complexes: application to ubiquitin binding

We develop coarse-grained models and effective energy functions for simulating thermodynamic and structural properties of multiprotein complexes with relatively low binding affinity (K_d > 1 μM) and apply them to binding of Vps27 to membrane-tethered ubiquitin. Folded protein domains are represented as rigid bodies. The interactions between the domains are treated at the residue level with amino-acid-dependent pair potentials and Debye-Hückel-type electrostatic interactions. Flexible linker peptides connecting rigid protein domains are represented as amino acid beads on a polymer with appropriate stretching, bending, and torsion-angle potentials. In simulations of membrane-attached protein complexes, interactions between amino acids and the membrane are described by residue-dependent short-range potentials and long-range electrostatics. We parameterize the energy functions by fitting the osmotic second virial coefficient of lysozyme and the binding affinity of the ubiquitin-CUE complex. For validation, extensive replica-exchange Monte Carlo simulations are performed of various protein complexes. Binding affinities for these complexes are in good agreement with the experimental data. The simulated structures are clustered on the basis of distance matrices between two proteins and ranked according to cluster population. In ∼ 70% of the complexes, the distance root-mean-square is less than 5 Å from the experimental structures. In ∼ 90% of the complexes, the binding interfaces on both proteins are predicted correctly, and in all other cases at least one interface is correct. Transient and nonspecifically bound structures are also observed. With the validated model, we simulate the interaction between the Vps27 multiprotein complex and a membrane-tethered ubiquitin. Ubiquitin is found to bind preferentially to the two UIM domains of Vps27, but transient interactions between ubiquitin and the VHS and FYVE domains are observed as well. These specific and nonspecific interactions are found to be positively cooperative, resulting in a substantial enhancement of the overall binding affinity beyond the ∼ 300 μM of the specific domains. We also find that the interactions between ubiquitin and Vps27 are highly dynamic, with conformational rearrangements enabling binding of Vps27 to diverse targets as part of the multivesicular-body protein-sorting pathway.     

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.     

Polyamines: Naturally occurring small molecule modulators of electrostatic protein-protein interactions

Modulations of protein-protein interactions are a key step in regulating protein function, especially in networks. Modulators of these interactions are supposed to be candidates for the development of novel drugs. Here, we describe the role of the small, polycationic and highly abundant natural polyamines that could efficiently bind to charged spots at protein interfaces as modulators of such protein-protein interactions. Using the mitochondrial cytochrome P45011A1 (CYP11A1) electron transfer system as a model, we have analyzed the capability of putrescine, spermidine, and spermine at physiologically relevant concentrations to affect the protein-protein interactions between adrenodoxin reductase (AdR), adrenodoxin (Adx), and CYP11A1. The actions of polyamines on the individual components, on their association/dissociation, on electron transfer, and on substrate conversion were examined. These studies revealed modulating effects of polyamines on distinct interactions and on the entire system in a complex way. Modulation via changed protein-protein interactions appeared plausible from docking experiments that suggested favourable high-affinity binding sites of polyamines (spermine > spermidine > putrescine) at the AdR-Adx interface. Our findings imply for the first time that small endogenous compounds are capable of interfering with distinct components of transient protein complexes and might control protein functions by modulating electrostatic protein-protein interactions.     

Non-Redundant Unique Interface Structures as Templates for Modeling Protein Interactions

Improvements in experimental techniques increasingly provide structural data relating to protein-protein interactions. Classification of structural details of protein-protein interactions can provide valuable insights for modeling and abstracting design principles. Here, we aim to cluster protein-protein interactions by their interface structures, and to exploit these clusters to obtain and study shared and distinct protein binding sites. We find that there are 22604 unique interface structures in the PDB. These unique interfaces, which provide a rich resource of structural data of protein-protein interactions, can be used for template-based docking. We test the specificity of these non-redundant unique interface structures by finding protein pairs which have multiple binding sites. We suggest that residues with more than 40% relative accessible surface area should be considered as surface residues in template-based docking studies. This comprehensive study of protein interface structures can serve as a resource for the community. The dataset can be accessed at http://prism.ccbb.ku.edu.tr/piface.     

Summarizing techniques that combine three non-parametric scores to detect disease-associated 2-way SNP-SNP interactions

Identifying susceptibility genes that influence complex diseases is extremely difficult because loci often influence the disease state through genetic interactions. Numerous approaches to detect disease-associated SNP-SNP interactions have been developed, but none consistently generates high-quality results under different disease scenarios. Using summarizing techniques to combine a number of existing methods may provide a solution to this problem. Here we used three popular non-parametric methods—Gini, absolute probability difference (APD), and entropy—to develop two novel summary scores, namely principle component score (PCS) and Z-sum score (ZSS), with which to predict disease-associated genetic interactions. We used a simulation study to compare performance of the non-parametric scores, the summary scores, the scaled-sum score (SSS; used in polymorphism interaction analysis (PIA)), and the multifactor dimensionality reduction (MDR). The non-parametric methods achieved high power, but no non-parametric method outperformed all others under a variety of epistatic scenarios. PCS and ZSS, however, outperformed MDR. PCS, ZSS and SSS displayed controlled type-I-errors (< 0.05) compared to GS, APDS, ES (> 0.05). A real data study using the genetic-analysis-workshop 16 (GAW 16) rheumatoid arthritis dataset identified a number of interesting SNP-SNP interactions.     

Regional and segmental flexibility of antibodies in interaction with antigens of different size

The interaction of antibodies (Abs) with protein antigens (Ags) of different size, such as hen egg white lysozyme, ovalbumin, and bovine serum albumin, was examined using analytical ultracentrifugation, electrospray ionization time-of-flight mass spectrometry, and surface plasmon resonance in order to estimate regional and segmental Ab flexibility. When both Abs and Ags were free in solution, sedimentation equilibrium and surface plasmon resonance analyses showed the formation of an Ag(2)Ab(1) complexes regardless of Ag size, suggesting that the Fab arms were able to move to avoid interference between Ags bound to Ab combining sites. The Ag(2)Ab(1) complex, as well as the Ag(1)Ab(1) complex, was observed by MS. However, when Abs were immobilized on the surface of a sensor chip through the Fc region, the stoichiometry of the Ag-Ab complex was dependent on the Ag size; Ag(2)Ab(1) forming with hen egg white lysozyme and Ag(1)Ab(1) with ovalbumin and bovine serum albumin. These results indicated that immobilization of the Fc region reduces the dynamic range of the Fab arms and results in interference from the first Ag bound to either combining site, which in turn prevents the binding of the second Ag to the other combining site. Our results allow us to propose that the Fab arms of B-cell receptors whose Fc regions are immobilized on cell surface have a reduced dynamic range.