Grafting of protein-protein interaction epitope

Transferring the biological function of one protein to another is a key issue in understanding the structure and function relationship of proteins. We have developed a strategy for grafting protein-protein interaction epitopes. As a first step, residues at the interface of the ligand protein which strongly interact with the receptor protein were identified. Then protein scaffolds were docked onto receptor protein based on geometric complementarity. Only high docking score matches were saved. For each saved match, the scaffold protein was accepted if it had suitable positions for grafting key interaction residues of the ligand protein. These candidate residues were mutated to corresponding residues in the ligand protein at each relevant position and the mutated scaffold protein was co-minimized with receptor protein. Finally, the minimized complexes were evaluated by a scoring function deduced from statistical analysis of rigid binding data sets. As a test case, the binding epitope of barstar, the inhibitor of barnase, was grafted onto smaller proteins. Pheromone Er-1 (PDB entry 1erc) has been found to be a good scaffold. The calculated binding free energy for mutated Pheromone Er-1 is equivalent to that of barstar.     

Amino acid mutagenesis of the ligand binding site and the dimer interface of the metabotropic glutamate receptor 1. Identification of crucial residues for setting the activated state

Previously, we determined the crystal structures of the dimeric ligand binding region of the metabotropic glutamate receptor subtype 1. Each protomer binds l-glutamate within the crevice between the LB1 and LB2 domains. We proposed that the two different conformations of the dimer interface between the two LB1 domains define the activated and resting states of the receptor protein. In this study, the residues in the ligand-binding site and the dimer interface were mutated, and the effects were analyzed in the full-length and truncated soluble receptor forms. The variations in the ligand binding activities of the purified truncated receptors are comparable with those of the full-length form. The mutated full-length receptors were also analyzed by inositol phosphate production and Ca(2+) response. The magnitude of the ligand binding capacities and the amplitude of the intracellular signaling were almost correlated. Alanine substitutions of four residues, Thr(188), Asp(208), Tyr(236), and Asp(318), which interact with the alpha-amino group of glutamate in the crystal, abolished their responses both to glutamate and quisqualate. The mutations of the Tyr(74), Arg(78), and Gly(293) residues, which interact with the gamma-carboxyl group of glutamate, lost their responsiveness to glutamate but not to quisqualate. Furthermore, a mutant receptor containing alanine instead of isoleucine at position 120 located within an alpha helix constituting the dimer interface showed no intracellular response to ligand stimulation. The results demonstrate the crucial role of the dimer interface in receptor activation.     

Structural determinants of ubiquitin-CXC chemokine receptor 4 interaction

Ubiquitin, a post-translational protein modifier inside the cell, functions as a CXC chemokine receptor (CXCR) 4 agonist outside the cell. However, the structural determinants of the interaction between extracellular ubiquitin and CXCR4 remain unknown. Utilizing C-terminal truncated ubiquitin and ubiquitin mutants, in which surface residues that are known to interact with ubiquitin binding domains in interacting proteins are mutated (Phe-4, Leu-8, Ile-44, Asp-58, Val-70), we provide evidence that the ubiquitin-CXCR4 interaction follows a two-site binding mechanism in which the hydrophobic surfaces surrounding Phe-4 and Val-70 are important for receptor binding, whereas the flexible C terminus facilitates receptor activation. Based on these findings and the available crystal structures, we then modeled the ubiquitin-CXCR4 interface with the RosettaDock software followed by small manual adjustments, which were guided by charge complementarity and anticipation of a conformational switch of CXCR4 upon activation. This model suggests three residues of CXCR4 (Phe-29, Phe-189, Lys-271) as potential interaction sites. Binding studies with HEK293 cells overexpressing wild type and CXCR4 after site-directed mutagenesis confirm that these residues are important for ubiquitin binding but that they do not contribute to the binding of stromal cell-derived factor 1α. Our findings suggest that the structural determinants of the CXCR4 agonist activity of ubiquitin mimic the typical structure-function relationship of chemokines. Furthermore, we provide evidence for separate and specific ligand binding sites on CXCR4. As exogenous ubiquitin has been shown to possess therapeutic potential, our findings are expected to facilitate the structure-based design of new compounds with ubiquitin-mimetic actions on CXCR4.     

Mutation of single hydrophobic residue I27, L35, F39, L58, L65, L67, or L71 in the N terminus of VP5 abolishes interaction with the scaffold protein and prevents closure of herpes simplex virus type 1 capsid shells

Protein-protein interactions drive the assembly of the herpes simplex virus type 1 (HSV-1) capsid. A key interaction occurs between the C-terminal tail of the scaffold protein (pre-22a) and the major capsid protein (VP5). Previously (Z. Hong, M. Beaudet-Miller, J. Durkin, R. Zhang, and A. D. Kwong, J. Virol. 70:533-540, 1996) it was shown that the minimal domain in the scaffold protein necessary for this interaction was composed of a hydrophobic amphipathic helix. The goal of this study was to identify the hydrophobic residues in VP5 important for this bimolecular interaction. Results from the genetic analysis of second-site revertant virus mutants identified the importance of the N terminus of VP5 for the interaction with the scaffold protein. This allowed us to focus our efforts on a small region of this large polypeptide. Twenty-four hydrophobic residues, starting at L23 and ending at F84, were mutated to alanine. All the mutants were first screened for interaction with pre-22a in the yeast two-hybrid assay. From this in vitro assay, seven residues, I27, L35, F39, L58, L65, L67, and L71, that eliminated the interaction when mutated were identified. All 24 mutants were introduced into the virus genome with a genetic marker rescue/marker transfer system. For this system, viruses and cell lines that greatly facilitated the introduction of the mutants into the genome were made. The same seven mutants that abolished interaction of VP5 with pre-22a resulted in an absolute requirement for wild-type VP5 for growth of the viruses. The viruses encoding these mutations in VP5 were capable of forming capsid shells comprised of VP5, VP19C, VP23, and VP26, but the closure of these shells into an icosahedral structure was prevented. Mutation at L75 did not affect the ability of this protein to interact with pre-22a, as judged from the in vitro assay, but this mutation specified a lethal effect for virus growth and abolished the formation of any detectable assembled structure. Thus, it appears that the L75 residue is important for another essential interaction of VP5 with the capsid shell proteins. The congruence of the data from the previous and present studies demonstrates the key roles of two regions in the N terminus of this large protein that are crucial for this bimolecular interaction. Thus, residues I27, L35, and F39 comprise the first subdomain and residues L58, L65, L67 and L71 comprise a second subdomain of VP5. These seven hydrophobic residues are important for the interaction of VP5 with the scaffold protein and consequently the formation of an icosahedral shell structure that encloses the viral genome.     

Investigation of Interactions at the Extracellular Loops of the Relaxin Family Peptide Receptor 1 (RXFP1)

Relaxin, an emerging pharmaceutical treatment for acute heart failure, activates the relaxin family peptide receptor (RXFP1), which is a class A G-protein-coupled receptor. In addition to the classic transmembrane (TM) domain, RXFP1 possesses a large extracellular domain consisting of 10 leucine-rich repeats and an N-terminal low density lipoprotein class A (LDLa) module. Relaxin-mediated activation of RXFP1 requires multiple coordinated interactions between the ligand and various receptor domains including a high affinity interaction involving the leucine-rich repeats and a predicted lower affinity interaction involving the extracellular loops (ELs). The LDLa is essential for signal activation; therefore the ELs/TM may additionally present an interaction site to facilitate this LDLa-mediated signaling. To overcome the many challenges of investigating relaxin and the LDLa module interactions with the ELs, we engineered the EL1 and EL2 loops onto a soluble protein scaffold, mapping specific ligand and loop interactions using nuclear magnetic resonance spectroscopy. Key EL residues were subsequently mutated in RXFP1, and changes in function and relaxin binding were assessed alongside the RXFP1 agonist ML290 to monitor the functional integrity of the TM domain of these mutant receptors. The outcomes of this work make an important contribution to understanding the mechanism of RXFP1 activation and will aid future development of small molecule RXFP1 agonists/antagonists.     

Apolipoprotein E-low density lipoprotein receptor binding: study of protein-protein interaction in rationally selected docked complexes

Apolipoprotein E (apoE) is an important protein involved in lipid metabolism due to its interaction with members of the low-density lipoprotein receptor (LDLR) family. To further understand the molecular basis for this receptor-binding activity, an apoE fragment containing the receptor binding region (residues 135-151) was docked onto the fifth LDLR ligand binding repeat (LR5) by computational methods. A subset of structures generated by the docking was rationally selected on the grounds of experimental data combined with modeling and was used for further analysis. The application and comparison of two different experimental structures for the apoE fragment underlines the local structural changes occurring in apoE when switching from a receptor-inactive to a receptor-active conformation. The body of interactions occurring at the interface between the two proteins is in very good agreement with the biochemical data available for both apoE and LDLR. Charged residues are involved in numerous ionic interactions and might therefore be important for the specificity of the interaction between apoE and LR5. In addition, the interface also features a tryptophan and a stacking of histidine residues, revealing that the association between the two proteins is not entirely governed by ionic interactions. In particular, the presence of histidine residues in the interface gives a structural basis for the pH-regulated release mechanism of apoE in the endosomes. The proposed molecular basis for apoE binding to LDLR could aid the design of strategies for targeting alterations in lipid transport and metabolism.     

Mapping of a yeast G protein betagamma signaling interaction.

The mating pathway of Saccharomyces cerevisiae is widely used as a model system for G protein-coupled receptor-mediated signal transduction. Following receptor activation by the binding of mating pheromones, G protein betagamma subunits transmit the signal to a MAP kinase cascade, which involves interaction of Gbeta (Ste4p) with the MAP kinase scaffold protein Ste5p. Here, we identify residues in Ste4p required for the interaction with Ste5p. These residues define a new signaling interface close to the Ste20p binding site within the Gbetagamma coiled-coil. Ste4p mutants defective in the Ste5p interaction interact efficiently with Gpa1p (Galpha) and Ste18p (Ggamma) but cannot function in signal transduction because cells expressing these mutants are sterile. Ste4 L65S is temperature-sensitive for its interaction with Ste5p, and also for signaling. We have identified a Ste5p mutant (L196A) that displays a synthetic interaction defect with Ste4 L65S, providing strong evidence that Ste4p and Ste5p interact directly in vivo through an interface that involves hydrophobic residues. The correlation between disruption of the Ste4p-Ste5p interaction and sterility confirms the importance of this interaction in signal transduction. Identification of the Gbetagamma coiled-coil in Ste5p binding may set a precedent for Gbetagamma-effector interactions in more complex organisms.     

Crystal structure of the ligand binding suppressor domain of type 1 inositol 1,4,5-trisphosphate receptor

Binding of inositol 1,4,5-trisphosphate (IP(3)) to the amino-terminal region of IP(3) receptor promotes Ca(2+) release from the endoplasmic reticulum. Within the amino terminus, the first 220 residues directly preceding the IP(3) binding core domain play a key role in IP(3) binding suppression and regulatory protein interaction. Here we present a crystal structure of the suppressor domain of the mouse type 1 IP(3) receptor at 1.8 A. Displaying a shape akin to a hammer, the suppressor region contains a Head subdomain forming the beta-trefoil fold and an Arm subdomain possessing a helix-turn-helix structure. The conserved region on the Head subdomain appeared to interact with the IP(3) binding core domain and is in close proximity to the previously proposed binding sites of Homer, RACK1, calmodulin, and CaBP1. The present study sheds light onto the mechanism underlying the receptor's sensitivity to the ligand and its communication with cellular signaling proteins.     

Mutagenesis and molecular modeling reveal the importance of the 5-HT3 receptor F-loop

The 5-HT(3) receptor is a member of the Cys-loop family of ligand-gated ion channels. The extracellular domains of these proteins contain six amino acid loops (A-F) that converge to form the ligand binding site. In this study we have mutated 21 residues in or close to the 5-HT(3) receptor F-loop (Ile(192) to Gly(212)) to Ala or to a residue with similar chemical properties. Mutant receptors were expressed in HEK293 cells, and binding affinity was measured using [(3)H]granisetron. Two regions displayed decreases in binding affinity when mutated to Ala (Ile(192)-Arg(196) and Asp(204)-Ser(206)), but only one region was sensitive when mutated to chemically similar residues (Ile(192)-Val(201)). Homology modeling using acetylcholine-binding protein crystal structures with a variety of different bound ligands suggests there may be distinct movements of Trp(195) and Asp(204) upon ligand binding, indicating that these residues and their immediate neighbors have the ability to interact differently with different ligands. The models suggest predominantly lateral movement around Asp(204) and rotational movement around Trp(195), indicating the former is in a more flexible region. Overall our results are consistent with a flexible 5-HT(3) receptor F-loop with two regions that have specific but distinct roles in ligand binding.     

A common site of the Fc receptor gamma subunit interacts with the unrelated immunoreceptors FcalphaRI and FcepsilonRI

The transmembrane (TM) region of the Fc receptor-gamma (FcRgamma) chain is responsible for the association of this ubiquitous signal transduction subunit with many immunoreceptor ligand binding chains, making FcRgamma key to a number of leukocyte activities in immunity and disease. Some receptors contain a TM arginine residue that interacts with Asp-11 of the FcRgamma subunit, but otherwise the molecular basis for the FcRgamma subunit interactions is largely unknown. This study reports residues in the TM region of the FcRgamma subunit are important for association with the high affinity IgE receptor FcepsilonRI and a leukocyte receptor cluster member, the IgA receptor FcalphaRI. FcRgamma residue Leu-21 was essential for surface expression of FcepsilonRIalpha/gamma2 and Tyr-8, Leu-14, and Phe-15 contributed to expression. Likewise, detergent-stable FcRgamma association with FcalphaRI was also dependent on Leu-14 and Leu-21 and in addition required residues Tyr-17, Tyr-25, and Cys-26. Modeling the TM regions of the FcRgamma dimer indicated these residues interacting with both FcalphaRI and FcepsilonRI are near the interface between the two FcRgamma TM helices. Furthermore, the FcRgamma residues interacting with FcalphaRI form a leucine zipper-like interface with mutagenesis confirming a complementary interface comprising FcalphaRI residues Leu-217, Leu-220, and Leu-224. The dependence of these two nonhomologous receptor interactions on FcRgamma Leu-14 and Leu-21 suggests that all the associated Fc receptors and the activating leukocyte receptor cluster members interact with this one site. Taken together these data provide a molecular basis for understanding how disparate receptor families assemble with the FcRgamma subunit.     

Geometric and chemical patterns of interaction in protein--ligand complexes and their application in docking

We present a new method for representing the binding site of a protein receptor that allows the use of the DOCK approach to screen large ensembles of receptor conformations for ligand binding. The site points are constructed from templates of what we called "attached points" (ATPTS). Each template (one for each type of amino acid) is composed of a set of representative points that are attached to side-chain and backbone atoms through internal coordinates, carry chemical information about their parent atoms and are intended to cover positions that might be occupied by ligand atoms when complexed to the protein. This method is completely automatic and proved to be extremely fast. With the aim of obtaining an experimental basis for this approach, the Protein Data Bank was searched for proteins in complex with small molecules, to study the geometry of the interactions between the different types of protein residues and the different types of ligand atoms. As a result, well-defined patterns of interaction were obtained for most amino acids. These patterns were then used for constructing a set of templates of attached points, which constitute the core of the ATPTS approach. The quality of the ATPTS representation was demonstrated by using this method, in combination with the DOCK matching and orientation algorithms, to generate correct ligand orientations for >1000 protein--ligand complexes.     

The roles of contact residue disorder and domain composition in characterizing protein-ligand binding specificity and promiscuity

Most protein chains interact with only one ligand but a small number of protein chains can bind several ligands, and many examples are available in the protein-ligand complex database of PDB. Among these proteins, some show preferences for the ligands or types of ligands they bind; however, so far we have only poor understanding of what determines protein-ligand binding and its specificity. Here we investigate the structural and functional properties of proteins in protein-ligand complexes. Analysis of the protein-ligand complex dataset from the PDB structure database reveals that proteins with more interactions have more disordered contact residues. Those proteins containing few disordered contact residues that bind multiple ligands have a tendency to consist of several domains. Analysis of physicochemical properties of hub contact residues binding multiple ligands indicates that they are enriched for hydrophilic, charged, polar and His-Asp catalytic triad residues. Finally, in order to differentiate proteins binding different classes of ligands, we mapped the three most prominent classes of ligands onto different superfamily domains. Our results demonstrate that contact residue disorder and ordered multiple domains are complementary factors that play a crucial role in determining ligand binding specificity and promiscuity.     

Structure-wise discrimination of cytosine,thymine, and uracil by proteins in terms of their nonbonded interactions

The molecular recognition and discrimination of very similar ligand moieties by proteins are important subjects in protein–ligand interaction studies. Specificity in the recognition of molecules is determined by the arrangement of protein and ligand atoms in space. The three pyrimidine bases, viz. cytosine, thymine, and uracil, are structurally similar, but the proteins that bind to them are able to discriminate them and form interactions. Since nonbonded interactions are responsible for molecular recognition processes in biological systems, our work attempts to understand some of the underlying principles of such recognition of pyrimidine molecular structures by proteins. The preferences of the amino acid residues to contact the pyrimidine bases in terms of nonbonded interactions; amino acid residue–ligand atom preferences; main chain and side chain atom contributions of amino acid residues; and solvent-accessible surface area of ligand atoms when forming complexes are analyzed. Our analysis shows that the amino acid residues, tyrosine and phenyl alanine, are highly involved in the pyrimidine interactions. Arginine prefers contacts with the cytosine base. The similarities and differences that exist between the interactions of the amino acid residues with each of the three pyrimidine base atoms in our analysis provide insights that can be exploited in designing specific inhibitors competitive to the ligands.     

AMPA receptors and bacterial periplasmic amino acid-binding proteins share the ionic mechanism of ligand recognition.

In order to identify key structural determinants for ligand recognition, we subjected the ligand-binding domain of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-selective glutamate receptor GluR-D subunit to site-directed mutagenesis. Based on the analysis of the [3H]AMPA-binding properties of the mutated binding sites, we constructed a revised three-dimensional model of the ligand-binding site, different in many respects from previously published models. In particular, our results indicate that the residues Arg507 and Glu727 represent the structural and functional correlates of Arg77 and Asp161 in the homologous bacterial lysine/ornithine/arginine-binding protein and histidine-binding protein, and directly interact with the alpha-carboxyl and alpha-amino group of the bound ligand, respectively. In contrast, Glu424, implicated previously in ionic interactions with the alpha-amino group of the agonist, is unlikely to have such a role in ligand binding. Our results indicate that glutamate receptors share with the bacterial polar amino acid-binding proteins the fundamental mechanism of amino acid recognition.     

Double-mutant cycle scanning of the interaction of a peptide ligand and its G protein-coupled receptor

The interaction between the yeast G protein-coupled receptor (GPCR), Ste2p, and its alpha-factor tridecapeptide ligand was subjected to double-mutant cycle scanning analysis by which the pairwise interaction energy of each ligand residue with two receptor residues, N205 and Y266, was determined. The mutations N205A and Y266A were previously shown to result in deficient signaling but cause only a 2.5-fold and 6-fold decrease, respectively, in the affinity for alpha-factor. The analysis shows that residues at the amine terminus of alpha-factor interact strongly with N205 and Y266 whereas residues in the center and at the carboxyl terminus of the peptide interact only weakly if at all with these receptor residues. Multiple-mutant thermodynamic cycle analysis was used to assess whether the energies of selected pairwise interactions between residues of the alpha-factor peptide changed upon binding to Ste2p. Strong positive cooperativity between residues 1 through 4 of alpha-factor was observed during receptor binding. In contrast, no thermodynamic evidence was found for an interaction between a residue near the carboxyl terminus of alpha-factor (position 11) and one at the N-terminus (position 3). The study shows that multiple-mutant cycle analyses of the binding of an alanine-scanned peptide to wild-type and mutant GPCRs can provide detailed information on contributions of inter- and intramolecular interactions to the binding energy and potentially prove useful in developing 3D models of ligand docked to its receptor.     

Molecular dynamics simulation study of AG10 and tafamidis binding to the Val122Ile transthyretin variant

Molecular dynamics (MD) simulations were used to investigate the binding of four ligands to the Val122Ile mutant of the protein transthyretin. Dissociation, misfolding, and subsequent aggregation of mutated transthyretin proteins are associated with the disease Familial Amyloidal Cardiomyopathy. The ligands investigated were the drug candidate AG10 and its decarboxy and N-methyl derivatives along with the drug tafamidis. These ligands bound to the receptor in two halogen binding pockets (HBP) designated AB and A’B’. Inter-ligand distances, solvent accessible surface areas, root mean squared deviation measurements, and extracted structures showed very little change in the AG10 ligands' conformations or locations within the HBP during the MD simulation. In addition, the AG10 ligands experienced stable, two-point interactions with the protein by forming hydrogen bonds with Ser-117 residues in both the AB and A’B’ binding pockets and Lysine-15 residues found near the surface of the receptor. Distance measurements showed these H-bonds formed simultaneously during the MD simulation. Removal of the AG10 carboxylate functional group to form decarboxy-AG10 disrupted this two-point interaction causing the ligand in the AB pocket to undergo a conformational change during the MD simulation. Likewise, addition of a methyl group to the AG10 hydrazone functional group also disrupted the two-point interaction by decreasing hydrogen bonding interactions with the receptor. Finally, MD simulations showed that the tafamidis ligands experienced fewer hydrogen bonding interactions than AG10 with the protein receptor. The tafamidis ligand in pocket A’B’ was also found to move deeper into the HBP during the MD simulation.     

Engineered oligomerization state of OmpF protein through computational design decouples oligomer dissociation from unfolding

Biogenesis of β-barrel membrane proteins is a complex, multistep, and as yet incompletely characterized process. The bacterial porin family is perhaps the best-studied protein family among β-barrel membrane proteins that allows diffusion of small solutes across the bacterial outer membrane. In this study, we have identified residues that contribute significantly to the protein-protein interaction (PPI) interface between the chains of outer membrane protein F (OmpF), a trimeric porin, using an empirical energy function in conjunction with an evolutionary analysis. By replacing these residues through site-directed mutagenesis either with energetically favorable residues or substitutions that do not occur in natural bacterial outer membrane proteins, we succeeded in engineering OmpF mutants with dimeric and monomeric oligomerization states instead of a trimeric oligomerization state. Moreover, our results suggest that the oligomerization of OmpF proceeds through a series of interactions involving two distinct regions of the extensive PPI interface: two monomers interact to form a dimer through the PPI interface near G19. This dimer then interacts with another monomer through the PPI interface near G135 to form a trimer. We have found that perturbing the PPI interface near G19 results in the formation of the monomeric OmpF only. Thermal denaturation of the designed dimeric OmpF mutant suggests that oligomer dissociation can be separated from the process of protein unfolding. Furthermore, the conserved site near G57 and G59 is important for the PPI interface and might provide the essential scaffold for PPIs.     

Targeting cysteine rich C1 domain of Scaffold protein Kinase Suppressor of Ras (KSR) with anthocyanidins and flavonoids – a binding affinity characterization study

Kinase Suppressor of Ras (KSR) is a molecular scaffold that interacts with the core kinase components of the ERK cascade, Raf, MEK, ERK to provide spatial and temporal regulation of Ras-dependent ERK cascade signaling. Interruption of this mechanism can have a high influence in inhibiting the downstream signaling of the mutated tyrosine kinase receptor kinase upon ligand binding. Still none of the studies targeted to prevent the binding of Raf, MEK binding on kinase suppressor of RAS. In that perspective the cysteine rich C1 domain of scaffold proteins kinase suppressor of Ras-1 was targeted rather than its ATP binding site with small ligand molecules like flavones and anthocyanidins and analyzed through insilico docking studies. The binding energy evaluation shows the importance of hydroxyl groups at various positions on the flavone and anthocyanidin nucleus. Over all binding interaction shows these ligands occupied the potential sites of cysteine rich C1 domain of scaffold protein KSR.     

Cross Talk between KGF and KITLG Proteins Implicated with Ovarian Folliculogenesis in Buffalo Bubalus bubalis

Molecular interactions between mesenchymal-derived Keratinocyte growth factor (KGF) and Kit ligand (KITLG) are essential for follicular development. These factors are expressed by theca and granulosa cells. We determined full length coding sequence of buffalo KGF and KITLG proteins having 194 and 274 amino acids, respectively. The recombinant KGF and KITLG proteins were solubilized in 10 mM Tris, pH 7.5 and 50 mM Tris, pH 7.4 and purified using Ni-NTA column and GST affinity chromatography, respectively. The purity and molecular weight of His-KGF (~23 kDa) and GST-KITLG (~57 kDa) proteins were confirmed by SDS-PAGE and western blotting. The co-immunoprecipitation assay accompanied with computational analysis demonstrated the interaction between KGF and KITLG proteins. We deduced 3D structures of the candidate proteins and assessed their binding based on protein docking. In the process, KGF specific residues, Lys123, Glu135, Lys140, Lys155 and Trp156 and KITLG specific ones, Ser226, Phe233, Gly234, Ala235, Phe236, Trp238 and Lys239 involved in the formation of KGF-KITLG complex were detected. The hydrophobic interactions surrounding KGF-KITLG complex affirmed their binding affinity and stability to the interacting interface. Additionally, in-silico site directed mutagenesis enabled the assessment of changes that occurred in the binding energies of mutated KGF-KITLG protein complex. Our results demonstrate that in the presence of KITLG, KGF mimics its native binding mode suggesting all the KGF residues are specific to their binding complex. This study provides an insight on the critical amino acid residues participating in buffalo ovarian folliculogenesis.     

Beauty Is in the Eye of the Beholder: Proteins Can Recognize Binding Sites of Homologous Proteins in More than One Way

Understanding the mechanisms of protein–protein interaction is a fundamental problem with many practical applications. The fact that different proteins can bind similar partners suggests that convergently evolved binding interfaces are reused in different complexes. A set of protein complexes composed of non-homologous domains interacting with homologous partners at equivalent binding sites was collected in 2006, offering an opportunity to investigate this point. We considered 433 pairs of protein–protein complexes from the ABAC database (AB and AC binary protein complexes sharing a homologous partner A) and analyzed the extent of physico-chemical similarity at the atomic and residue level at the protein–protein interface. Homologous partners of the complexes were superimposed using Multiprot, and similar atoms at the interface were quantified using a five class grouping scheme and a distance cut-off. We found that the number of interfacial atoms with similar properties is systematically lower in the non-homologous proteins than in the homologous ones. We assessed the significance of the similarity by bootstrapping the atomic properties at the interfaces. We found that the similarity of binding sites is very significant between homologous proteins, as expected, but generally insignificant between the non-homologous proteins that bind to homologous partners. Furthermore, evolutionarily conserved residues are not colocalized within the binding sites of non-homologous proteins. We could only identify a limited number of cases of structural mimicry at the interface, suggesting that this property is less generic than previously thought. Our results support the hypothesis that different proteins can interact with similar partners using alternate strategies, but do not support convergent evolution.