Characterization and prediction of the binding site in DNA-binding proteins: improvement of accuracy by combining residue composition, evolutionary conservation and structural parameters

We present a set of four parameters that in combination can predict DNA-binding residues on protein structures to a high degree of accuracy. These are the number of evolutionary conserved residues (N(cons)) and their spatial clustering (ρ(e)), hydrogen bond donor capability (D(p)) and residue propensity (R(p)). We first used these parameters to characterize 130 interfaces in a set of 126 DNA-binding proteins (DBPs). The applicability of these parameters both individually and in combination, to distinguish the true binding region from the rest of the protein surface was then analyzed. R(p) shows the best performance identifying the true interface with the top rank in 83% cases. Importantly, we also used the unbound-bound test cases of the protein-DNA docking benchmark to test the efficacy of our method. When applied to the unbound form of the DBPs, R(p) can distinguish 86% cases. Finally, we have applied the SVM approach for recognizing the interface region using the above parameters along with the individual amino acid composition as attributes. The accuracy of prediction is 90.5% for the bound structures and 93.6% for the unbound form of the proteins.     

Structural insights into human GPCR protein OA1: a computational perspective

Human ocular albinism type 1 protein (OA1)—a member of the G-protein coupled receptor (GPCR) superfamily—is an integral membrane glycoprotein expressed exclusively by intracellular organelles known as melanocytes, and is responsible for the proper biogenesis of melanosomes. Mutations in the Oa1 gene are responsible for the disease ocular albinism. Despite its clinical importance, there is a lack of in-depth understanding of its structure and mechanism of activation due to the absence of a crystal structure. In the present study, homology modeling was applied to predicting OA1 structure following thorough sequence analysis and secondary structure predictions. The predicted model had the signature residues and motifs expected of GPCRs, and was used for carrying out molecular docking studies with an endogenous ligand, l-DOPA and an antagonist, dopamine; the results agreed quite well with the available experimental data. Finally, three sets of explicit molecular dynamics simulations were carried out in lipid bilayer, the results of which not only confirmed the stability of the predicted model, but also helped witness some differences in structural features such as rotamer toggle switch, helical tilts and hydrogen bonding pattern that helped distinguish between the agonist- and antagonist-bound receptor forms. In place of the typical “D/ERY”-motif-mediated “ionic lock”, a hydrogen bond mediated by the “DAY” motif was observed that could be used to distinguish the agonist and antagonist bound forms of OA1. In the absence of a crystal structure, this study helped to shed some light on the structural features of OA1, and its behavior in the presence of an agonist and an antagonist, which might be helpful in the future drug discovery process for ocular albinism.     

Biochemical and Biophysical Characterisation of Higher Oligomeric Structure of Rat Nucleosome Assembly Protein 1

Nucleosome assembly protein 1 (NAP1) is a histone chaperone that exchanges histone H2A-H2B dimer from chromatin templates. Studies with yeast NAP1 (yNAP1) have revealed its existence as multiple oligomeric species in solution. Here, rat NAP1 (rNAP1), which is 98% identical to the human NAP1 (hNAP1) was used as a model to characterize the oligomeric structures of this protein in higher eukaryotes. Gel filtration chromatography and Dynamic light scattering of recombinant rNAP1 indicated that the protein exists as a complex mixture of multimeric species even at 500 mM ionic strength. The solution-state complexity remains unchanged even at higher ionic strengths. Equilibrium unfolding (ΔG 14.6 kcal mol^??1) shows that rNAP1, both dimeric and oligomeric, follow the two-state model of unfolding with no detectable intermediates. Homology modelling suggests that rat and yeast NAP1 share an overall similar structure with conserved domains. However, dissimilar substitutions like threonine and lysine with glycine in the β-hairpin involved in oligomerization, possibly leads to the observed differences in the oligomerization propensity of the two proteins. Molecular dynamic simulation (MDS) of the two structures also revealed that rNAP1 dimer is more stable owing to the extensive hydrogen bonding in comparison to yNAP1. Further, in vitro kinase assay showed that the phosphorylation of rNAP1 favors oligomerization with no effect on its histone binding capacity. Our results clearly suggest that there are differences in the in-solution behavior of rNAP1 compared to yNAP1 which may have in vivo functional implications for the regulation of these complexes during chromatin assembly and rearrangement.     

Cavities in protein–DNA and protein–RNA interfaces

An analysis of cavities present in protein–DNA and protein–RNA complexes is presented. In terms of the number of cavities and their total volume, the interfaces formed in these complexes are akin to those in transient protein–protein heterocomplexes. With homodimeric proteins protein–DNA interfaces may contain cavities involving both the protein subunits and DNA, and these are more than twice as large as cavities involving a single protein subunit and DNA. A parameter, cavity index, measuring the degree of surface complementarity, indicates that the packing of atoms in protein–protein/DNA/RNA is very similar, but it is about two times less efficient in the permanent interfaces formed between subunits in homodimers. As within the tertiary structure and protein–protein interfaces, protein–DNA interfaces have a higher inclination to be lined by β-sheet residues; from the DNA side, base atoms, in particular those in minor grooves, have a higher tendency to be located in cavities. The larger cavities tend to be less spherical and solvated. A small fraction of water molecules are found to mediate hydrogen-bond interactions with both the components, suggesting their primary role is to fill in the void left due to the local non-complementary nature of the surface patches.