Structure-based function prediction of uncharacterized protein using binding sites comparison.

A challenge in structural genomics is prediction of the function of uncharacterized proteins. When proteins cannot be related to other proteins of known activity, identification of function based on sequence or structural homology is impossible and in such cases it would be useful to assess structurally conserved binding sites in connection with the protein's function. In this paper, we propose the function of a protein of unknown activity, the Tm1631 protein from Thermotoga maritima, by comparing its predicted binding site to a library containing thousands of candidate structures. The comparison revealed numerous similarities with nucleotide binding sites including specifically, a DNA-binding site of endonuclease IV. We constructed a model of this Tm1631 protein with a DNA-ligand from the newly found similar binding site using ProBiS, and validated this model by molecular dynamics. The interactions predicted by the Tm1631-DNA model corresponded to those known to be important in endonuclease IV-DNA complex model and the corresponding binding free energies, calculated from these models were in close agreement. We thus propose that Tm1631 is a DNA binding enzyme with endonuclease activity that recognizes DNA lesions in which at least two consecutive nucleotides are unpaired. Our approach is general, and can be applied to any protein of unknown function. It might also be useful to guide experimental determination of function of uncharacterized proteins

Top-ranked similar binding sites in proteins of different folds found using the predicted binding site in Tm1631 as query to the binding site comparison approach.^a

a<p>The entire list of similar binding sites is in Table S1 in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003341#pcbi.1003341.s001" target="_blank">Text S1</a>.</p

Tm1631-DNA model after 90 ns of MD.

<p>Reactive phosphate group in DNA is marked with a red asterisk. (a) Tm1631-DNA model, residues that interact with the DNA are marked. (b) Magnified view of the Tm1631-DNA interface. DNA phosphate groups and residues that interact with the DNA are represented as sticks; black dashed lines denote putative hydrogen bonds and salt bridges. (c) and (d) Schematic picture of Tm1631-DNA and endonuclease IV-DNA interactions. Similar residues in Tm1631 and endonuclease IV binding sites are in white and blue ellipses, respectively. Hydrogen bonds with DNA are shown for amino acid side chains (solid black arrows) and backbone atoms (solid cyan arrows). Stacking interactions with DNA nucleotides are dashed black lines.</p

Tm1631-DNA model based on comparison of Tm1631 protein (1vpq) to known endonuclease IV-DNA complex (2nqj) from PDB.

<p>Tm1631 is white, endonuclease IV is blue, DNA is green and light-blue cartoons, sulfate ions are CPK sticks, crescent-shaped grooves in both proteins are shaded areas. Initial Tm1631-DNA model; Tyr47 and Tyr48 penetrate the DNA's extra-helical region (left). Endonuclease IV-DNA complex (right).</p

Tm1631 protein surface conservation analysis by ProBiS.

<p>Tm1631 is shown in surface representation, which is colored by degrees of structural conservation from unconserved (white) to conserved (red). The predicted binding site is encircled by a yellow dashed line.</p

Workflow of the function prediction for the Tm1631 protein structure of unknown function.

<p>Workflow of the function prediction for the Tm1631 protein structure of unknown function.</p

Similar evolutionary patterns in nucleotide binding sites found in PDB using ProBiS.

<p>Predicted Tm1631 binding site (left) is similar to (right): (a) active site in DNA binding site of polymerase X (2w9m) with DNA ligand that was transposed from homologous protein structure 3au6; (b) allosteric site in uridine monophosphate kinase (2jjx); (c) active site of DNA-glycosylase (3fhf) with DNA ligand transposed from homologous protein structure 3knt.</p

Structurally Conserved Binding Sites of Hemagglutinin as Targets for Influenza Drug and Vaccine Development

ProBiS is a new method to identify the binding site of protein through local structural alignment against the nonredundant Protein Data Bank (PDB), which may result in unique findings compared to the energy-based, geometry-based, and sequence-based predictors. In this work, binding sites of Hemagglutinin (HA), which is an important target for drugs and vaccines in influenza treatment, have been revisited by ProBiS. For the first time, the identification of conserved binding sites by local structural alignment across all subtypes and strains of HA available in PDB is presented. ProBiS finds three distinctive conserved sites on HA’s structure (named Site 1, Site 2, and Site 3). Compared to other predictors, ProBiS is the only one that accurately defines the receptor binding site (Site 1). Apart from that, Site 2, which is located slightly above the TBHQ binding site, is proposed as a potential novel conserved target for membrane fusion inhibitor. Lastly, Site 3, located around Helix A at the stem domain and recently targeted by cross-reactive antibodies, is predicted to be conserved in the latest H7N9 China 2013 strain as well. The further exploration of these three sites provides valuable insight in optimizing the influenza drug and vaccine development