Structure-Based Function Prediction of Uncharacterized Protein Using Binding Sites Comparison

Abstract

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. For a substantial proportion of proteins, their functions are not known since these proteins are not related in sequence to any other known proteins. Binding sites are evolutionarily conserved across very distant protein families, and finding similar binding sites between known and unknown proteins can provide clues as to functions of the unknown proteins. We choose one of the “unknown function” proteins, and found, using a novel strategy of binding site comparison to construct a hypothetical protein-ligand complex, subsequently validated by molecular dynamics that this protein most likely binds and repairs the damaged DNA similar to known DNA-repair enzymes. Our methodology is general and enables one to determine functions of other proteins currently labelled as “unknown function”. We envision that the methodology presented herein, the binding sites comparisons enhanced by molecular dynamics, will stimulate the function prediction of other uncharacterized proteins with structures in the Protein Data Bank and boost experimental functional studies of proteins of unknown functions.