Effective function annotation through catalytic residue conservation

Abstract

Because of the extreme impact of genome sequencing projects, protein sequences without accompanying experimental data now dominate public databases. Homology searches, by providing an opportunity to transfer functional information between related proteins, have become the de facto way to address this. Although a single, well annotated, close relationship will often facilitate sufficient annotation, this situation is not always the case, particularly if mutations are present in important functional residues. When only distant relationships are available, the transfer of function information is more tenuous, and the likelihood of encountering several well annotated proteins with different functions is increased. The consequence for a researcher is a range of candidate functions with little way of knowing which, if any, are correct. Here, we address the problem directly by introducing a computational approach to accurately identify and segregate related proteins into those with a functional similarity and those where function differs. This approach should find a wide range of applications, including the interpretation of genomics/proteomics data and the prioritization of targets for high-throughput structure determination. The method is generic, but here we concentrate on enzymes and apply high-quality catalytic site data. In addition to providing a series of comprehensive benchmarks to show the overall performance of our approach, we illustrate its utility with specific examples that include the correct identification of haptoglobin as a nonenzymatic relative of trypsin, discrimination of acid-d-amino acid ligases from a much larger ligase pool, and the successful annotation of BioH, a structural genomics target.

Fig. 1.

Improvement in function assignment accuracy of iCSA compared with sequence homology alone. (a) Results are shown for the accuracy of EC assignment to Swiss-Prot homologues obtained by using the iCSA filter (black bars) and compared, at each psi-blast iteration up to four, with results obtained by using just psi-blast (gray bars). (b) Percentage improvement in function assignment accuracy with iCSA compared with psi-blast only.

Fig. 2.

Trypsin catalytic site and trypsin sequence aligned to haptoglobin sequence. (a) Trypsin 3D structure (PDB ID code 1A0J³³, chain A). Catalytic residues are shown in red (His-57), green (Asp-102), and blue (Ser-195). (b) Trypsin (PDB ID code 1A0J, chain A, residues 1–196) aligned to haptoglobin (Swiss-Prot sequence P19006, residues 85–303). Catalytic residue positions in trypsin are marked by an asterisk. 1A0J retrieved P19006 with a blast-level search; the sequences have 26% pairwise sequence identity.