title: An analysis approach to identify specific functional sites in orthologous proteins using sequence and structural information: application to neuroserpin reveals regions that differentially regulate inhibitory activity.
abstract: The analysis of sequence conservation is commonly used to predict functionally important sites in proteins. We have developed an approach that first identifies highly conserved sites in a set of orthologous sequences using a weighted substitution-matrix-based conservation score and then filters these conserved sites based on the pattern of conservation present in a wider alignment of sequences from the same family and structural information to identify surface-exposed sites. This allows us to detect specific functional sites in the target protein and exclude regions that are likely to be generally important for the structure or function of the wider protein family. We applied our method to two members of the serpin family of serine protease inhibitors. We first confirmed that our method successfully detected the known heparin binding site in antithrombin while excluding residues known to be generally important in the serpin family. We next applied our sequence analysis approach to neuroserpin and used our results to guide site-directed polyalanine mutagenesis experiments. The majority of the mutant neuroserpin proteins were found to fold correctly and could still form inhibitory complexes with tissue plasminogen activator (tPA). Kinetic analysis of tPA inhibition, however, revealed altered inhibitory kinetics in several of the mutant proteins, with some mutants showing decreased association with tPA and others showing more rapid dissociation of the covalent complex. Altogether, these results confirm that our sequence analysis approach is a useful tool that can be used to guide mutagenesis experiments for the detection of specific functional sites in proteins.
authors.affiliation: School of Biological Sciences and Centre for Brain Research, University of Auckland, Auckland, New Zealand.
authors.forename: Tet Woo
authors.initials: TW
authors.lastname: Lee
authors.forename: Annie Shu-Ping
authors.initials: AS
authors.lastname: Yang
authors.forename: Thomas
authors.initials: T
authors.lastname: Brittain
authors.forename: Nigel P
authors.initials: NP
authors.lastname: Birch
mesh_terms: D000818:Animals; D000991:Antithrombins / Q000737:chemistry; D017124:Conserved Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016296:Mutagenesis / Q000235:genetics; D050505:Mutant Proteins / Q000737:chemistry; D009154:Mutation / Q000235:genetics; D009479:Neuropeptides / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D020539:Sequence Analysis, Protein / Q000379:methods*; D017386:Sequence Homology, Amino Acid; D015843:Serpins / Q000737:chemistry* / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000378:metabolism; D000097522:Neuroserpin
chemical_list: D000991:Antithrombins; D050505:Mutant Proteins; D009479:Neuropeptides; D015843:Serpins; D010959:Tissue Plasminogen Activator; D000097522:Neuroserpin
keywords: amino acid conservation score; antithrombin; consensus residues; evolutionary conservation; multiple sequence alignment; protease inhibition; serpin superfamily; site-directed mutagenesis; specifically-conserved sites; tissue plasminogen activator