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.

Neonatal Hypoxia Ischaemia: Mechanisms, Models, and Therapeutic Challenges

“…Neuroserpin was first identified in the secreted product of cultured neuronal axons (Osterwalder et al, 1996). Protein expression is primarily localized to neurons (Osterwalder et al, 1996; Hastings et al, 1997; Lee et al, 2015b). The protein neuroserpin is encoded in mouse and human by the gene Serpini1.…”

Neuroserpin Differentiates Between Forms of Tissue Type Plasminogen Activator via pH Dependent Deacylation

“…An intriguing correlation may be made with recent findings by Lee et al (2015) , who showed that NSP-t-PA complex stability is dependent on evolutionarily conserved residues in NSP, and their findings will inform design of future NSP-mutants to identify the amino acid residues that are ultimately responsible for the pH dependency of NSP-t-PA complex deacylation.…”

Physiological and pathological roles of tissue plasminogen activator and its inhibitor neuroserpin in the nervous system

“…Other results, however, indicate that neuroserpin does not behave as a classical inhibitory serpin toward tPA. Unlike most covalent serpin:protease complexes, tPA:neuroserpin is unstable and dissociates within minutes to release cleaved neuroserpin and active tPA (Barker-Carlson et al, 2002 ; Ricagno et al, 2009 ; Lee et al, 2015 ). As complex dissociation is expected to occur prior to clearance (Barker-Carlson et al, 2002 ), these data suggest that neuroserpin is likely to function as a transient inhibitor of tPA in vivo .…”

“…As complex dissociation is expected to occur prior to clearance (Barker-Carlson et al, 2002 ), these data suggest that neuroserpin is likely to function as a transient inhibitor of tPA in vivo . Interestingly, evolutionarily conserved residues in neuroserpin regulate the half-life of tPA:neuroserpin complexes, suggesting that the precise half-life of the complexes may be physiologically important (Lee et al, 2015 ). tPA:neuroserpin interactions may involve other players, such as an unknown co-factor that stabilizes the complex (Barker-Carlson et al, 2002 ).…”