title: Identification through combinatorial random and rational mutagenesis of a substrate-interacting exosite in the gamma domain of streptokinase.
abstract: To identify new structure-function correlations in the γ domain of streptokinase, mutants were generated by error-prone random mutagenesis of the γ domain and its adjoining region in the β domain followed by functional screening specifically for substrate plasminogen activation. Single-site mutants derived from various multipoint mutation clusters identified the importance of discrete residues in the γ domain that are important for substrate processing. Among the various residues, aspartate at position 328 was identified as critical for substrate human plasminogen activation through extensive mutagenesis of its side chain, namely D328R, D328H, D328N, and D328A. Other mutants found to be important in substrate plasminogen activation were, namely, R319H, N339S, K334A, K334E, and L335Q. When examined for their 1:1 interaction with human plasmin, these mutants were found to retain the native-like high affinity for plasmin and also to generate amidolytic activity with partner plasminogen in a manner similar to wild type streptokinase. Moreover, cofactor activities of the mutants precomplexed with plasmin against microplasminogen as the substrate as well as in silico modeling studies suggested that the region 315-340 of the γ domain interacts with the serine protease domain of the macromolecular substrate. Overall, our results identify the presence of a substrate specific exosite in the γ domain of streptokinase.
authors.affiliation: Institute of Microbial Technology (Council of Scientific and Industrial Research), Chandigarh 160036, India.
authors.forename: Suman
authors.initials: S
authors.lastname: Yadav
authors.forename: Rachna
authors.initials: R
authors.lastname: Aneja
authors.forename: Prakash
authors.initials: P
authors.lastname: Kumar
authors.forename: Manish
authors.initials: M
authors.lastname: Datt
authors.forename: Sonali
authors.initials: S
authors.lastname: Sinha
authors.forename: Girish
authors.initials: G
authors.lastname: Sahni
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005343:Fibrinolytic Agents / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D016296:Mutagenesis; D020125:Mutation, Missense; D017434:Protein Structure, Tertiary; D013291:Streptococcus / Q000201:enzymology* / Q000235:genetics; D013300:Streptokinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D001426:Bacterial Proteins; D005343:Fibrinolytic Agents; D013300:Streptokinase