title: Discovery of amino acid motifs for thrombin cleavage and validation using a model substrate.
abstract: Understanding the active site preferences of an enzyme is critical to the design of effective inhibitors and to gaining insights into its mechanisms of action on substrates. While the subsite specificity of thrombin is understood, it is not clear whether the enzyme prefers individual amino acids at each subsite in isolation or prefers to cleave combinations of amino acids as a motif. To investigate whether preferred peptide motifs for cleavage could be identified for thrombin, we exposed a phage-displayed peptide library to thrombin. The resulting preferentially cleaved substrates were analyzed using the technique of association rule discovery. The results revealed that thrombin selected for amino acid motifs in cleavage sites. The contribution of these hypothetical motifs to substrate cleavage efficiency was further investigated using the B1 IgG-binding domain of streptococcal protein G as a model substrate. Introduction of a P(2)-P(1)' LRS thrombin cleavage sequence within a major loop of the protein led to cleavage of the protein by thrombin, with the cleavage efficiency increasing with the length of the loop. Introduction of further P(3)-P(1) and P(1)-P(1)'-P(3)' amino acid motifs into the loop region yielded greater cleavage efficiencies, suggesting that the susceptibility of a protein substrate to cleavage by thrombin is influenced by these motifs, perhaps because of cooperative effects between subsites closest to the scissile peptide bond.
authors: Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.
authors: Natasha M
authors: NM
authors: Ng
authors: James D
authors: JD
authors: Pierce
authors: Geoffrey I
authors: GI
authors: Webb
authors: Boris I
authors: BI
authors: Ratnikov
authors: Lakshmi C
authors: LC
authors: Wijeyewickrema
authors: Renee C
authors: RC
authors: Duncan
authors: Amy L
authors: AL
authors: Robertson
authors: Stephen P
authors: SP
authors: Bottomley
authors: Sarah E
authors: SE
authors: Boyd
authors: Robert N
authors: RN
authors: Pike
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D017104:Bacteriophage M13 / Q000737:chemistry / Q000235:genetics; D006868:Hydrolysis; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019151:Peptide Library; D015202:Protein Engineering / Q000379:methods; D011897:Random Allocation; D015203:Reproducibility of Results; D013291:Streptococcus; D013379:Substrate Specificity / Q000235:genetics; D013917:Thrombin / Q000737:chemistry* / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D019151:Peptide Library; D014764:Viral Proteins; C081255:gene 3 protein, Enterobacteria phage Ike; D013917:Thrombin