Discovery of Amino Acid Motifs for Thrombin Cleavage and Validation Using a Model Substrate

详细信息    查看全文

• 作者：Natasha M. Ng ; James D. Pierce ; Geoffrey I. Webb ; Boris I. Ratnikov ; Lakshmi C. Wijeyewickrema ; Renee C. Duncan ; Amy L. Robertson ; Stephen P. Bottomley ; Sarah E. Boyd ; Robert N. Pike
• 刊名：Biochemistry
• 出版年：2011
• 出版时间：December 6, 2011
• 年：2011
• 卷：50
• 期：48
• 页码：10499-10507
• 全文大小：292K
• 年卷期：v.50,no.48(December 6, 2011)
• ISSN：1520-4995

文摘

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 P₂鈥揚₁鈥?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 P₃鈥揚₁ and P₁鈥揚₁鈥测€揚₃鈥?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.