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Nonpolar interactions of thrombin S' subsites with its bivalent inhibitor: methyl scan of the inhibitor linker.
Biochemistry. 1997 Nov 04; 36(44):13494-502.B

Abstract

We have designed bivalent thrombin inhibitors, consisting of a nonsubstrate type active site blocking segment, a hirudin-based fibrinogen recognition exosite blocking segment, and a linker connecting these segments. The inhibition provided by the bivalent inhibitors with various linker lengths revealed that a minimum of 15 atoms was required for simultaneous binding of the two blocking segments of the inhibitor to thrombin without significant distortion. The crystal structure of the inhibitors with a 16-atom linker showed some conformational flexibility in the linker portion which still lies deep in the groove joining the active site and the fibrinogen recognition exosite. Since the thrombin S' subsites are not well characterized, we designed a new strategy to search for possible nonpolar interactions between the linker and the thrombin S' subsites. This strategy, the "methyl scan", is based on the incorporation of a methyl side chain at each atom position of the linker by using sarcosine, D,L-alanine, D,L-3-aminoisobutyric acid, or N-methyl-beta-alanine. The methyl groups on the second and the eighth atom positions of the linker, which correspond to the side chains of the P1' and the P3' residues, respectively, improved the affinity of the inhibitors significantly. Further study of the stereospecificity showed that L-Ala at the P1' residue and D-Ala at the P3' residue preferably improved the affinity of the inhibitors 20- and 25-fold, respectively. Molecular modeling calculations using a methyl probe were also carried out to identify favorable nonpolar interacting sites on the thrombin surface. Two sites were identified in the vicinity of the P1' and the P3' residues, supporting the validity of the methyl scan method. Thus, this study has improved our understanding of the interactions taking place in this groove. In particular, we have been able to show that some specific structural features, such as hydrophobic complementarity between the linker and the thrombin S' subsites, could be exploited and make these inhibitors trivalent.

Authors+Show Affiliations

National Research Council of Canada, Biotechnology Research Institute, Montreal, Quebec, Canada H4P 2R2.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

9354617

Citation

Slon-Usakiewicz, J J., et al. "Nonpolar Interactions of Thrombin S' Subsites With Its Bivalent Inhibitor: Methyl Scan of the Inhibitor Linker." Biochemistry, vol. 36, no. 44, 1997, pp. 13494-502.
Slon-Usakiewicz JJ, Purisima E, Tsuda Y, et al. Nonpolar interactions of thrombin S' subsites with its bivalent inhibitor: methyl scan of the inhibitor linker. Biochemistry. 1997;36(44):13494-502.
Slon-Usakiewicz, J. J., Purisima, E., Tsuda, Y., Sulea, T., Pedyczak, A., Féthière, J., Cygler, M., & Konishi, Y. (1997). Nonpolar interactions of thrombin S' subsites with its bivalent inhibitor: methyl scan of the inhibitor linker. Biochemistry, 36(44), 13494-502.
Slon-Usakiewicz JJ, et al. Nonpolar Interactions of Thrombin S' Subsites With Its Bivalent Inhibitor: Methyl Scan of the Inhibitor Linker. Biochemistry. 1997 Nov 4;36(44):13494-502. PubMed PMID: 9354617.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Nonpolar interactions of thrombin S' subsites with its bivalent inhibitor: methyl scan of the inhibitor linker. AU - Slon-Usakiewicz,J J, AU - Purisima,E, AU - Tsuda,Y, AU - Sulea,T, AU - Pedyczak,A, AU - Féthière,J, AU - Cygler,M, AU - Konishi,Y, PY - 1997/11/14/pubmed PY - 1997/11/14/medline PY - 1997/11/14/entrez SP - 13494 EP - 502 JF - Biochemistry JO - Biochemistry VL - 36 IS - 44 N2 - We have designed bivalent thrombin inhibitors, consisting of a nonsubstrate type active site blocking segment, a hirudin-based fibrinogen recognition exosite blocking segment, and a linker connecting these segments. The inhibition provided by the bivalent inhibitors with various linker lengths revealed that a minimum of 15 atoms was required for simultaneous binding of the two blocking segments of the inhibitor to thrombin without significant distortion. The crystal structure of the inhibitors with a 16-atom linker showed some conformational flexibility in the linker portion which still lies deep in the groove joining the active site and the fibrinogen recognition exosite. Since the thrombin S' subsites are not well characterized, we designed a new strategy to search for possible nonpolar interactions between the linker and the thrombin S' subsites. This strategy, the "methyl scan", is based on the incorporation of a methyl side chain at each atom position of the linker by using sarcosine, D,L-alanine, D,L-3-aminoisobutyric acid, or N-methyl-beta-alanine. The methyl groups on the second and the eighth atom positions of the linker, which correspond to the side chains of the P1' and the P3' residues, respectively, improved the affinity of the inhibitors significantly. Further study of the stereospecificity showed that L-Ala at the P1' residue and D-Ala at the P3' residue preferably improved the affinity of the inhibitors 20- and 25-fold, respectively. Molecular modeling calculations using a methyl probe were also carried out to identify favorable nonpolar interacting sites on the thrombin surface. Two sites were identified in the vicinity of the P1' and the P3' residues, supporting the validity of the methyl scan method. Thus, this study has improved our understanding of the interactions taking place in this groove. In particular, we have been able to show that some specific structural features, such as hydrophobic complementarity between the linker and the thrombin S' subsites, could be exploited and make these inhibitors trivalent. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/9354617/Nonpolar_interactions_of_thrombin_S'_subsites_with_its_bivalent_inhibitor:_methyl_scan_of_the_inhibitor_linker_ L2 - https://doi.org/10.1021/bi970857h DB - PRIME DP - Unbound Medicine ER -