Probing the Hirudin-Thrombin Interaction by Incorporation of Noncoded Amino Acids and Molecular Dynamics Simulation
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- 作者：Vincenzo De Filippis ; Giorgio Colombo ; Ilaria Russo ; Barbara Spadari ; and Angelo Fontana
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：November 19, 2002
- 年：2002
- 卷：41
- 期：46
- 页码：13556 - 13569
- 全文大小：228K
- 年卷期：v.41,no.46(November 19, 2002)
- ISSN：1520-4995
文摘
Thrombin is a primary target for the development of novel anticoagulants, since it plays twoimportant and opposite roles in hemostasis: procoagulant and anticoagulant. All thrombin functions areinfluenced by Na+ binding, which triggers the transition of this enzyme from an anticoagulant (slow)form to a procoagulant (fast) form. In previous studies, we have conveniently produced by chemicalsynthesis analogues of the N-terminal fragment 1-47 of hirudin HM2 containing noncoded amino acidsand displaying up to ~2700-fold more potent antithrombin activity, comparable to that of full-lengthhirudin. In the work presented here, we have exploited the versatility of chemical synthesis to probe thestructural and energetic properties of the S3 site of thrombin through perturbations introduced in thestructure of hirudin fragment 1-47. In particular, we have investigated the effects of systematic replacementof Tyr3 with noncoded amino acids retaining the aromatic nucleus of Tyr, as well as similar hydrophobicand steric properties, but possessing different electronic (e.g., p-fluoro-, p-iodo-, or p-nitro-Phe), charge(p-aminomethyl-Phe), or conformational (homo-Phe) properties. Our results indicate that the affinity offragment 1-47 for thrombin is proportional to the desolvation free energy change upon complex formation,and is inversely related to the electric dipole moment of the amino acid side chain at position 3 of hirudin.In this study, we have also identified the key features that are responsible for the preferential binding ofhirudin to the procoagulant (fast) form of thrombin. Strikingly, shaving at position 3, by Tyr 
Alaexchange, abolishes the differences in the affinity for thrombin allosteric forms, whereas a bulkier sidechain (e.g., 
-naphthylalanine) improves binding preferentially to the fast form. These results providestrong, albeit indirect, evidence that the procoagulant (fast) form of thrombin is in a more open andaccessible conformation with respect to the less forgiving structure it acquires in the slow form. Thisview is also supported by the results of molecular dynamics simulations conducted for 18 ns on freethrombin in full explicit water, showing that after ~5 ns thrombin undergoes a significant conformationaltransition, from a more open conformation (which we propose can be related to the fast form) to a morecompact and closed one (which we propose can be related to the slow form). This transition mainlyinvolves the Trp148 and Trp60D loop, the S3 site, and the fibrinogen binding site, whereas the S1 site,the Na+-binding site, and the catalytic pocket remain essentially unchanged. In particular, our data indicatethat the S3 site of the enzyme is less accessible to water in the putative slow form. This structural pictureprovides a reasonable molecular explanation for the fact that physiological substrates related to theprocoagulant activity of thrombin (fibrinogen, thrombin receptor 1, and factor XIII) orient a bulky sidechain into the S3 site of the enzyme. Taken together, our results can have important implications for thedesign of novel thrombin inhibitors, of practical utility in the treatment of coagulative disorders.
Alaexchange, abolishes the differences in the affinity for thrombin allosteric forms, whereas a bulkier sidechain (e.g., -naphthylalanine) improves binding preferentially to the fast form. These results providestrong, albeit indirect, evidence that the procoagulant (fast) form of thrombin is in a more open andaccessible conformation with respect to the less forgiving structure it acquires in the slow form. Thisview is also supported by the results of molecular dynamics simulations conducted for 18 ns on freethrombin in full explicit water, showing that after ~5 ns thrombin undergoes a significant conformationaltransition, from a more open conformation (which we propose can be related to the fast form) to a morecompact and closed one (which we propose can be related to the slow form). This transition mainlyinvolves the Trp148 and Trp60D loop, the S3 site, and the fibrinogen binding site, whereas the S1 site,the Na+-binding site, and the catalytic pocket remain essentially unchanged. In particular, our data indicatethat the S3 site of the enzyme is less accessible to water in the putative slow form. This structural pictureprovides a reasonable molecular explanation for the fact that physiological substrates related to theprocoagulant activity of thrombin (fibrinogen, thrombin receptor 1, and factor XIII) orient a bulky sidechain into the S3 site of the enzyme. Taken together, our results can have important implications for thedesign of novel thrombin inhibitors, of practical utility in the treatment of coagulative disorders.