Probing the hirudin-thrombin interaction by incorporation of noncoded amino acids and molecular dynamics simulation

Thrombin is a primary target for the development of novel
anticoagulants, since It plays two important and opposite roles in
hemostasis: procoagulant and anticoagulant. All thrombin functions are
influenced 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 chemical synthesis
analogues of the N-terminal fragment 1-47 of hirudin HM2 containing
noncoded amino acids and displaying up to similar to2700-fold more
potent antithrombin activity, comparable to that of full-length hirudin.
In the work presented here, we have exploited the versatility of
chemical synthesis to probe the structural and energetic properties of
the S3 site of thrombin through perturbations introduced in the
structure of hirudin fragment 1-47. In particular, we have investigated
the effects of systematic replacement of Tyr3 with noncoded amino acids
retaining the aromatic nucleus of Tyr, as well as similar hydrophobic
and 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 of fragment 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 of hirudin to
the procoagulant (fast) form of thrombin. Strikingly, shaving at
position 3, by Tyr --> Ala exchange, abolishes the differences in the
affinity for thrombin allosteric forms, whereas a bulkier side chain
(e.g., beta-naphthylalanine) improves binding preferentially to the fast
form. These results provide strong, albeit indirect, evidence that the
procoagulant (fast) form of thrombin is in a more open and accessible
conformation with respect to the less forgiving structure it acquires in
the slow form. This view is also supported by the results of molecular
dynamics simulations conducted for 18 ns on free thrombin in full
explicit water, showing that after similar to5 ns thrombin undergoes a
significant conformational transition, from a more open conformation
(which we propose can be related to the fast form) to a more compact and
closed one (which we propose can be related to the slow form). This
transition mainly involves 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 indicate that the S3 site of the enzyme is less accessible to
water in the putative slow form. This structural picture provides a
reasonable molecular explanation for the fact that physiological
substrates related to the procoagulant activity of thrombin (fibrinogen,
thrombin receptor 1, and factor XIII) orient a bulky side chain into the
S3 site of the enzyme. Taken together, our results can have important
implications for the design of novel thrombin inhibitors, of practical
utility in the treatment of coagulative disorders.