NMR solution structure of a novel hirudin variant HM2, N-terminal 1-47 and N64 → V + G mutant

Abstract

The 64 amino acid hirudin‐like peptide HM2 (Hirudinaria manillensis) is one of the agents known to specifically block the blood‐clotting enzyme thrombin, and therefore is used as a potential pharmacological tool for the treatment of arterial and venous thrombosis. This peptide and its derivatives provide a new set of probes for studies aimed at elucidating the structural basis of the inhibition of α‐thrombin. We used 581, 699, and 492 nmr‐derived constraints respectively in a protocol employing simulated annealing, followed by restrained molecular dynamics and restrained energy minimization to derive the three‐dimensional structures of HM2 and its mutants the HM2(V + G) and the HM2(1–47). HM2 consists of a well‐defined core region of two double‐stranded β‐sheet and a disordered C‐terminus. These features are shared by other members of the hirudin family. The same type of folding has also been observed for recombinant hirudins whose structure has been determined in solution by nmr spectroscopy and in the structure of the complex hirudin‐thrombin determined by x‐ray diffraction. Molecular dynamics (MD) simulation methods were applied in the study of the structural and dynamic fluctuation properties of the hirudin derivatives solvated by 1625 and 1276 water molecules with periodic boundary conditions for HM2 and HM2(1–47), respectively. Trajectories of 100 and 50 ps for the two unconstrained systems were generated at constant temperature and pressure. Analysis of the MD simulation shows that the structure of the peptide core is fairly rigid and stable in itself while the conformation of the C‐terminal tail, which is involved in the inhibitory mechanism of thrombin, fluctuates and appears as a disordered region. © 1997 John Wiley & Sons, Inc. Biopoly 41: 731–749, 1997

3‐Nitrotyrosine as a spectroscopic probe for investigating protein–protein interactions

“…Due to its important pharmacological implications, the hirudin thrombin pair has been the object of thorough biochemical and structural studies. Indeed, the high‐resolution crystallographic structures of both free (Pineda et al 2004; Johnson et al 2005) and hirudin‐bound forms of thrombin are available (Rydel et al 1991), together with several NMR structures of full‐length and truncated hirudin forms (Haruyama and Wuthrich 1989; Szyperski et al 1992; Nicastro et al 1997). Hence, taking advantage of the synthetic procedure previously established (De Filippis et al 1995, 1998), we synthesized two NT‐containing analogs of hirudin fragment 1 47: the Y3NT analog, in which Tyr3 was replaced by NT, and the S2R/Y3NT analog, containing the double substitution Ser2 → Arg and Tyr3 → NT.…”

Protein engineering by chemical methods: Incorporation of nonnatural amino acids as a tool for studying protein folding, stability, and function

“…Hirudin is the most potent and specific inhibitor of αT (K d = 20‐200 fM) and is now used as an anticoagulant drug (Refludan). Hirudin HM2 variant from Hirudinaria manillensis , an hematophagous parasite of bovines, is formed by a compact N‐terminal region (amino acids 1‐47) and a flexible negatively charged C‐terminal tail (amino acids 48‐64) (4htc.pdb) . The N‐terminal domain is stabilized by three disulfide bonds and covers the protease active site by extensively penetrating into the enzyme specificity sites through its N‐terminal tripeptide (Figure C‐E).…”

“…A, RP‐HPLC analysis of the time‐course kinetics of fully reduced HM2(1‐47) (R) to yield the folded species with the native disulfide bond topology (N). (Inset) Tube representation of the NMR solution structure of HM2(1‐47) . Disulfide bonds are indicated by yellow sticks.…”

“…The inhibitory potency of the synthetic Hir(1‐47) analogs toward the procoagulant ( fast ) and anticoagulant ( slow ) form of αT was measured at 25°C by monitoring the release of p ‐nitroanilide ( p ‐NA) from the synthetic substrate (D)‐Phe‐Pro‐Arg‐ p ‐NA under salt conditions stabilizing the fast (0.2 M NaCl) or the slow (0.2 M ChCl) form of the protease (Figure ). The amino acid exchanges were designed starting from the NMR solution structure of HM2 and the crystallographic structure of hirudin‐αT complex …”

“…Position 3 . Tyr‐3 in HM2(1‐47) has a well‐defined conformation both in the free and αT‐bound state . Tyr‐3 partially fills the apolar S3 site of the protease, which is shaped by Leu‐99, Ile‐174, and Trp‐215, and is highly conserved in the hirudin family .…”