Proton Nuclear Magnetic Resonance Study of Hirudin: Resonance Assignment and Secondary Structure?

The 'H NMR spectrum of the 65-residue protein hirudin is assigned in a sequential manner by using a combination of two-dimensional nuclear magnetic resonance techniques to demonstrate through-bond and through-space (<5-i() connectivities. The secondary structure of hirudin is deduced from a qualitative interpretation of the nuclear Overhauser effects involving the backbone NH, CaH, and CPH protons. It is shown that hirudin has two P-sheets and no a-helices. H i r u d i n , a small protein of 65 residues from the leech Hirudo medicinalis, is the most potent natural inhibitor of coagulation known (Haycraft, 1884; Markwardt, 1970). Hirudin acts by binding specifically and very tightly (K,,,oc 2 X 1O'O M-l) to a-thrombin, thereby inactivating it and abolishing its ability to cleave fibrinogen (Magnusson, 1972; Markwardt, 1985; Stone & Hofsteenge, 1986). These remarkable properties have generated considerable interest with regard to the possible clinical use of hirudin as an antithrombotic agent (Nowak & Markwardt, 1980; Ishikawa et al., 1980; Walsmann & Markwardt, 1981; Markwardt et al., 1982; Kloss & Mittman, 1982). In a series of recent papers the complete amino acid sequence of hirudin and the location of the three disulfide bridges have been determined (Badgy et al., 1976; Dodt et al., 1984, 1985, 1986). Further, the structural genes of two hirudin variants have been cloned into expression vectors and successfully expressed in Escherichia coli (Harvey et al., 1986; Bergmann et al., 1986). At the present time, however, nothing is known about either the secondary or tertiary structure of hirudin. Moreover, hirudin itself fails to crystallize, and the crystals of the thrombin-hirudin complex that have been obtained were unsuitable for X-ray crystallographic studies (W. Bode and R. Huber, personal communication). An alternative approach to solving the three-dimensional structure of hirudin is therefore called for. This involves the application of NMR' spectroscopy in solution (Wiithrich et al., 1982) and, in par+ This work was supported by the Max-Planck Gesellschaft (G.M.C. and A.M.G.). ticular, the use of the nuclear Overhauser effect (NOE; Noggle & Schirmer, 1971) to obtain a large set of approximate interproton distances that can then be used as the basis of a three-dimensional structure determination using either distance geometry algorithms (Have1 & Wiithrich, 1985; Braun & Go, 1985) and/or restrained molecular dynamics (Clore et al., 1985, 1986a,b; Kaptein et al., 1985; Briinger et al., 1986). In this paper, we present the first step toward this goal, namely, the assignment of the 'H NMR spectrum of hirudin using a combination of two-dimensional NMR techniques and the delineation of its secondary structure from a qualitative interpretation of the NOE data. EXPERIMENTAL PROCEDURES Hirudin (batch CH.B.H3/5-720) was obtained from Plantorgan Werk (Bad Zwischenahn, FRG) as a gift from Dr. R. Maschler and Prof. E. Fink (Plantorgan) and Dr. D. Tripier (Hoechst) and was isolated and purified from the whole body of leeches as described previously (Bagdy et al., 1976). Purity was ascertained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal residue analysis, amino acid analysis, and high-pressure liquid chromatography (D. Tripier, personal communication). The preparation contained one major species comprising 85% of the total and several minor Abbreviations: NMR, nuclear magnetic resonance; NOE, nuclear Overhauser effect; NOESY, two-dimensional NOE spectroscopy; HOHAHA, homonuclear Hartmann-Hahn spectroscopy; DQF-COSY, double quantum filtered homonuclear correlated spectroscopy. 0006-2960/87/0426-0333$01 SO10