Solution structure of recombinant hirudin and the Lys-47 .fwdarw. Glu mutants: a nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing study

Abstract

The solution structure of recombinant wild-type hirudin and of the putative active site mutant Lys-47 .fwdarw. Glu has been investigated by nuclear magnetic resonance (MNR) spectroscopy at 600 MHz. The 1H NMR spectra of the two hirudin variants are assigned in a sequential manner with a combination of two-dimensional NMR techniques. Some assignments made in our previous paper [Sukumaran, D. K., Clore, G. M. Preuss, A., Zarbock, J., and Gronenborn, A, M. (1987) Biochemstry 26, 333-338] were found to be incorrect and are now corrected. Analysis of the NOE data indicates that hirudin consists of an N-terminal compact domain (residues 1-49) held together by three disulfide linkages and a disordered C-terminal tail (residues 50-65) which does not fold back on the rest of the protein. This last observation corrects conclusions drawn by us previously on hirudin extracted from its natural source, the leech Hirudo medicinalis. The improved sensitivity of the 600-MHz spectrometer relatie to that of our old 500-MHz spectrometer, the availability of two variants with slightly different chemical shifts, and the additional information arising from stereospecific assignments of methylene .beta.-protons and methyl protons of valine have permitted the determination of the solution structure of hirudin with much greater precision than before. Structure calculations on the N-terminal domain using the hybrid distance geometry-dynamical simulated annealing method were based on 685 and 661 approximate interproton distance restraints derived from nuclear Overhauser enhancement (NOE) data for the wild-type and mutant hirudin, respectively, together with 16 distance restraints for 8 backbone hydrogen bonds identified on the basis of NOE and amide NH exchange data and 26 .vphi. backbone and 18 .chi.1 side-chain torsion angle restraints derived from NOE and three-bond coupling constant data. A total of 32 structures were computed for both the wild-type and mutant hirudin. The structure of residues 2-30 and 37-48 which form the core of the N-terminal domain is well determined in both cases with an average atomic rms difference between the individual structures and the respective mean structures of .apprx. 0.7 .ANG. for the backbone atoms and .apprx. 1 .ANG. for all atoms. As found previously the orientation of the exposed finger of antiparallel .beta.-sheet (residues 31-36) with respect to the core could not be determined on the basis of the present data due to the absence of any long-range NOEs between the exposed finger and the core. The Lys-47 .fwdarw. Glu mutation has only a small, but clearly discernible, effect on the structure which can be attributed to the larger space requirement for the longer Lys side chain relative to that of the shorter Glu side chain.