The conformations of hirudin in solution: a study using nuclear magnetic resonance, distance geometry and restrained molecular dynamics

Abstract

The solution conformations of the protein hirudin have been investigated by the combined use of distance geometry and restrained molecular dynamics calculations. The basis for the structure determination comprised 359 approximate interproton distance restraints and 10 4 backbone torsion angle restraints derived from n.m.r. measurements. It is shown that hirudin is composed of three domains: a central core made up of residues 3-30, 37-46 and 56-57; a protruding 'finger' (residues 31-36) consisting of the tip of an antiparaliel 3 sheet, and an exposed loop (residues 47-55). The structure of each individual domain is relatively well defined with average backbone atomic r.m.s. differences of <2 A between the final seven converged restrained dynamic structures and the mean structure obtained by averaging their coordinates. The orientation of the two minor domains relative to the central core, however, could not be determined as no long-range (I i-jI >5) interdomain proton-proton contacts could be observed in the two-dimensional nuclear Overhauser enhancement spectra. From the restrained molecular dynamics calculations it appears that the two minor domains exhibit large rigid-body motions relative to the central core.

Three-dimensional structure of potato carboxypeptidase inhibitor in solution. A study using nuclear magnetic resonance, distance geometry, and restrained molecular dynamics

“…The computation of the tertiary structure employed the same two-stage approach that we previously used for crl-purothionin (Clore et al, 1986a,b), phoratoxin (Clore et al, 1987a), hirudin (Clore et al, 1987b), and the globular domain of histone H5 (Clore et al, 1987c), namely, a structure generation phase using the metric matrix distance geometry program DISGEO (Havel, 1986), followed by a refinement phase using a combination of restrained energy…”

Changes in interactions in complexes of hirudin derivatives and human α‐thrombin due to different crystal forms

“…It consists of a polypeptide chain of 65 or 66 residues with three disulfide bridges (Bagdy et al, 1976; Dodt et al, 1984Dodt et al, , 1985. Its three-dimensional struc-ture has been determined by two-dimensional NMR techniques (Clore et al, 1987;Folkers et al, 1989;Haruyama & Wuthrich, 1989). Hirudin forms a compact N-terminal domain (residues 3-49) containing three disulfide bridges between cysteine residues 6 and 14, 16 and 28, and 22 and 39 and a long C-terminal tail composed of residues 50-65 that is disordered in solution.…”

A simple method for delineating well‐defined and variable regions in protein structures determined from interproton distance data

“…In recent years considerable progress has been made towards determining three-dimensional structures of proteins in solution using NMR spectroscopy [1][2][3][4][5][6][7][8][9]. This generally involves three stages: (i) the sequential assignment of proton resonances by means of two-dimensional NMR techniques identifying through-bond and throughspace (<5 ./~) connectivities [10]; (ii) the assignment of as many cross-peaks as possible in the NOESY spectra in order to obtain a large set of approximate interproton distance restraints; and (iii) the determination of a 3D-structure on the basis of these restraints using a suitable computer algorithm, for example metric matrix distance Abbreviations: NMR, nuclear magnetic resonance; NOE, nuclear Overhauser effect; NOESY, twodimensional NOE spectroscopy; rms, root mean square geometry [11][12][13][14], restrained least squares minimization in torsion angle space with a variable target function [15] and restrained molecular dynamics [5,[16][17][18].…”

“…The recently determined structure of the protein hirudin [8] provides a typical example of the latter case. Hirudin, a 63 residue protein, has a well defined core and two minor domains consisting of a finger of antiparallel fl-sheet (residues 31-36) and an exposed loop (residues 47-55).…”