Nuclear magnetic resonance solution structure of hirudin(1–51) and comparison with corresponding three-dimensional structures determined using the complete 65-residue hirudin polypeptide chain

doi:10.1016/0022-2836(92)90325-E

Journal of Molecular Biology

Volume 228, Issue 4, 20 December 1992, Pages 1193-1205

https://doi.org/10.1016/0022-2836(92)90325-E Get rights and content

Abstract

The three-dimensional structure of the N-terminal 51-residue domain of recombinant hirudin in aqueous solution was determined by ¹H nuclear magnetic resonance (NMR) spectroscopy, and the resulting high-quality solution structure was compared with corresponding structures obtained from studies with the intact, 65-residue polypeptide chain of hirudin. On the basis of 580 distance constraints derived from nuclear Overhauser effects and 109 dihedral angle constraints, a group of 20 conformers representing the solution structure of hirudin(1–51) was computed with the program DIANA and energyminimized with a modified version of the program AMBER. Residues 3 to 30 and 37 to 48 form a well-defined molecular core with two antiparallel β-sheets composed of residues 14 to 16 and 20 to 22, and 27 to 31 and 36 to 40, and three reverse turns at residues 8 to 11 (type II), 17 to 20 (type II′) and 23 to 26 (type II). The average root-mean-square deviation of the individual NMR conformers relative to their mean co-ordinates is 0.38 Å for the backbone atoms and 0.77 Å for all heavy atoms of these residues. Increased structural disorder was found for the N-terminal dipeptide segment, the loop at residues 31 to 36, and the C-terminal tripeptide segment. The solution structure of hirudin(1–51) has the same molecular architecture as the corresponding polypeptide segment in natural hirudin and recombinant desulfatohirudin. It is also closely similar to the crystal structure of the N-terminal 51-residue segment of hirudin in a hirudin-thrombin complex, with root-mean-square deviations of the crystal structure relative to the mean solution structure of 0.61 Å for the backbone atoms and 0.91 Å for all heavy atoms of residues 3 to 30 and 37 to 48. Further coincidence is found for the loop formed by residues 31 to 36, which shows increased structural disorder in all available solution structures of hirudin, and of which residues 32 to 35 are not observable in the electron density map of the thrombin complex. Significant local structural differences between hirudin(1–51) in solution and hirudin in the crystalline thrombin complex were identified mainly for the N-terminal tripeptide segment and residues 17 to 21. These are further analyzed in an accompanying paper.

References (59)

M. Billeter et al.
Sequential resonance assignments in protein ¹H nuclear magnetic resonance spectra. Computation of sterically allowed proton-proton distances and statistical analysis of proton-proton distances in single crystal protein conformations
J. Mol. Biol.
(1982)
M. Billeter et al.
Comparison of the high-resolution structures of the α-amylase inhibitor tendamistat determined by nuclear magnetic resonance in solution and by X-ray diffraction in single crystals
J. Mol. Biol.
(1989)
P.Y. Chou et al.
β-Turns in proteins
J. Mol. Biol.
(1977)
A. De Marco et al.
Digital filtering with a sinusoidal window function: an alternative technique for resolution enhancement in FT NMR
J. Magn. Reson.
(1976)
P. Güntert et al.
A new procedure for high-quality baseline correction of two- and higher-dimensional NMR spectra
J. Magn. Reson.
(1992)
P. Güntert et al.
Efficient computation of three-dimensional protein structures in solution from nuclear magnetic resonance data using the program DIANA and the supporting programs CALIBA, HABAS and GLOMSA
J. Mol. Biol.
(1991)
P. Güntert et al.
Structure determination of the Antp(C39 → S) homeodomain from nuclear magnetic resonance data in solution using a novel strategy for the structure calculation with the programs DIANA, CALIBA, HABAS and GLOMSA
J. Mol. Biol.
(1991)
D. Marion et al.
Application of phase sensitive two-dimensional correlated spectroscopy (COSY) for measurements of ¹H-¹H spin-spin coupling constants in proteins
Biochem. Biophys. Res. Commun.
(1983)
D. Marion et al.
Improved solvent suppression in one- and two-dimensional NMR spectra by convolution of time-domain data
J. Magn. Reson.
(1989)
D. Marion et al.
Rapid recording of 2D NMR spectra without phase cycling: application to the study of hydrogen exchange in proteins
J. Magn. Reson.
(1989)

F. Markwardt

Hirudin as an inhibitor of thrombin

Methods Enzymol.

(1970)

G. Otting

Zero-quantum suppression in NOESY and experiments with a z filter

J. Magn. Reson.

(1990)

G. Otting et al.

Suppression of zero-quantum coherence in NOESY and soft-NOESY

J. Magn. Reson.

(1990)

M. Rance et al.

Improved spectral resolution in COSY ¹H NMR spectra of proteins via double quantum filtering

Biochem. Biophys. Res. Commun.

(1983)

T.J. Richmond

Solvent accessible surface area and excluded volume in proteins. Analytical equations for overlapping spheres and implications for the hydrophobic effect

J. Mol. Biol.

(1984)

T.J. Rydel et al.

Refined structure of the hirudin-thrombin complex

J. Mol. Biol.

(1991)

M. Scharf et al.

Primary structures of new ‘Iso-hirudins’

FEBS Letters

(1989)

T. Szyperski et al.

Impact of protein-protein contacts on the conformation of hirudin in the X-ray crystal structure of a complex with thrombin studied by comparison with the NMR solution structure of hirudin(1–51)

J. Mol. Biol.

(1992)

G. Wagner et al.

Truncated driven nuclear overhauser effect (TOE). A new technique for studies of selective ¹H-¹H Overhauser effects in the presence of spin diffusion

J. Magn. Reson.

(1979)

G. Wagner et al.

Sequential resonance assignments in protein ¹H nuclear magnetic resonance spectra, Basic pancreatic trypsin inhibitor

J. Mol. Biol.

(1982)

G. Wider et al.

Sequential resonance assignments in protein ¹H nuclear magnetic resonance spectra. Glucagon bound to perdeuterated dodecylphosphocholine micelles

J. Mol. Biol.

(1982)

M.P. Williamson et al.

Solution conformation of proteinase inhibitor IIa from bull seminal plasma by ¹H nuclear magnetic resonance and distance geometry

J. Mol. Biol.

(1985)

K. Wüthrich et al.

Sequential resonance assignments as a basis for determination of spatial protein structures by high resolution proton nuclear magnetic resonance

J. Mol. Biol.

(1982)

K. Wüthrich et al.

Pseudo-structures for the 20 common amino acids for use in studies of protein conformations by measurements of intramolecular proton-proton distance constraints with nuclear magnetic resonance

J. Mol. Biol.

(1983)

A. Yonath et al.

Polymers of tripeptides as collagen models. IV. Structure of poly(l-prolyl-glycyl-l-proline)

J. Mol. Biol.

(1969)

Anil-Kumar et al.

A two-dimensional nuclear Overhauser enhancement (2D NOE) experiment for the elucidation of complete proton-proton cross-relaxation networks in biological macromolecules

Biochem. Biophys. Res. Commun.

(1980)

Anil-Kumar et al.

Buildup rates of the nuclear Overhauser effect measured by two-dimensional proton magnetic resonance spectroscopy: implications for studies of protein conformation

J. Amer. Chem. Soc.

(1981)

M. Billeter et al.

Restrained energy refinement with two different algorithms and force fields of the structure of the α-amylase inhibitor tendamistat determined by NMR in solution

Biopolymers

(1990)

W. Bode et al.

The refined 1.9 Å crystal structure of human α-thrombin: interaction with D-Phe-Pro-Arg chloromethylketone and significance of the Tyr-Pro-Pro-Trp insertion segment

EMBO J.

(1989)

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Journal of Molecular Biology

ArticleNuclear magnetic resonance solution structure of hirudin(1–51) and comparison with corresponding three-dimensional structures determined using the complete 65-residue hirudin polypeptide chain

Abstract

J. Mol. Biol.

J. Mol. Biol.

J. Mol. Biol.

J. Magn. Reson.

J. Magn. Reson.

J. Mol. Biol.

J. Mol. Biol.

Biochem. Biophys. Res. Commun.

J. Magn. Reson.

J. Magn. Reson.

Methods Enzymol.

J. Magn. Reson.

J. Magn. Reson.

Biochem. Biophys. Res. Commun.

J. Mol. Biol.

J. Mol. Biol.

FEBS Letters

J. Mol. Biol.

J. Magn. Reson.

J. Mol. Biol.

J. Mol. Biol.

J. Mol. Biol.

J. Mol. Biol.

J. Mol. Biol.

J. Mol. Biol.

A two-dimensional nuclear Overhauser enhancement (2D NOE) experiment for the elucidation of complete proton-proton cross-relaxation networks in biological macromolecules

Biochem. Biophys. Res. Commun.

Buildup rates of the nuclear Overhauser effect measured by two-dimensional proton magnetic resonance spectroscopy: implications for studies of protein conformation

J. Amer. Chem. Soc.

Restrained energy refinement with two different algorithms and force fields of the structure of the α-amylase inhibitor tendamistat determined by NMR in solution

Biopolymers

The refined 1.9 Å crystal structure of human α-thrombin: interaction with D-Phe-Pro-Arg chloromethylketone and significance of the Tyr-Pro-Pro-Trp insertion segment

EMBO J.

Article
Nuclear magnetic resonance solution structure of hirudin(1–51) and comparison with corresponding three-dimensional structures determined using the complete 65-residue hirudin polypeptide chain