Contribution of interactions with the core domain of hirudin to the stability of its complex with thrombin.
The importance of interactions with residues 15-21 in the core domain of hirudin for the formation of the complex with thrombin has been investigated by site-directed mutagenesis. Contacts made by Leu-15 were found to be particularly important; replacement of this residue by alanine led to a decrease in the binding energy (delta delta Gbo) of 15 kJ.mol-1. Comparison with effects obtained in previous mutagenesis studies indicate that interactions with the side chain of Leu-15 contribute more to the stability of the complex than those of any other hirudin residues. Interactions with the side chains of Glu-17, Asn-20 and Val-21 also contributed significantly to binding energy; the delta delta Gbo value for these mutations was between 3 and 6 kJ.mol-1. Examination of the crystal structure of the thrombin-hirudin complex suggested the possibility that ionic interactions that would increase binding energy could be engineered by mutating Ser-19, Asn-20 and Gln-49 to acidic residues. The stability of the thrombin-hirudin complex was not, however, increased by these substitutions. The results obtained are discussed in terms of the crystal structure of the thrombin-hirudin complex.
Betz
A
A
Department of Haematology, University of Cambridge, U.K.
Hopkins
P C
PC
Le Bonniec
B F
BF
Stone
S R
SR
D000595:Amino Acid Sequence; D001665:Binding Sites; D006629:Hirudins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007477:Ions; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013816:Thermodynamics; D013917:Thrombin / Q000737:chemistry* / Q000378:metabolism
D006629:Hirudins; D007477:Ions; D013917:Thrombin