Protein Engineering vol. 7 no. 7 pp. 895-903, 1994
© 1994 Oxford University Press
RESEARCH-ARTICLE |
Mutational analysis and molecular modeling of the N-terminal kringle-containing domain of hepatocyte growth factor identifies amino acid side chains important for interaction with the c-Metreceptor
Depanroenls of Cell Genetics 2Protein Engineering, Genentech, Inc. 460 Point San Bruno Boulevard, South San Francisco, CA 94080, USA
3To whom correspondence should be addressed
The hepatocyte growth factor receptor (HGFr) transduces a wide range of biological signals, including mitogenesis, motogenesis and morphogenesis. We recently localized a region within the N-terminal 175 amino acids of hepatocyte growth factor (HGF), termed HGF/NK1, that is necessary and sufficient for binding to the HGFr. HGF/NK1 contains an as-yet structurally undefined N-terminal region followed by the first of four HGF kringles. We have used a combination of molecular modeling and mutagenesis to dissect the function of this region of HGF. Two mutation-sensitive patches on the proposed surface regions of HGF kringle one (K1) were identified. The first patch consists of residues E159, S161, E195 and R197, all of which are predicted to be close to each other in the tertiary structure of Kl. The second patch, lying on the opposite side of the kringle, consists of residues D171 and Q173. Mutational analysis of the Nterminal region of HGF identified residue D117 which also appeared to influence receptor binding. We also investigated the properties of a naturally occurring HGF variant (
5-HGF) that arises from an alternatively spiked transcript and therefore lacks five residues within K1. Our data suggest that in wild-type HGF, F162 is crucial in maintaining the hydrophobk core of the kringle. In 5-HGF, the loss of this residue is compensated for by a functional substitution of F162 with Y167, which is predicted to occupy the 5-HGF K1 core. Comparison of the models of wild-type and 5 kringles reveals that the positions of the presumed receptor binding determinants remain unchanged. These studies suggest a structural basis for the receptor binding properties of wild-type and 5-HGF and provide important clues as to the mechanism of kringle–mediated protein-protein interactions.
Keywords: hepatocyte growth factor/kringle/molecular modeling/receptor binding site/structure -function study
Received January 13, 1994; accepted March 30, 1994.
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