Abstract
Macrophage-stimulating protein (MSP) and hepatocyte growth factor/scatter factor (HGF/SF) are plasminogen-related growth and motility factors that interact with cell-surface protein tyrosine kinase receptors. Each one is a heterodimeric protein comprising a disulfide-linked alpha chain and a serine protease-like beta chain. Despite structural similarities between MSP and HGF, the primary receptor binding site is located on the alpha chain of HGF/SF but on the beta chain of MSP. To obtain insight into the structural basis for MSP beta chain binding, beta chain structure was modeled from coordinates of an existing model of the HGF beta chain. The model revealed that the region corresponding to the S1 specificity pocket in trypsin is filled by the Asn(682)/Glu(648) interacting pair, leaving a shallow cavity for possible beta chain interaction with the receptor. Mutants in this region were created, and their binding characteristics were determined. A double mutation of Asn(682)/Glu(648) caused diminished binding of the beta chain to the MSP receptor, and a single mutation of neighboring Arg(683) completely abolished binding. Thus, this region of the molecule is critical for binding. We also found that at equimolar concentrations of free alpha and beta chains, alpha chain binding to receptor was detectable, at levels considerably lower than beta chain binding. The EC(50) values determined by quantitative enzyme-linked immunosorbent assay are 0.25 and 16.9 nM for beta and alpha chain, respectively. The data suggest that MSP has two independent binding sites with high and low affinities located in beta and alpha chain, respectively, and that the two sites together mediate receptor dimerization and subsequent activation.
Highlights
From the ‡Laboratory of Immunobiology, NCI-Frederick Cancer Research and Development Center and ¶Macromolecular Structure Laboratory, NCI-Frederick Cancer Research and Development Center, ABL-Basic Research Program, Frederick, Maryland 21702
Macrophage-stimulating protein (MSP) and hepatocyte growth factor/scatter factor (HGF/SF) are plasminogen-related growth and motility factors that interact with cell-surface protein tyrosine kinase receptors
Despite loss of enzymatic activity because of catalytic triad mutations, the geometry of the regions of MSP that correspond to the active site of serine proteases has been conserved
Summary
Vol 274, No 42, Issue of October 15, pp. 29937–29943, 1999 Printed in U.S.A. RESIDUES CRITICAL FOR  CHAIN BINDING AND EVIDENCE FOR INDEPENDENT ␣ CHAIN BINDING*. The model revealed that the region corresponding to the S1 specificity pocket in trypsin is filled by the Asn682/Glu648 interacting pair, leaving a shallow cavity for possible  chain interaction with the receptor Mutants in this region were created, and their binding characteristics were determined. Features of the ␣ chain of MSP and HGF include an N-terminal domain (N domain) corresponding to the plasminogen preactivation peptide, four kringles, and a segment that terminates in the cleavage site for activation; the  chain is the serine protease-like domain. MSP and HGF correspond to enzymes, and their receptors correspond to enzyme substrates From this viewpoint, regions of the MSP serine protease-like domain of particular interest for binding to receptor would include the catalytic site and several surface. The result supports a recent hypothesis that ligand-induced RON dimerization by MSP is mediated by a single ligand molecule, which binds to a receptor via the  chain, after which a second receptor is engaged by the ␣ chain [27]
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