PDBsum entry 1yc0

Hydrolase/inhibitor

PDB id: 1yc0

Contents

Protein chains

247 a.a.

66 a.a.

Ligands

PO4

Waters ×119

References listed in PDB file

Key reference

Title

Conformational lability in serine protease active sites: structures of hepatocyte growth factor activator (hgfa) alone and with the inhibitory domain from hgfa inhibitor-1b.

Authors

S.Shia, J.Stamos, D.Kirchhofer, B.Fan, J.Wu, R.T.Corpuz, L.Santell, R.A.Lazarus, C.Eigenbrot.

Ref.

J Mol Biol, 2005, 346, 1335-1349. [DOI no: 10.1016/j.jmb.2004.12.048]

PubMed id

15713485

Abstract

Hepatocyte growth factor activator (HGFA) is a serine protease that converts hepatocyte growth factor (HGF) into its active form. When activated HGF binds its cognate receptor Met, cellular signals lead to cell growth, differentiation, and migration, activities which promote tissue regeneration in liver, kidney and skin. Intervention in the conversion of HGF to its active form has the potential to provide therapeutic benefit where HGF/Met activity is associated with tumorigenesis. To help identify ways to moderate HGF/Met effects, we have determined the molecular structure of the protease domain of HGFA. The structure we determined, at 2.7 A resolution, with no pseudo-substrate or inhibitor bound is characterized by an unconventional conformation of key residues in the enzyme active site. In order to find whether this apparently non-enzymatically competent arrangement would persist in the presence of a strongly-interacting inhibitor, we also have determined, at 2.6 A resolution, the X-ray structure of HGFA complexed with the first Kunitz domain (KD1) from the physiological inhibitor hepatocyte growth factor activator inhibitor 1B (HAI-1B). In this complex we observe a rearranged substrate binding cleft that closely mirrors the cleft of other serine proteases, suggesting an extreme conformational dynamism. We also characterize the inhibition of 16 serine proteases by KD1, finding that the previously reported enzyme specificity of the intact extracellular region of HAI-1B resides in KD1 alone. We find that HGFA, matriptase, hepsin, plasma kallikrein and trypsin are potently inhibited, and use the complex structure to rationalize the structural basis of these results.

Figure 1.
Figure 1. Uninhibited HGFA. Schematic representation of the crystallized HGFA 34 kDa fragment (starting at residue 373), looking into the substrate binding/active site region. Disulfide links are labeled. Cys168 was modeled with two side-chain conformations, only one of which makes a bond to Cys182. Cys187 is unpaired. An eight-residue section of the HGFA light chain (green) can be seen in the rear, including its disulfide link to the protease domain (Cys394/Cys122). Loops colored pink differ structurally among homologous enzymes and help determine inhibitor and substrate specificity. The crystallized construct in the context of intact HGFA is depicted at the bottom. Molecular images produced using PyMOL (Delano Scientific, San Carlos CA).

Figure 5.
Figure 5. The HGFA/KD1 complex. (a) The conventional conformation of the HGFA substrate binding region from the HAI-1-KD1 complex (light brown) compared to the unconventional conformation from uninhibited HGFA (blue). (b) Overall view with HGFA surface (light brown/red) and KD1 (green/yellow), including side-chains form Arg13(258), Arg15(260), and Phe18(263). The (N-terminal affinity tag + KD1) construct in the context of HAI-1B is depicted at the bottom. Prominent residues from the HGFA 37-loop, 60-loop and 99-loop are labeled. Red and yellow colors are residues of HGFA (red) and KD1 (yellow) with an atom within 3.5 Å of the other protein. (c) Details of the interaction between HGFA and HAI-1-KD1, in the same orientation as above. Portions of the KD1 domain (yellow) are depicted in the HGFA (light brown) active site. H-bonds between KD1 and HGFA are grey/black dotted lines. Inhibitor residue labels are underlined.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2005, 346, 1335-1349) copyright 2005.