2R0K

Protease domain of HGFA with inhibitor Fab58

Summary for 2R0K

• Entry DOI: 10.2210/pdb2r0k/pdb
• Related: 1YBW 1YC0 2R0L
• Descriptor: Hepatocyte growth factor activator, antibody light chain of Fab58, antibody heavy chain of Fab58, Fab portion only (3 entities in total)
• Functional Keywords: serine protease, antibody, inhibitor, egf-like domain, glycoprotein, hydrolase, kringle, secreted, zymogen, immune system
• Biological source: Homo sapiens (human); More
• Cellular location: Secreted: Q04756
• Total number of polymer chains: 3
• Total formula weight: 77861.43

Authors

Eigenbrot, C.,Shia, S. (deposition date: 2007-08-20, release date: 2007-12-25, Last modification date: 2023-08-30)

Primary citation

Wu, Y.,Eigenbrot, C.,Liang, W.C.,Stawicki, S.,Shia, S.,Fan, B.,Ganesan, R.,Lipari, M.T.,Kirchhofer, D.
Structural insight into distinct mechanisms of protease inhibition by antibodies.
Proc.Natl.Acad.Sci.Usa, 104:19784-19789, 2007

PubMed Abstract: To better understand how the relatively flat antigen-combining sites of antibodies interact with the concave shaped substrate-binding clefts of proteases, we determined the structures of two antibodies in complex with the trypsin-like hepatocyte growth-factor activator (HGFA). The two inhibitory antibodies, Ab58 and Ab75, were generated from a human Fab phage display library with synthetic diversity in the three complementarity determining regions (H1, H2, and H3) of the heavy chain, mimicking the natural diversity of the human Ig repertoire. Biochemical studies and the structures of the Fab58:HGFA (3.5-A resolution) and the Fab75:HGFA (2.2-A resolution) complexes revealed that Ab58 obstructed substrate access to the active site, whereas Ab75 allosterically inhibited substrate hydrolysis. In both cases, the antibodies interacted with the same protruding element (99-loop), which forms part of the substrate-binding cleft. Ab58 inserted its H1 and H2 loops in the cleft to occupy important substrate interaction sites (S3 and S2). In contrast, Ab75 bound at the backside of the cleft to a region corresponding to thrombin exosite II, which is known to interact with allosteric effector molecules. In agreement with the structural analysis, binding assays with active site inhibitors and enzymatic assays showed that Ab58 is a competitive inhibitor, and Ab75 is a partial competitive inhibitor. These results provide structural insight into antibody-mediated protease inhibition. They suggest that unlike canonical inhibitors, antibodies may preferentially target protruding loops at the rim of the substrate-binding cleft to interfere with the catalytic machinery of proteases without requiring long insertion loops.

PubMed: 18077410
DOI: 10.1073/pnas.0708251104

Experimental method

X-RAY DIFFRACTION (3.51 Å)