Structural insight into distinct mechanisms of protease inhibition by antibodies
- 11 December 2007
- journal article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences of the United States of America
- Vol. 104 (50), 19784-19789
- https://doi.org/10.1073/pnas.0708251104
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.Keywords
This publication has 40 references indexed in Scilit:
- The Mechanism of Inhibition of Antibody-based Inhibitors of Membrane-type Serine Protease 1 (MT-SP1)Journal of Molecular Biology, 2007
- Function Blocking Antibodies to Neuropilin-1 Generated from a Designed Human Synthetic Antibody Phage LibraryJournal of Molecular Biology, 2007
- Heparin Modulates the 99-Loop of Factor IXaPublished by Elsevier BV ,2006
- Conformational Lability in Serine Protease Active Sites: Structures of Hepatocyte Growth Factor Activator (HGFA) Alone and with the Inhibitory Domain from HGFA Inhibitor-1BJournal of Molecular Biology, 2005
- High-affinity Human Antibodies from Phage-displayed Synthetic Fab Libraries with a Single Framework ScaffoldJournal of Molecular Biology, 2004
- Degenerate interfaces in antigen-antibody complexesJournal of Molecular Biology, 2001
- Influence of cofactor binding and active site occupancy on the conformation of the macromolecular substrate exosite of factor VIIaJournal of Molecular Biology, 1998
- Refinement of Macromolecular Structures by the Maximum-Likelihood MethodActa Crystallographica Section D-Biological Crystallography, 1997
- [20] Processing of X-ray diffraction data collected in oscillation modeMethods in Enzymology, 1997
- The CCP4 suite: programs for protein crystallographyActa Crystallographica Section D-Biological Crystallography, 1994