High temperature requirement A1 (HtrA1) is a trypsin-fold serine protease implicated in the progression of age-related macular degeneration (AMD). Our interest in an antibody therapy to neutralize HtrA1 faces the complication that the target adopts a trimeric arrangement, with three active sites in close proximity. In the present study, we describe antibody 94, obtained from a human antibody phage display library, which forms a distinct macromolecular complex with HtrA1 and inhibits the enzymatic activity of recombinant and native HtrA1 forms. Using biochemical methods and negative-staining EM we were able to elucidate the molecular composition of the IgG94 and Fab94 complexes and the associated inhibition mechanism. The 246-kDa complex between the HtrA1 catalytic domain trimer (HtrA1_Cat) and Fab94 had a propeller-like organization with one Fab bound peripherally to each protomer. Low-resolution EM structures and epitope mapping indicated that the antibody binds to the surface-exposed loops B and C of the catalytic domain, suggesting an allosteric inhibition mechanism. The HtrA1_Cat–IgG94 complex (636 kDa) is a cage-like structure with three centrally located IgG94 molecules co-ordinating two HtrA1_Cat trimers and the six active sites pointing into the cavity of the cage. In both complexes, all antigen-recognition regions (paratopes) are found to bind one HtrA1 protomer and all protomers are bound by a paratope, consistent with the complete inhibition of enzyme activity. Therefore, in addition to its potential therapeutic usefulness, antibody 94 establishes a new paradigm of multimeric serine protease inhibition.
The trimeric serine protease HtrA1 forms a cage-like inhibition complex with an anti-HtrA1 antibody
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Claudio Ciferri, Michael T. Lipari, Wei-Ching Liang, Alberto Estevez, Julie Hang, Scott Stawicki, Yan Wu, Paul Moran, Mike Elliott, Charles Eigenbrot, Kenneth J. Katschke, Menno van Lookeren Campagne, Daniel Kirchhofer; The trimeric serine protease HtrA1 forms a cage-like inhibition complex with an anti-HtrA1 antibody. Biochem J 1 December 2015; 472 (2): 169–181. doi: https://doi.org/10.1042/BJ20150601
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