Structure of an Fab–Protease Complex Reveals a Highly Specific Non-canonical Mechanism of Inhibition

Abstract

SummaryThe vast majority of protein protease inhibitors bind their targets in a substrate-like manner. This is a robust and efficient mechanism of inhibition, but due to the highly conserved architecture of protease active sites, these inhibitors often exhibit promiscuity. Inhibitors that show strict specificity for one protease usually achieve this selectivity by combining substrate-like binding in the active site with exosite binding on the protease surface. The development of new, specific inhibitors can be greatly aided by binding to non-conserved regions of proteases if potency can be maintained. Due to their ability to bind specifically to nearly any antigen, antibodies provide an excellent scaffold for creating inhibitors targeted to a single member of a family of highly homologous enzymes. The 2.2 Å resolution crystal structure of an Fab antibody inhibitor in complex with the serine protease membrane-type serine protease 1 (MT-SP1/matriptase) reveals the molecular basis of its picomolar potency and specificity. The inhibitor has a distinct mechanism of inhibition; it gains potency and specificity through interactions with the protease surface loops, and inhibits by binding in the active site in a catalytically non-competent manner. In contrast to most naturally occurring protease inhibitors, which have diverse structures but converge to a similar inhibitory archetype, antibody inhibitors provide an opportunity to develop divergent mechanisms of inhibition from a single scaffold.

Cross-domain inhibition of TACE ectodomain

“…Existing inhibitory antibodies against the serine protease MT-SP1 were isolated by competitively selecting for scFv in the presence of a macromolecular antigen antagonist (30). This approach yielded a steric hindrance antibody antagonist (E2) that sourced antigen specificity through MT-SP1 noncatalytic surface loops and inhibited by binding the active site (31). However, unlike D1(A12), the epitope of E2 resides within the single catalytic domain of the MT-SP1 antigen.…”

Imaging a functional tumorigenic biomarker in the transformed epithelium

“…Central to the detection of active matriptase, and delineation of the mechanism behind matriptase activity emerging from the epithelium, is the antibodybased probe A11. A11 is a fully human monoclonal antibody discovered previously by phage display (26). Analysis of the crystal structure of A11 bound to matriptase showed that the binding face of the antibody did not contain lysine residues (26).…”

“…A11 is a fully human monoclonal antibody discovered previously by phage display (26). Analysis of the crystal structure of A11 bound to matriptase showed that the binding face of the antibody did not contain lysine residues (26). This allows for the modification of lysine residues for imaging without abrogating the function of the antibody.…”

“…E-mail: charles.craik@ucsf.edu or henry. a human fragment of antigen binding (Fab) phage display library, a human monoclonal antibody that could selectively recognize the active form of matriptase was discovered (25,26). The lead antibody, A11, was converted from the Fab to the larger and more potent IgG with a K i of 35 pM and showed no cross-reactivity with matriptase zymogen, HAI-1-bound matriptase, or the murine matriptase ortholog epithin (27).…”

Antigen recognition by single-domain antibodies: structural latitudes and constraints

“…Naturally-occurring competitive inhibitors of protease enzymes are convex, and this appears to be a difficult geometry for the paratopes of conventional antibodies to achieve (see below): even in cases of near-true competitive inhibition, conventional antibodies use a flat or concave V H /V L interface to bind protruding regions on enzymes and partially insert one or more CDRs into the active site cleft in a non-substrate-like manner. 66,67 This hypothesis is supported by experiments using purified polyclonal immunoglobulin (Ig)Gs from enzyme-immunized dromedaries showing that competitive inhibition was a feature of heavy chain-only IgGs, but not of conventional IgGs. 11,32 It remains unclear why immunization with some enzymes yields mostly sdAbs with planar paratopes and bind outside the active site, achieving allosteric or no inhibition, although tolerance mechanisms may play a role.…”