A "knob-in-hole" gate in the antibody CH1 domain underlies subclass-independent IgA hinge cleavage by the commensal metallopeptidase IgAse.

Human immunoglobulin (Ig) A (IgA) protects ≈400 m2 of mucosae from microbes. Bacterial IgA-peptidases subvert this defense by cleaving the Fab-Fc hinge of subclass IgA1 while sparing the hinge-divergent, colon-dominant IgA2 subclass. The exception is IgAse, a ≈140-kDa, seven-domain, membrane-bound metallopeptidase from the colonic commensal Thomasclavelia ramosa that targets both subclasses but no other protein. We previously narrowed the minimal functional assembly down to the IgAse catalytic and wrapping domains (CD and WD) plus the IgA1/2 Fab', i.e., the hinge and a Fab of light and heavy chains (LC, HC), each with a variable and a constant Ig domain (VL + CL, VH + CH1). The transient interaction culminates in cleavage of a proline-valine bond within the first two hinge residues, downstream of a conserved C-terminal CH1 disulfide (SS2). Here, single-particle cryoelectron microscopy of a disulfide-stabilized minimal IgAse:IgA2 Fab' complex yielded high-quality density strongly supporting two states along the cleavage reaction coordinate, a "recognition complex" and a subsequent "precatalytic complex," in which Fab' binds frontally to the IgAse CD via a CH1 β-sheet and overhang loop LβEβF. This loop binds like a knob to a preformed hole in the active-site cleft. Binding rearranges the knob and rotates SS2 ≈ 180°, enabling access of the hinge into the cleft of the otherwise unaffected precatalytic complex. Together, this work defines a unique, CH1-mediated hinge-delivering "gate" mechanism, validated by a crystal structure of unbound IgA2 Fab, point mutations disrupting the CH1:CD interface, and molecular dynamics calculations, which provide the structural framework for subclass-independent IgA cleavage.