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 F_ab-F_c 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 F_ab', i.e., the hinge and a F_ab of light and heavy chains (LC, HC), each with a variable and a constant Ig domain (V_L + C_L, V_H + C_H1). The transient interaction culminates in cleavage of a proline-valine bond within the first two hinge residues, downstream of a conserved C-terminal C_H1 disulfide (SS2). Here, single-particle cryoelectron microscopy of a disulfide-stabilized minimal IgAse:IgA2 F_ab' complex yielded high-quality density strongly supporting two states along the cleavage reaction coordinate, a "recognition complex" and a subsequent "precatalytic complex," in which F_ab' binds frontally to the IgAse CD via a C_H1 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, C_H1-mediated hinge-delivering "gate" mechanism, validated by a crystal structure of unbound IgA2 F_ab, point mutations disrupting the C_H1:CD interface, and molecular dynamics calculations, which provide the structural framework for subclass-independent IgA cleavage.