Dynamic gating by ϕ-clamp loop controls peptide translocation through the anthrax toxin nanopore.

The ϕ clamp, a key active site in the anthrax toxin protective antigen (PA) nanopore, governs the complex process of peptide translocation, yet its precise mechanism remains debated. A key question is how might the nanopore mitigate the formation of too stable/static of a ϕ-clamp interaction with the polymer it is attempting to translocate. One hypothesis is that it would "gate" to bind and "ungate" to release. Here, we dissect this mechanism using single-channel electrophysiology on wild-type PA and two ϕ-clamp mutants (F427A and F427Y). Thermodynamic analysis reveals the wild-type F427 residue creates a deep, energetically stable "hydrophobic trap" (state 0). Kinetic analysis further establishes F427 as a "specific chemical sensor"; the strong correlations between translocation kinetics and peptide hydrophobicity seen in wild-type PA are abolished in the F427A mutant. Critically, partially blocked intermediate states persist even upon F427A side-chain ablation. This finding supports a refined structural model where these states arise not from populating discrete F427 rotamers, but rather from a larger-scale, dynamic dilation of the entire clamp-containing loop. Overall, this model resolves the paradox of how a peptide clamp can facilitate transport: translocation proceeds via a dynamic equilibrium between a constricted, high-affinity "gated/clamped" state (the trap) and a dilated, lower-affinity "ungated/unclamped" state (the intermediates), allowing the pore to mitigate its own energetic traps through conformational gating. This detailed mechanistic insight provides a framework for rationally engineering next-generation dynamical nanopore biosensors.