Fast folding of anthrax lethal factor N-terminal domain independent of proline isomerization

The N-terminal domain of anthrax lethal factor (LF_N) comprises the first 263 amino acids of lethal factor and is required for binding to oligomeric protective antigen (PA) and subsequent translocation into the host cell cytosol. During entry, LF_N must completely unfold to traverse the narrow PA pore φ-clamp and refold in the cytosol to elicit toxicity. Despite containing six proline residues, including one (Pro166) in the native cis conformation, we demonstrate that LF_N refolds rapidly from an equilibrium denatured state, independent of slow proline isomerization. Equilibrium urea denaturation experiments monitored by tyrosine fluorescence and circular dichroism at pH 8°C and 20°C revealed reversible two-state unfolding with a conformational stability (ΔG°_H2O) of ∼4 kcal/mol. Stopped-flow kinetic studies showed single-phase refolding/unfolding at pH 8, completing in less than 1 s. Chevron analysis exhibited rollover in the unfolding and refolding arms that, when fitted to a two-state mechanism with a broad transition barrier, gave a ΔG° of ∼4-5 kcal/mol, consistent with equilibrium results. The lower ΔG° was not due to folding/unfolding to an intermediate state or partial structure, as ¹⁵N-HSQC results show complete unfolding at 4 M urea. At pH 5, we observe two phases, a fast phase and a slow phase with activation energies of 11.4 and 18.1 kcal/mol, respectively. Chevron analysis at pH 5 is consistent with the four-state model (U ↔ J ↔ I ↔ N) described by Krantz, with the slow phase we ascribe to slow coupling of subdomains rather than proline isomerization. Our results suggest that the PA-binding domain of LF is optimized for swift refolding upon cytosolic entry, potentially bypassing the need for host chaperones or prolyl isomerases.