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

The N-terminal domain of anthrax lethal factor (LFN) 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, LFN 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 LFN 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.