The Minimal Folding Motif of the Repeat-in-Toxin Domain of Adenylate Cyclase Toxin

. The folding of its repeats-in-toxin (RTX) domain is critical to CyaA's toxin activity and virulence. The RTX domain contains five RTX blocks (RTX-i to RTX-v), and its folding follows a strict vectorial, series folding pathway that starts from the folding of the C-terminal RTX-v and proceeds sequentially toward its N-terminal RTX blocks. Thus, a folded RTX-v or its equivalent is required for engineering any artificial RTX domain for biophysical and biochemical studies of other RTX blocks. By combining single-molecule optical tweezers, protein engineering, and nuclear magnetic resonance spectroscopy, here, we characterized the folding pathway of RTX-v in detail and aimed to identify the minimal folding unit of RTX-v that can serve as a folding nucleus to template the folding of its N-terminal RTX blocks. Our results revealed that RTX-v folds via an on-pathway intermediate. Residues 1580-1681 constitute the minimal folding motif but with marginal stability, while residues 1574-1681 represent a more stable folding motif and serve as the on-pathway folding intermediate of RTX-v. This folding intermediate bears important implications for the design and engineering of β-roll fusion proteins.