Fig. 1.

Conservation and covariance within pore lining loops. (a) An alignment of the PA homologues by using clustalx (24). Abbreviations are defined in Supporting Text, which is published as supporting information on the PNAS web site. Shown is the alignment between residues T390 and Y436 according to protective antigen. Shown above are the secondary structural features including the loop regions, residues 395–408 (i.e., the 397 loop) and residues 421–431 (i.e., the 426 loop). Residues with an asterisk denote residues that were identified from mutagenesis studies as being important for mediating toxicity to CHO-K1 cells (20, 21). F427 (‡; colored red) is the translocation active site residue required for PA-mediated toxicity (2, 21, 22). Highlighted in blue, red, and gray are residues that showed an identical conservation pattern and, thus, are referred to as correlated mutations. (b) Side-view of a single subunit of PA taken from the PA₈₃-CMG2 costructure (PDB ID code 1T6B) (12), showing the position of the 397 and 426 loops, in blue and red, respectively, and the side chains of K397, D426, and F427. (c) Cutaway side view surface rendering of the PA prepore heptamer (1TZO) showing loop positions relative to the putative 14-stranded β-barrel. (d) Normalized planar lipid bilayer translocation records of LF_N through PA₆₃ channels. After PA₆₃ channel formation reached steady state, 10 nM LF_N was added to the cis chamber (at ΔΨ = 10 mV; symmetric pH 5.5 conditions), resulting in a rapid decrease in conductance. Once conductance block reached a steady state, the cis chamber was perfused, and translocation was initiated by adding a fixed amount of KOH to the trans compartment to bring the final pH to 7.2. (e) Cellular translocation assay. PA₈₃ was titrated, mixed with a fixed concentration of LF_N-DTA (100 pM), added to CHO-K1 cells, and incubated overnight. Cell death is quantitated by measuring the ability of cells to take up and cleave WST-1.