Mapping the PA- and LF-binding sites by site-directed mutagenesis. (a) The seven residues important for binding PA (D182, D187, L188, Y223, H229, L235, and Y236) cluster on the surface of LF_N and are shown in green and red (15). (The D184A mutant did not show a binding defect but is depicted in pink, because it is likely to contribute to a net negative charge in this region.) Approximate dimensions of the site are indicated by the D187 Cα-H229 Cα distance (15 Å). The N terminus of the domain (E27) is shown in blue. (b) A surface rendering of the N-terminal domain 1′ (residues 175-258) from two PA₆₃ subunits (yellow and pink) as viewed from the top of the heptameric ring. LF_N binding studies to dimeric PA₆₃ suggested two clusters of important residues shown in green and blue (8). One cluster contains residues P205, I207, I210, K214, K197, and R200 (subsite I). The second cluster contains R178 and the K197 and R200 residues from the neighboring PA₆₃ subunit (subsite II). (Note: The P205A mutant was not tested in the dimeric background but is likely part of the first subsite because of its proximity to I207. K197 and R200, which were originally tested only in the context of subsite II, have recently been shown to participate in subsite I as well; H.C.L., unpublished work.) Approximate dimensions of the site are indicated by distances of the K214 Cα to the R200 Cα of the same subunit (16 Å) and the neighboring subunit (30 Å). Coordinates for LF_N and the PA₆₃-PA₆₃ dimer were obtained from the 1J7N (12) and 1TZO (25) crystal structures, respectively. (c)LF_N was manually docked on the PA dimer in two orientations that differed by ≈180°.

The Rosetta-Dock model of the LF_N-PA dimer complex lies in a deep energy funnel. Three thousand independent trajectories were carried out, starting from the manually docked model. The energy for each structure (arbitrary units) is plotted against the rmsd between LF_N molecules when the PA subunits from the manually docked and final models are aligned. There is a dramatic energy funnel around 20 Å from the starting model. The lowest energy structure also is the center of the largest cluster of low-energy models, and it is our most reliable model for this complex.

Identification of a disulfide crosslink between LF_N Y108C and PA N209C. LF_N Y108C and PA₆₃ N209C form a disulfide-linked complex under oxidizing conditions (lane 1), which is disrupted in the presence of 10 mM DTT (lane 2).

A cell-surface binding assay shows that the LF_N D187K-PA K213D/K213E and LF_N E142K-PA K218E pairs can rescue binding defects. Data represent the fraction of mutant ³⁵S-labeled LF_N bound specifically to PA on cells relative to that of wild-type LF_N. Error bars represent SEM.

The model of LF_N bound to PA. (a) In this depiction of LF_N (gray) bound to the surface of dimeric PA₆₃ (light pink and yellow), the LF_N E135, E142, and D187 residues are shown in red; the PA K197, K213 and K218 residues are shown in blue; and the LF_N Y108 -PA N209 pair from the disulfide crosslinking experiment is shown in green. (Note: The E135 and K197 residues are predicted to interact based on the model but were not able to complement each other in a charge-reversal experiment.) As modeled, the bulk of LF_N's contacts are with a single PA₆₃ subunit (light pink), but contacts do exist with the neighboring PA₆₃ subunit (yellow). The N- and C-terminal helices of LF_N are shown in green and bright pink, respectively. (b) A close-up of the modeled interface between LF_N and PA subsite I suggests a large number of electrostatic interactions and a buried LF_N His residue. Residues from LF_N and PA that may form electrostatic interactions are shown in red and blue, respectively. LF_N H229 and Y236 were identified as the two most important residues for binding PA (15) and are shown in green, whereas the three important hydrophobic residues from the PA ligand-binding site (8) are shown in purple. (c) An aerial view of the PA heptamer-LF_N complex in which three LF_N molecules are bound. The footprint for LF_N contacts on the PA dimer is shown in gray. LF_N does not make contacts with residues R178 and R200 at subsite II. The three experimentally obtained PA contact points (K213, N209, and K218) are shown in red, green, and purple, respectively. The N- and C-terminal helices of LF_N are shown in green and bright pink, respectively. (d) A cartoon side view of the LF_N-PA complex in which only one LF_N (blue) is bound. PA is depicted as a cartoon cutaway to emphasize the interior lumen of the heptameric ring. As modeled, the N-terminal helix of LF_N (green) points toward the interior of the heptameric ring, whereas the C-terminal helix (bright pink) points upward and away from the heptamer, thereby allowing room for the LF catalytic domain. The N-terminal 26 residues of LF_N are drawn in cartoon format as a black line and can potentially insert into the prepore lumen. LF translocation is thought to be initiated by the LF N terminus and occur through the lumen of the heptameric ring (11, 24).