Figure 4. The N-terminal strands of LptD interacts with BamA during assembly.

(A) β-strands four, five, and six of substrate LptD interact with BamA. Crosslinking was tested as described in Figure 3, but with pBPA substitutions in the N-terminal portion of substrate LptD/LptD4213. ‘Membrane’ and ‘lumen’ specify where the indicated residues would face in the mature barrel. (B) Top-down view of LptD showing that residues in at least 3 β-strands in the N-terminal region of the LptD barrel interact with BamA. Residues in LptD4213 that form strong crosslinks to BamA are shown in blue, while residues that form weak crosslinks are shown in cyan. The N- and C-terminal strands of LptD are indicated in tan, and LptE is shown in green. This color scheme is maintained in the rest of the figure. (C) Side view of LptD showing crosslinking positions as depicted in (B). Additional crosslinking experiments at residues in the first three strands of substrate LptD are shown in Figure 4—figure supplement 1. Additional views that include residues that do not form crosslinks are shown in Figure 4—figure supplement 2. Note that only crosslinks within a blot can be compared, and each blot includes only proximal residues.

Figure 4—figure supplement 1. Crosslinking of the first three strands of substrate LptD to BamA.

(A) β-strands one, two, and three of substrate LptD interact with BamA. Crosslinking was tested as described in Figure 4. (B) Top-down view of the region near the N- and C-termini of LptD showing residues in LptD4213 that form crosslinks to BamA during assembly, but are important in mediating closure of LptD in the folded form. Residues K234, T238, and Y244 interact with L4 in folded LptD (within the region in pink, which is deleted in LptD4213).

Figure 4—figure supplement 2. Crosslinking of the N-terminal region of substrate LptD to BamA.

(A) Sequence alignment of the N-terminal region of LptD across representative Gram-negative bacteria. Alignments were performed as in Figure 3—figure supplement 1. Residues that crosslink strongly or weakly to BamA are presented in blue or cyan, respectively, while residues that do not form crosslinks are presented in black. (B) Top-down view mapping the residues in the N-terminal strands of substrate LptD that interact with BamA. Residues in blue and cyan crosslink strongly and weakly to BamA, respectively. Residues that do not form crosslinks are presented in the same color as secondary structure. The first and last β-strands of LptD are shown in tan, the deletion in LptD4213 is shown in pink, and LptE is shown in green. (C) As in (B), but presented in a side view.