Volume 25, Issue 1, Pages 94-106 (January 2017) Flexibility in the Periplasmic Domain of BamA Is Important for Function  Lisa R. Warner, Petia Z. Gatzeva-Topalova, Pamela A. Doerner, Arthur Pardi, Marcelo C. Sousa  Structure  Volume 25, Issue 1, Pages 94-106 (January 2017) DOI: 10.1016/j.str.2016.11.013 Copyright © 2016 Elsevier Ltd Terms and Conditions

Structure 2017 25, 94-106DOI: (10.1016/j.str.2016.11.013) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 Orientations of BamA POTRA Domains (A) Crystal structures of BamA POTRA3–5 domains superimposed on POTRA4. (B) Crystal structures of BamA POTRA1–2 domains superimposed on POTRA2. (C) Crystal structures of BamA POTRA1-3 domains superimposed on POTRA3 displaying conformational flexibility in the hinge between POTRA2 and POTRA3 that gives rise to “compact” and “extended” conformations. The POTRA domains shown are from the following crystal structures: E coli BamA POTRA1–4 (PDB: 2QZC), red; POTRA1–4 (PDB: 2QDF), green; POTRA1–4 (PDB: 3EFC), blue; POTRA4–5 (PDB: 3OG5), gray; POTRA4–5 (PDB: 3Q6B), magenta; BamACDE complex (PDB: 5EKQ), yellow; Neisseria gonorrhoeae BamA (PDB: 4K3B), cyan; and Haemophilus ducreyi BamA POTRA4–5-barrel (PDB: 4K3C), orange. Structure 2017 25, 94-106DOI: (10.1016/j.str.2016.11.013) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 Interdomain Interactions in BamA POTRA1–5 (A) The absolute value of the 1H/15N chemical-shift perturbations (Δδ 1H,15N), which were obtained by comparing POTRA1–2 or POTRA4–5 with POTRA1–5. POTRA2 has small chemical-shift perturbations upon addition of POTRA3 compared with those in POTRA4. (B) The large chemical-shift changes observed in the POTRA4 domain are consistent with the domain packing observed in the crystal structures 3EFC (Gatzeva-Topalova et al., 2008) and 2QDF (Kim et al., 2007). The Δδ 1H,15N values are mapped on the spliced model for POTRA1–5 (see Experimental Procedures) with perturbations between 0.1 and 0.2 ppm in yellow, >0.2 to 0.4 ppm in orange, and >0.4 in red. The larger Δδ 1H,15N values tend to cluster to the linker connecting POTRA3 and POTRA4, the α2 helix and loop L2, and sections of strands β2 and β3 and loop L4 in POTRA4. For residues with perturbations >0.1 their heavy atoms are shown as spheres. Structure 2017 25, 94-106DOI: (10.1016/j.str.2016.11.013) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 15N Relaxation Data Show that POTRA1–5 Tumbles as Two Subdomains (A) The {1H}-15N heteronuclear NOEs values for individual amide residues, where the dashed line shows an NOE threshold value of 0.70. Error bars were calculated from measurement of volumes in regions with no peaks and standard error propagation. (B) The residue-specific rotational correlation times, τc, are shown as a function of sequence and were calculated from backbone 15N relaxation measurements as described in Experimental Procedures. Error bars were calculated from the nonlinear least square fitting of the relaxation data and standard error propagation. The average rotational correlation times, <τc>, were calculated for each of the individual domains and are shown as horizontal lines. The secondary structure of POTRA1–5 is shown above. Structure 2017 25, 94-106DOI: (10.1016/j.str.2016.11.013) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 Bootstrap Analysis of Alignment Tensors of Individual Domains of POTRA1–5 Alignment tensors were calculated from RDCs collected in (A) 10 mg/mL Pf1 phage alignment medium and (B) 3% C12E5/hexanol alignment medium. Histogram plots of the distribution of Da from the bootstrap analysis are shown for the individual POTRA domains, where the bootstrap consisted of 4,000 datasets generated with random resampling as described in Experimental Procedures. The histograms were generated with bins of 0.5 Hz and fit to a Gaussian (solid lines). Bimodal distributions were observed in some of the domains, especially in the 3% C12E5/hexanol medium. Structure 2017 25, 94-106DOI: (10.1016/j.str.2016.11.013) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 5 The Effects of BamA Double-Cysteine Mutants on Cell Growth Two double-cysteine mutants of BamA, S143C-A220C and V144C-D211C, were designed such that disulfide bond formation would stabilize the (A) compact or (B) extended conformation of BamA, respectively. The E. coli BamA depletion strain JCM166 transformed with a plasmid encoding for wild-type BamA (black), BamA(S143C-A220C) (green), BamA(V144C-D211C) (red), or a GFP-negative control (blue), were grown in the presence of arabinose and then switched at time = 0 to cultures containing (C) fucose, (D) fucose + TCEP, (E) arabinose, or (F) arabinose + TCEP. Structure 2017 25, 94-106DOI: (10.1016/j.str.2016.11.013) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 6 BamA Double-Cysteine Mutants Form Intramolecular Disulfides In Vivo The E. coli BamA depletion strain JCM166 expressing His-tagged BamA cysteine free (Cysless), the double-cysteine mutants (V144C-D211C and S143C-A220C) and, where indicated, a single-cysteine mutant (V144C), were grown in the presence of arabinose. (A) The cells were cultured with (+) or without (−) TCEP and when the OD reached ≈0.6, the cells were treated with biotin-maleimide to label available cysteines. The HISBamA mutants were purified by Ni-nitrilotriacetic acid (NTA) and analyzed by western blotting probed with BamA antibodies (α-BamA) or streptavidin-HRP (Strep-HRP) to reveal biotin labeling. (B) The cells were cultured without TCEP and when the OD reached ≈0.6, the cells were treated with N-ethylmaleimide to block reactive cysteines. The HISBamA mutants were purified by Ni-NTA and analyzed by western blotting probed with BamA antibodies (α-BamA) under reducing (+DTT) or non-reducing (−DTT) conditions. Structure 2017 25, 94-106DOI: (10.1016/j.str.2016.11.013) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 7 The Unassigned Residues in POTRA3 Cluster Near the POTRA2–3 Interface The unassigned residues in POTRA3 are highlighted in red on the structure of POTRA3 in the (A) extended and (B) compact forms of POTRA1–4. These residues are mainly in the L2 loop and α2 helix as well as the L3 loop and portions of the β2 and β3 strand. The gray dashed ovals highlight areas of structural differences for POTRA3 in the two crystal structures, and the dashed red line in (B) indicates zero occupancy for the residues 201–214. Structure 2017 25, 94-106DOI: (10.1016/j.str.2016.11.013) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 8 Structure of the BAM Complex (A) Side view, parallel to the membrane plane, of the BAM complex with BamA in green, BamB in yellow, BamC in light cyan, BamD in orange, and BamE in blue. POTRA domains 1–3 are labeled P1 to P3 and the hinge between POTRA2 and POTRA3 is indicated with an arrow. (B) Bottom view of the BAM complex, looking perpendicular to the membrane plane from the periplasmic side. POTRA domains 1–5 are labeled P1 to P5 with colors and labels as in (A). Structure 2017 25, 94-106DOI: (10.1016/j.str.2016.11.013) Copyright © 2016 Elsevier Ltd Terms and Conditions