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Solution structure and elevator mechanism of the membrane electron transporter CcdA

Abstract

Membrane oxidoreductase CcdA plays a central role in supplying reducing equivalents from the bacterial cytoplasm to the envelope. It transports electrons across the membrane using a single pair of cysteines by a mechanism that has not yet been elucidated. Here we report an NMR structure of the Thermus thermophilus CcdA (TtCcdA) in an oxidized and outward-facing state. CcdA consists of two inverted structural repeats of three transmembrane helices (2 × 3-TM). We computationally modeled and experimentally validated an inward-facing state, which suggests that CcdA uses an elevator-type movement to shuttle the reactive cysteines across the membrane. CcdA belongs to the LysE superfamily, and thus its structure may be relevant to other LysE clan transporters. Structure comparisons of CcdA, semiSWEET, Pnu, and major facilitator superfamily (MFS) transporters provide insights into membrane transporter architecture and mechanism.

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Fig. 1: CcdA reducing pathway and NMR spectra of TtCcdA.
Fig. 2: Structure of oxidized TtCcdA.
Fig. 3: Inward-facing model of oxidized TtCcdA.
Fig. 4: Elevator-type movement of the transport helices.
Fig. 5: Proposed reducing-equivalent transport mechanism of CcdA.
Fig. 6: Structural comparison of CcdA, semiSWEET, Pnu, and MFS.

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Acknowledgements

We thank J. F. Ellena for support and advice on NMR. We thank D. S. Cafiso for scientific discussions. This work was supported by grant R01GM078296 to J.H.B. from the National Institutes of Health.

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Contributions

Y.Z. and J.H.B. designed the research; Y.Z. performed the experiments and analyzed the data; Y.Z. and J.H.B. wrote the manuscript.

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Correspondence to Yunpeng Zhou or John H. Bushweller.

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Integrated supplementary information

Supplementary Figure 1 TtCcdA is monodisperse, active, and shows internal sequence repeats

a, Alignment of the internal sequence repeats of TtCcdA (30% identity and 57% similarity). The PCxxP motifs are colored yellow. b, Elution profiles of oxidized TtCcdA in 4.2 mM n-decyl-β-D-maltoside (DM) and 1.5 mM n-dodecyl phosphocholine (DPC) and Bio-Rad gel filtration standard run on S200 size exclusion column (GE Healthcare). Based on the protein standard, the estimated molecular weight of TtCcdA-detergent complex is 70 kDa. c, Time course of the thiol-disulfide exchange reactions between 50 μM reduced TtTrx and 10 μM oxidized TtCcdA in DM and in DPC.

Supplementary Figure 2 Structural restraints, calculation, and validation

a, Paramagnetic relaxation enhancement restraints were recorded as the signal intensity reduction comparing the nitroxide spin labeled paramagnetic sample (right, in this case label on V78C) and the ascorbic acid reduced diamagnetic sample (left). Residues in the red circles show significant signal intensity reductions due to line-broadening. b, Superimposition of the 10 lowest energy structures of oxidized TtCcdA. Green spheres show the locations of the 8 paramagnetic labels. c, 1DHN residual dipolar couplings from neutral gel (black) and positively charged gel (red) are plotted against the TtCcdA sequence. For a straight helix, residual dipolar couplings oscillate periodically in a certain range. The kinks and bends of the helices can be seen in the shift of the data range. d, The backbone RMSD vs. Xplor-NIH energy scatter plot of the fold and refine steps of structural calculation. RMSD is to the lowest energy structure of each calculation step. The green dots represent structures of the correct fold. The dark green dots represent the 10 lowest energy structures of the 200 structures calculated. The red dots represent structures of the mirror image fold. e, The structure of TtCcdA was determined using redundant restraints. Four structural ensembles (10 structures each) were calculated using partial long-range PRE and/or RDC restraints (see methods for the restraints used in each case). Each ensemble was compared with the reported representative structure. The Qfree factors were calculated using the withheld RDC data from the neutral gel. The mean and standard deviation of the pairwise RMSD and Qfree factors are shown as blue bars and red dots, respectively. The structures calculated using seven sets of PRE restraints without RDC restraints (PRE x 7, using ~70% the long-range restraints) are close to the reported structure. The structures calculated using six sets of PRE restraints without RDC restraints (PRE x 6, using ~60% the long-range restraints) could be separated into two clustered groups of equal populations. One has the correct fold and the other has the mirror image fold.

Supplementary Figure 3 Solvent accessibility, Gx6G motifs, dynamics, and membrane topology of TtCcdA

a, The ratio of peak intensity changes upon titration of 20 mM gadolinium paramagnetic relaxation reagent. b, Mapping the intensity change onto the structure of TtCcdA. Residues showing large intensity changes (Ipara/Idia < 0.2) are colored green. Residues showing small changes (Ipara/Idia > 0.5) are colored purple. c, The Gx6G motifs between TM3/TM6 and TM2/TM5. Cartoon representations of the antiparallel left-handed TM3/TM6 (left) and TM2/TM5 (right) pairs, viewed in the membrane from the outside of the protein. “G” is glycine or other small amino acids such as alanine or serine (red). They form complementary surfaces with the large hydrophobic residues (green) from the opposite TM helix. The Gx6G motifs form tight packing between TM helices. d, The random coil index derived order parameters (RCI-S2) are plotted against the TtCcdA sequence. Higher values up to 1 indicate restricted motion on the ns-ps time scale. e, TtCcdA assumes an Nout-Cout membrane topology. The residues of positive charge (arginine and lysine) and negative charge (aspartic acid and glutamic acid) are shown as blue and red sticks, respectively.

Supplementary Figure 4 The structure, sequence alignments, and mapped solvent accessibility of AfCcdA

a, Cartoon representations of AfCcdA (PDB 2N4X) as viewed in the membrane plane (left) and from the periplasmic side (right). The two sequence repeats, the non-repeating region, and the two PCxxP motifs are colored red/blue, white, and yellow, respectively. The TM helices are numbered and the horizontal helices are labeled h and h’. The yellow spheres show the locations that correspond to the two essential cysteines. b, The solvent accessibility profile of EcDsbD is mapped onto the structure of AfCcdA (green: solvent accessible, purple: solvent inaccessible). c, AfCcdA displays the internal sequence repeats (red versus blue, 23% identity, 55% similarity). d, Multiple sequence alignment of CcdA. The sequences are from Thermus thermophilus HB8 (YP_144675.1, wild-type TtCcdA in this study), Meiothermus rufus (WP_027881202.1), Marinithermus hydrothermalis (WP_013703256.1), Oceanithermus profundus (WP_013457257.1), Rhodobacter capsulatus (WP_013067872.1), and Archaeoglobus fulgidus DSM 4304 (AAB90190.1, wild-type AfCcdA). The conserved PCxxP, Gx6G and other glycine-containing motifs are colored yellow, cyan, and green, respectively. The six TM helices of AfCcdA are labeled using red letters.

Supplementary Figure 5 Sequence alignment for the repeat-swap homology modeling

a, Schematic of model and template alignment: TM 1-3 (blue) is modeled using the TM 4–6 (red) structure as template, L3 (grey) is modeled de novo, TM 4–6 is modeled using the TM 1–3 structure as template, and H1-H2 (grey) is modeled by itself. b, Model and template alignment used by the MODELLER program to generate the homology models. Blue and red indicate the repeats modeled by each other. Grey indicates the non-repeating sequences modeled using their own structures as templates. The rest are modeled de novo. Open boxes indicate the helices of the model and the template.

Supplementary Figure 6 TROSY-HSQC spectra of the mutants of oxidized TtCcdA

a, 0.27 mM F45C/A113C mutant. b, 0.27 mM F45C/A113C mutant with 0.30 mM HgCl2. c, Overlay of the two previous spectra plotted at the same contour level. d, G154A mutant.

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Supplementary Figures 1–6 and Supplementary Note

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Supplementary Dataset 1

Original gel image of Figure 3b showing the cross-linking of the TtCcdA F45C A113C mutant.

Supplementary Video 1

Video showing conformational change of CcdA going to and from the inward- and outward-facing states.

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Zhou, Y., Bushweller, J.H. Solution structure and elevator mechanism of the membrane electron transporter CcdA. Nat Struct Mol Biol 25, 163–169 (2018). https://doi.org/10.1038/s41594-018-0022-z

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