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. 2013 May 27;425(10):1670-82.
doi: 10.1016/j.jmb.2013.02.009. Epub 2013 Feb 14.

Structure of the Disulfide Bond Generating Membrane Protein DsbB in the Lipid Bilayer

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Free PMC article

Structure of the Disulfide Bond Generating Membrane Protein DsbB in the Lipid Bilayer

Ming Tang et al. J Mol Biol. .
Free PMC article

Abstract

The integral membrane protein DsbB in Escherichia coli is responsible for oxidizing the periplasmic protein DsbA, which forms disulfide bonds in substrate proteins. We have developed a high-resolution structural model by combining experimental X-ray and solid-state NMR with molecular dynamics (MD) simulations. We embedded the high-resolution DsbB structure, derived from the joint calculation with X-ray reflections and solid-state NMR restraints, into the lipid bilayer and performed MD simulations to provide a mechanistic view of DsbB function in the membrane. Further, we revealed the membrane topology of DsbB by selective proton spin diffusion experiments, which directly probe the correlations of DsbB with water and lipid acyl chains. NMR data also support the model of a flexible periplasmic loop and an interhelical hydrogen bond between Glu26 and Tyr153.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
SSNMR chemical shift assignments of DsbB (Cys41Ser). (a) Strip plot of NCACX (blue) and NCOCX (red) of DsbB (Cys41Ser) acquired at −12 °C, showing the residues at the active site of DsbB. (b) Topology view of DsbB with assigned residues highlighted in blue and cyan. Residues in blue are at the active site of DsbB. The four rectangles represent the transmembrane helices predicted by secondary structure analysis based on chemical shifts.
Figure 2
Figure 2
Structures of DsbB (Cys41Ser). (a) Overlay of ten lowest energy structures of DsbB (Cys41Ser) calculated with only X-ray reflections. (b) Overlay of ten lowest energy structures of DsbB (Cys41Ser) calculated with X-ray reflections and SSNMR restraints (PDB ID: 2LTQ). The colors represent the secondary structure elements (magenta: α-helix, cyan and white: coil and turn).
Figure 3
Figure 3
Site-specific lipid or water correlations to DsbB. (a) 13C-13C 2D correlation spectrum with proton spin diffusion from lipid acyl chains to DsbB, (b) 13C-13C 2D correlation spectrum with proton spin diffusion from water to DsbB and (c) 13C-13C 2D correlation spectrum of DsbB. (d) Overlay of expansions of Ala region in 2D spectra of lipid-DsbB (red), water-DsbB (blue) and DsbB (black).
Figure 4
Figure 4
Structural model of DsbB in the membrane. The structure of DsbB (purple) was calculated jointly with X-ray and SSNMR restraints, and then put into the lipid bilayers through solvation, energy minimization and equilibrium. The highlighted residues are those that correlate with water (blue) and lipids (orange), as observed in selective proton spin diffusion experiments. They are consistent with the structural model in terms of their proximity to water (cyan) and lipid (gray) molecules. Some lipid and water molecules are not displayed to reveal DsbB more clearly.
Figure 5
Figure 5
Glu26 and Tyr153 hydrogen bond in DsbB. (a) Overview of DsbB structural model from the membrane plane. The hydrogen bond between Glu26 and Tyr153, the ubiquinone cofactor (UQ) and the sidechains of the four significant Cys residues (yellow) are highlighted. The dashed line indicates the kink in TM1 due to Glu26 and Tyr153 hydrogen bond. (b) Overlay of DsbB models viewed from the periplasmic side. The structure from joint calculation (cyan) shows that Glu26 in TM1 and Tyr153 in TM4 are in close proximity. The model from MD (purple) is consistent with that Glu26 and Tyr153 are linked through hydrogen bonding. The crystal structure (orange) and solution NMR structure (green) have Glu26 relatively distant from Tyr153 (> 8 Å). (c) 13C-13C 2D correlation spectrum with 15N REDOR dephasing and 500 ms DARR mixing of [U-13C, 15N]DsbB(Cys41Ser), showing the crosspeaks between the sidechains of Glu26 and Tyr153.

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