Evidence for conformational changes within DsbD: possible role for membrane-embedded proline residues - PubMed (original) (raw)

Evidence for conformational changes within DsbD: possible role for membrane-embedded proline residues

Annie Hiniker et al. J Bacteriol. 2006 Oct.

Abstract

The mechanism by which DsbD transports electrons across the cytoplasmic membrane is unknown. Here we provide evidence that DsbD's conformation depends on its oxidation state. Our data also suggest that four highly conserved prolines surrounding DsbD's membrane-embedded catalytic cysteines may have an important functional role, possibly conferring conformational flexibility to DsbD.

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Figures

FIG. 1.

FIG. 1.

Protease sensitivities of reduced (lanes R) and oxidized (lanes O) DsbD. A. Digestion pattern of a DsbD construct presenting two thrombin sites inserted between α and β and between β and γ. After reduction of the protein with dithiothreitol, followed by gel filtration to remove the reductant, DsbD is cut into three polypeptides by thrombin (α and γ, which have the same molecular mass and are therefore visible as a single band of 15 kDa [marked α/γ], and β) (3). B. A significant amount of reduced DsbD (asterisk) is still uncut after a 10-min incubation at pH 7.8 with endoGlu-C, a protease which cleaves after glutamate residues under these conditions. There are 15 glutamate residues in DsbD.

FIG. 2.

FIG. 2.

Multiple-sequence alignments of DsbDβ. The sequences of transmembrane segments 1 and 4 of E. coli DsbD (as predicted previously [5]) were aligned with the corresponding segments of DsbD homologues from Shewanella baltica, Idiomarina loihiensis, Methylobacillus flagellatus, Chlorobium tepidum, Campylobacter lari, Desulfovibrio desulfuricans, Chlamydophila abortus, Photobacterium profundum, and Rhodospirillum rubrum by using ClustalW. The conserved residues are shaded, an asterisk indicates that the residues in that column are identical in all sequences aligned, a colon indicates that conserved substitutions are present, and a period indicates that semiconserved substitutions are present.

FIG. 3.

FIG. 3.

A. Spot titers of wild-type (wt) and mutant DsbDs on copper plates. Strains were grown on plates containing copper (6 mM), ampicillin (200 μg/ml), and 40 μM IPTG to induce expression. A dsbA dsbD double mutant was used to express DsbD variants Pro289A, Pro284A, Pro166A, and Pro162A and wild-type DsbD from pTrc. B. Expression levels of the mutants and wild-type DsbD. BL21 cells expressing wild-type DsbD and variants were grown in LB, and protein expression was induced with IPTG. After a 4-h induction, cells were collected. Membrane pellets were prepared, and proteins were solubilized in 1% Triton. Expression levels were assessed by Western blot analysis using an anti-His tag antibody. Lanes: 1, wild type; 2, P162A, 3, P166A; 4, P284A; 5, P289A.

FIG. 4.

FIG. 4.

Redox states of wild-type DsbD* and variants. Cells expressing wild-type DsbD* and variants were grown in LB, and protein expression was induced with IPTG. After a 4-h induction, cells were collected and disrupted and membrane pellets were prepared. Proteins were solubilized in 1% Triton and free thiols were alkylated with MalPEG as described previously (10). Proteins were separated by SDS-polyacrylamide gel electrophoresis and visualized by Western blotting using anti-His tag antibodies. Oxidized protein (bands 1 and 2) and reduced protein (bands 3 and 4) are indicated. DTT, dithiothreitol.

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