Abstract
Electron paramagnetic resonance (EPR)-based hybrid experimental and computational approaches were applied to determine the structure of a full-length E. coli integral membrane sulfurtransferase, dimeric YgaP, and its structural and dynamic changes upon ligand binding. The solution NMR structures of the YgaP transmembrane domain (TMD) and cytosolic catalytic rhodanese domain were reported recently, but the tertiary fold of full-length YgaP was not yet available. Here, systematic site-specific EPR analysis defined a helix-loop-helix secondary structure of the YagP-TMD monomers using mobility, accessibility and membrane immersion measurements. The tertiary folds of dimeric YgaP-TMD and full-length YgaP in detergent micelles were determined through inter- and intra-monomer distance mapping and rigid-body computation. Further EPR analysis demonstrated the tight packing of the two YgaP second transmembrane helices upon binding of the catalytic product SCN−, which provides insight into the thiocyanate exportation mechanism of YgaP in the E. coli membrane.
Highlights
Membrane proteins play essential roles in material transportation, signal transduction, and energy metabolism across the cell membrane
The circular dichroism (CD) spectra of cysless variant (YgaP C64S&C159S) and spin-labeled variants are highly similar to the wild type protein in the 198, 208 and 223 nm region (Supplementary Figure 2), indicating that there are no significant structure perturbations caused by mutagenesis
The tertiary structure of dimeric full-length YgaP was still unknown in the nuclear magnetic resonance (NMR) studies reported recently[21], owing to the lack of long distance constrains between the transmembrane domain and cytosolic catalytic rhodanese domain
Summary
Membrane proteins play essential roles in material transportation, signal transduction, and energy metabolism across the cell membrane. In the report of Eichmann group, the tertiary fold of full-length YgaP is not available, due to the lack of long distance constraints between the rhodanese domain and the transmembrane domain, the structures of these two domains were both determined using solution NMR. Chemical shift perturbations of full-length YgaP with different concentrations of sodium thiosulfate (NaSSO4) were observed for the 15N-1H moieties of residues located at the second transmembrane helix, whereas no obvious chemical shift perturbations were observed in the YgaP rhodanese domain upon the addition of SCN− These observations indicated that the YgaP-TMD may participate in the exportation of SCN− after the cyanate detoxification process[21]. In conjunction with the solution NMR structure of the rhodanese domain determined in our laboratory and rigid-body computational methods, the dimeric full-length YgaP structural fold was achieved in DPC micelles using the EPR distance constraints. The CW-EPR mobility, accessibility and distance analyses of YgaP in the absence or presence of SCN− lead to a hypothesized transmembrane helical packing model of the dimeric full-length YgaP-TMD upon SCN− binding
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