TY - JOUR
T1 - Ligand-induced conformational changes in the Bacillus subtilis chemoreceptor McpB determined by disulfide crosslinking in vivo
AU - Szurmant, Hendrik
AU - Bunn, Michael W.
AU - Cho, Stephen H.
AU - Ordal, George W.
N1 - Funding Information:
We thank the rest of the Ordal lab for intellectual contributions to this work. The National Institutes of Health grant RO1 GM54365 supported this research.
PY - 2004/12/3
Y1 - 2004/12/3
N2 - Previously, we characterized the organization of the transmembrane (TM) domain of the Bacillus subtilis chemoreceptor McpB using disulfide crosslinking. Cysteine residues were engineered into serial positions along the two helices through the membrane, TM1 and TM2, as well as double mutants in TM1 and TM2, and the extent of crosslinking determined to characterize the organization of the TM domain. In this study, the organization of the TM domain was studied in the presence and absence of ligand to address what ligand-induced structural changes occur. We found that asparagine caused changes in crosslinking rate on all residues along the TM1-TM1′ helical interface, whereas the crosslinking rate for almost all residues along the TM2-TM2′ interface did not change. These results indicated that helix TM1 rotated counterclockwise and that TM2 did not move in respect to TM2′ in the dimer on binding asparagine. Interestingly, intramolecular crosslinking of paired substitutions in 34/280 and 38/273 were unaffected by asparagine, demonstrating that attractant binding to McpB did not induce a "piston-like" vertical displacement of TM2 as seen for Trg and Tar in Escherichia coli. However, these paired substitutions produced oligomeric forms of receptor in response to ligand. This must be due to a shift of the interface between different receptor dimers, within previously suggested trimers of dimers, or even higher order complexes. Furthermore, the extent of disulfide bond formation in the presence of asparagine was unaffected by the presence of the methyl-modification enzymes, CheB and CheR, or the coupling proteins, CheW and CheV, demonstrating that these proteins must have local structural effects on the cytoplasmic domain that is not translated to the entire receptor. Finally, disulfide bond formation was also unaffected by binding proline to McpC. We conclude that ligand-binding induced a conformational change in the TM domain of McpB dimers as an excitation signal that is likely propagated within the cytoplasmic region of receptors and that subsequent adaptational events do not affect this new TM domain conformation.
AB - Previously, we characterized the organization of the transmembrane (TM) domain of the Bacillus subtilis chemoreceptor McpB using disulfide crosslinking. Cysteine residues were engineered into serial positions along the two helices through the membrane, TM1 and TM2, as well as double mutants in TM1 and TM2, and the extent of crosslinking determined to characterize the organization of the TM domain. In this study, the organization of the TM domain was studied in the presence and absence of ligand to address what ligand-induced structural changes occur. We found that asparagine caused changes in crosslinking rate on all residues along the TM1-TM1′ helical interface, whereas the crosslinking rate for almost all residues along the TM2-TM2′ interface did not change. These results indicated that helix TM1 rotated counterclockwise and that TM2 did not move in respect to TM2′ in the dimer on binding asparagine. Interestingly, intramolecular crosslinking of paired substitutions in 34/280 and 38/273 were unaffected by asparagine, demonstrating that attractant binding to McpB did not induce a "piston-like" vertical displacement of TM2 as seen for Trg and Tar in Escherichia coli. However, these paired substitutions produced oligomeric forms of receptor in response to ligand. This must be due to a shift of the interface between different receptor dimers, within previously suggested trimers of dimers, or even higher order complexes. Furthermore, the extent of disulfide bond formation in the presence of asparagine was unaffected by the presence of the methyl-modification enzymes, CheB and CheR, or the coupling proteins, CheW and CheV, demonstrating that these proteins must have local structural effects on the cytoplasmic domain that is not translated to the entire receptor. Finally, disulfide bond formation was also unaffected by binding proline to McpC. We conclude that ligand-binding induced a conformational change in the TM domain of McpB dimers as an excitation signal that is likely propagated within the cytoplasmic region of receptors and that subsequent adaptational events do not affect this new TM domain conformation.
KW - chemotaxis
KW - conformational change
KW - disulfide crosslinking
KW - receptor
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U2 - 10.1016/j.jmb.2004.09.093
DO - 10.1016/j.jmb.2004.09.093
M3 - Article
C2 - 15544802
AN - SCOPUS:8544279966
SN - 0022-2836
VL - 344
SP - 919
EP - 928
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 4
ER -