Signalling substitutions in the periplasmic domain of chemoreceptor Trg induce or reduce helical sliding in the transmembrane domain.
We used in vivo oxidative cross-linking of engineered cysteine pairs to assess conformational changes in the four-helix transmembrane domain of chemoreceptor Trg. Extending previous work, we searched for and found a fourth cross-linking pair that spanned the intrasubunit interface between transmembrane helix 1 (TM1) and its partner TM2. We determined the effects of ligand occupancy on cross-linking rate constants for all four TM1-TM2 diagnostic pairs in conditions that allowed the formation of receptor-kinase complexes for the entire cellular complement of Trg. Occupancy altered all four rates in a pattern that implicated sliding of TM2 relative to TM1 towards the cytoplasm as the transmembrane signalling movement in receptor-kinase complexes. Transmembrane signalling can be reduced or induced by single amino acid substitutions in the ligand-binding region of the periplasmic domain of Trg. We determined the effects of these substitutions on conformation in the transmembrane domain and on ligand-induced changes using the diagnostic TM1-TM2 cysteine pairs. Effects on rates of in vivo cross-linking showed that induced signalling substitutions altered the relative positions of TM1 and TM2 in the same way as ligand binding, and reduced signalling substitutions blocked or attenuated the ligand-induced shift. These results provide strong support for the helical sliding model of transmembrane signalling.
Beel
B D
BD
School of Molecular Biosciences, Washington State University, Pullman, WA 99164-4660, USA.
Hazelbauer
G L
GL
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002633:Chemotaxis; D003432:Cross-Linking Reagents / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D029968:Escherichia coli Proteins; D008565:Membrane Proteins / Q000378:metabolism; D000072236:Methyl-Accepting Chemotaxis Proteins; D010084:Oxidation-Reduction; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction
D001426:Bacterial Proteins; C489103:CheW protein, E coli; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; D011956:Receptors, Cell Surface; C041476:Trg protein, E coli; C051236:CheW protein, Bacteria; D003545:Cysteine