Abstract:
:The Bacillus subtilis chemoreceptor McpB is a dimer of identical subunits containing two transmembrane (TM) segments (TM1, residues 17-34: TM2, residues 280-302) in each monomer with a 2-fold axis of symmetry. To study the organization of the TM domains, the wild-type receptor was mutated systematically at the membrane bilayer/extracytoplasmic interface with 15 single cysteine (Cys) substitutions in each of the two TM domains. Each single Cys substitution was capable of complementing a null allele in vivo, suggesting that no significant perturbation of the native tertiary or quaternary structure of the chemoreceptor was introduced by the mutations. On the basis of patterns of disulfide crosslinking between subunits of the dimeric receptor, an alpha-helical interface was identified between TM1 and TM1' (containing residues 32, 36, 39, and 43) and between TM2 and TM2' (containing residues 276, 277, 280, 283 and 286). Pairs of cysteine substitutions (positions 34/280 and 38/273) in TM1 and TM2 were used to further elucidate specific contacts within a monomer subunit, enabling a model to be constructed defining the organization of the TM domain. Crosslinking of residues that were 150-180 degrees removed from position 32 (positions 37, 41, and 44) suggested that the receptors may be organized as an array of trimers of dimers in vivo. All crosslinking was unaffected by deletion of cheB and cheR (loss of receptor demethylation/methylation enzymes) or by deletion of cheW and cheV (loss of proteins that couple receptors with the autophosphorylating kinase). These findings indicate that the organization of the transmembrane region and the stability of the quaternary complex of receptors are independent of covalent modifications of the cytoplasmic domain and conformations in the cytoplasmic domain induced by the coupling proteins.
journal_name
J Mol Bioljournal_title
Journal of molecular biologyauthors
Bunn MW,Ordal GWdoi
10.1016/s0022-2836(03)00834-9keywords:
subject
Has Abstractpub_date
2003-08-22 00:00:00pages
941-9issue
4eissn
0022-2836issn
1089-8638pii
S0022283603008349journal_volume
331pub_type
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