Cysteine-scanning mutagenesis and disulfide mapping studies of the conserved domain of the twin-arginine translocase TatB component.
The cytoplasmic membrane protein TatB is an essential component of the Escherichia coli twin-arginine (Tat) protein translocation pathway. Together with the TatC component it forms a complex that functions as a membrane receptor for substrate proteins. Structural predictions suggest that TatB is anchored to the membrane via an N-terminal transmembrane alpha-helix that precedes an amphipathic alpha-helical section of the protein. From truncation analysis it is known that both these regions of the protein are essential for function. Here we construct 31 unique cysteine substitutions in the first 42 residues of TatB. Each of the substitutions results in a TatB protein that is competent to support Tat-dependent protein translocation. Oxidant-induced disulfide cross-linking shows that both the N-terminal and amphipathic helices form contacts with at least one other TatB protomer. For the transmembrane helix these contacts are localized to one face of the helix. Molecular modeling and molecular dynamics simulations provide insight into the possible structural basis of the transmembrane helix interactions. Using variants with double cysteine substitutions in the transmembrane helix, we were able to detect cross-links between up to five TatB molecules. Protein purification showed that species containing at least four cross-linked TatB molecules are found in correctly assembled TatBC complexes. Our results suggest that the transmembrane helices of TatB protomers are in the center rather than the periphery of the TatBC complex.
Lee
Philip A
PA
Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, United Kingdom.
Orriss
George L
GL
Buchanan
Grant
G
Greene
Nicholas P
NP
Bond
Peter J
PJ
Punginelli
Claire
C
Jack
Rachael L
RL
Sansom
Mark S P
MS
Berks
Ben C
BC
Palmer
Tracy
T
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001120:Arginine / Q000378:metabolism*; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D003198:Computer Simulation; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000254:growth & development / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D033902:Periplasmic Binding Proteins / Q000235:genetics; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D021381:Protein Transport
D004220:Disulfides; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D033902:Periplasmic Binding Proteins; C473116:TatB protein, E coli; D001120:Arginine; D003545:Cysteine