title: Structural analysis of substrate binding by the TatBC component of the twin-arginine protein transport system.
abstract: The Tat system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. In Escherichia coli substrate proteins initially bind to the integral membrane TatBC complex which then recruits the protein TatA to effect translocation. Overproduction of TatBC and the substrate protein SufI in the absence of TatA led to the accumulation of TatBC-SufI complexes that could be purified using an affinity tag on the substrate. Three-dimensional structures of the TatBC-SufI complexes and unliganded TatBC were obtained by single-particle electron microscopy and random conical tilt reconstruction. Comparison of the structures shows that substrate molecules bind on the periphery of the TatBC complex and that substrate binding causes a significant reduction in diameter of the TatBC part of the complex. Although the TatBC complex contains multiple copies of the signal peptide-binding TatC protomer, purified TatBC-SufI complexes contain only 1 or 2 SufI molecules. Where 2 substrates are present in the TatBC-SufI complex, they are bound at adjacent sites. These observations imply that only certain TatC protomers within the complex interact with substrate or that there is a negative cooperativity of substrate binding. Similar TatBC-substrate complexes can be generated by an alternative in vitro reconstitution method and using a different substrate protein.
authors: Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.
authors: Michael J
authors: MJ
authors: Tarry
authors: Eva
authors: E
authors: Schäfer
authors: Shuyun
authors: S
authors: Chen
authors: Grant
authors: G
authors: Buchanan
authors: Nicholas P
authors: NP
authors: Greene
authors: Susan M
authors: SM
authors: Lea
authors: Tracy
authors: T
authors: Palmer
authors: Helen R
authors: HR
authors: Saibil
authors: Ben C
authors: BC
authors: Berks
mesh_terms: D001120:Arginine / Q000378:metabolism*; D001692:Biological Transport; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D008958:Models, Molecular; D010088:Oxidoreductases / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; C473632:SufI protein, E coli; C473116:TatB protein, E coli; C461379:TatC protein, E coli; D001120:Arginine; D010088:Oxidoreductases; C499679:cueO protein, E coli