A Large Conformational Change Couples the ATP Binding Site of SecA to the SecY Protein Channel Alice Robson, Antonia E. G. Booth, Vicki A. M. Gold Anthony R. Clarke and Ian Collinson⁎ Department of Biochemistry, University of Bristol, University Walk, Bristol, BS8 1TD, UK Received 1 August 2007; received in revised form 26 September 2007; accepted 27 September 2007 Available online 4 October 2007 In bacteria, the SecYEG protein translocation complex employs the cytosolic ATPase SecA to couple the energy of ATP binding and hydrolysis to the mechanical force required to push polypeptides through the membrane. The molecular basis of this energy transducing reaction is not well understood. A peptide-binding array has been employed to identify sites on SecYEG that interact with SecA. These results along with fluorescence spectroscopy have been exploited to characterise a long-distance conformational change that connects the nucleotide-binding fold of SecA to the transmembrane polypeptide channel in SecY. These movements are driven by binding of non-hydrolysable ATP analogues to a monomer of SecA in association with the SecYEG complex. We also determine that interaction with SecYEG simultaneously decreases the affinity of SecA for ATP and inhibitory magne- sium, favouring a previously identified active state of the ATPase. Mutants of SecA capable of binding but not hydrolysing ATP do not elicit this con- formationally active state, implicating residues of the Walker B motif in the early chain of events that couple ATP binding to the mobility of the channel. © 2007 Elsevier Ltd. All rights reserved. Edited by I. B. Holland Keywords: ATPase; conformational changes; membrane transport; protein- translocation Introduction Proteins rely on specific translocation machines if they are to cross or enter the lipid bilayer. Protein secretion and membrane insertion usually occurs through the ubiquitous SecYEG/Sec61 protein con- ducting complex. 1 In bacteria, proteins can be trans- located during or after they have been fully trans- lated; in the latter case they are maintained in an unfolded conformation by the action of chaperones such as SecB. 2 A heterotrimeric membrane-bound complex SecYEG and a soluble protein factor SecA are necessary and sufficient for this reaction to proceed. 3 The free energy released during the ATP hydrolytic cycle by SecA is used to push the trans- locating polypeptide substrate through SecYEG. 4–6 Important advances in our knowledge have begun to provide clues about the mechanics of this com- plicated reaction. The high-resolution structure of an archael homologue of the channel revealed the site of the signal sequence binding pocket, the protein- conducting pore and the cytosolic loops responsible for the interaction with partner proteins. 7 Whilst the X-ray structure was crystallised in monomeric form, the SecYEG complex is known to form dimers in the membrane and when active. 8–11 Proteins are trans- located through one of the two protein channels that are formed by a dimer, one at the centre of each protomer. 7,12 Low-resolution structures of the active and inactive forms of the complex indicate that it must undergo large conformational changes during transport. 11 The SecA partner protein is a large soluble pro- tein of 102 kDa, which is primarily dimeric in solu- tion. 13–17 However, monomers have also been implicated as the active species. 12,18–21 Complexes containing either one or two SecA molecules bound to a SecYEG dimer, depending on the nucleotide present, have been observed by analytical ultracen- trifugation and blue native gel electrophoresis. 8,22 Several different high-resolution structures of SecA have been determined. Most of them are dimers 23–27 and one of them is a monomer. 28 Despite *Corresponding author. E-mail address: ian.collinson@bristol.ac.uk. Abbreviations used: NBF, nucleotide-binding fold; TM, trans-membrane helix; DDM, n-dodecyl-β-D-maltoside. doi:10.1016/j.jmb.2007.09.086 J. Mol. Biol. (2007) 374, 965–976 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2007 Elsevier Ltd. All rights reserved.