Two Cooperating Helices Constitute the Lipid-binding Domain of the Bacterial SRP Receptor David Braig 1 , Constance Bär 1 , Jörg-Oliver Thumfart 2 and Hans-Georg Koch 1 ⁎ 1 Institut für Biochemie und Molekularbiologie, ZBMZ, Stefan-Meier-Str. 17, Albert-Ludwigs-Universität Freiburg, D-79104 Freiburg, Germany 2 Physiologisches Institut, Hermann-Herder-Str. 7, Albert-Ludwigs-Universität Freiburg, D-79104 Freiburg, Germany Received 19 December 2008; received in revised form 2 April 2009; accepted 28 April 2009 Available online 3 May 2009 Protein targeting by the bacterial signal recognition particle requires the specific interaction of the signal recognition particle (SRP)–ribosome– nascent chain complex with FtsY, the bacterial SRP receptor. Although FtsY in Escherichia coli lacks a transmembrane domain, the membrane-bound FtsY displays many features of an integral membrane protein. Our data reveal that it is the cooperative action of two lipid-binding helices that allows this unusually strong membrane contact. Helix I comprises the first 14 amino acids of FtsY and the second is located at the interface between the A- and the N-domain of FtsY. We show by site-directed cross-linking and binding assays that both helices bind to negatively charged phospholipids, with a preference for phosphatidyl glycerol. Despite the strong lipid binding, helix I does not seem to be completely inserted into the lipid phase, but appears to be oriented parallel with the membrane surface. The two helices together with the connecting linker constitute an independently folded domain, which maintains its lipid binding even in the absence of the conserved NG-core of FtsY. In summary, our data reveal that the two consecutive lipid-binding helices of FtsY can provide a membrane contact that does not differ significantly in stability from that provided by a transmembrane domain. This explains why the bacterial SRP receptor does not require an integral β-subunit for membrane binding. © 2009 Elsevier Ltd. All rights reserved. Edited by W. Baumeister Keywords: FtsY; protein–lipid interaction; cotranslational protein targeting; phospholipids; amphipathic helices; signal recognition particle Introduction Integral membrane proteins account for approxi- mately 30% of the coding capacity of the genome of an average organism and they are critically impor- tant for many essential cell functions, such as energy transduction, signalling and cell division. In addition to this large number of permanently lipid-integrated proteins, several proteins have been shown to associate with the membrane only transiently via contact to integral membrane proteins or to the lipids directly. 1 Membrane association of these amphitropic proteins is usually reversible, and binding affinity is subject to regulation, resulting in a dual localization of these proteins to both the cytosol and the membrane. 2 Prototypes of these proteins in Escher- ichia coli are the cell division protein MinD, 3 and the bacterial signal recognition particle (SRP) receptor FtsY, 4,5 which both execute essential functions within the cell. FtsY has a key role in membrane protein targeting in bacteria because it provides the essential link between the soluble SRP–ribosome–nascent chain complex (SRP–RNC) and the membrane- bound SecYEG translocon. The interaction between the SRP–RNC and FtsY induces hydrolysis of GTP by both SRP and FtsY, 6,7 which subsequently leads to the dissociation of SRP from the RNC, thereby exposing the translocon binding site of the ribosome. 8 One puzzling feature of FtsY is that it does not contain a *Corresponding author. E-mail address: Hans-Georg.Koch@biochemie.uni-freiburg.de. Abbreviations used: CCT, CTP-phosphocholine cytidyltransferase; GMP-PNP, guanosine 5′ (β,γ-imido) triphosphate; INV, inner membrane vesicles; Mal-PEG, mono-methyl polyethylene glycol-5000 2-maleimidoethyl ether; m/z, mass over charge; P, pellet; pBpa, p-benzoyl-L- phenylalanine; PL, phospholipase; RNC, ribosome nascent chain complexes; S, supernatant; SRP, signal recognition particle; wt, wild type; PG, phosphatidylglycerol; CL, cardiolipin. doi:10.1016/j.jmb.2009.04.061 J. Mol. Biol. (2009) 390, 401–413 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2009 Elsevier Ltd. All rights reserved.