The Hydrophobic Core of Twin-Arginine Signal Sequences Orchestrates Specific Binding to Tat-Pathway Related Chaperones Anitha Shanmugham¤a, Adil Bakayan¤b, Petra Vo¨ ller¤c, Joost Grosveld, Holger Lill, Yves J. M. Bollen* Department of Molecular Cell Biology, VU University Amsterdam, Amsterdam, The Netherlands Abstract Redox enzyme maturation proteins (REMPs) bind pre-proteins destined for translocation across the bacterial cytoplasmic membrane via the twin-arginine translocation system and enable the enzymatic incorporation of complex cofactors. Most REMPs recognize one specific pre-protein. The recognition site usually resides in the N-terminal signal sequence. REMP binding protects signal peptides against degradation by proteases. REMPs are also believed to prevent binding of immature pre-proteins to the translocon. The main aim of this work was to better understand the interaction between REMPs and substrate signal sequences. Two REMPs were investigated: DmsD (specific for dimethylsulfoxide reductase, DmsA) and TorD (specific for trimethylamine N-oxide reductase, TorA). Green fluorescent protein (GFP) was genetically fused behind the signal sequences of TorA and DmsA. This ensures native behavior of the respective signal sequence and excludes any effects mediated by the mature domain of the pre-protein. Surface plasmon resonance analysis revealed that these chimeric pre- proteins specifically bind to the cognate REMP. Furthermore, the region of the signal sequence that is responsible for specific binding to the corresponding REMP was identified by creating region-swapped chimeric signal sequences, containing parts of both the TorA and DmsA signal sequences. Surprisingly, specificity is not encoded in the highly variable positively charged N-terminal region of the signal sequence, but in the more similar hydrophobic C-terminal parts. Interestingly, binding of DmsD to its model substrate reduced membrane binding of the pre-protein. This property could link REMP-signal peptide binding to its reported proofreading function. Citation: Shanmugham A, Bakayan A, Vo¨ ller P, Grosveld J, Lill H, et al. (2012) The Hydrophobic Core of Twin-Arginine Signal Sequences Orchestrates Specific Binding to Tat-Pathway Related Chaperones. PLoS ONE 7(3): e34159. doi:10.1371/journal.pone.0034159 Editor: Paul C. Driscoll, MRC National Institute for Medical Research, United Kingdom Received June 30, 2011; Accepted February 27, 2012; Published March 30, 2012 Copyright: 2012 Shanmugham et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. * E-mail: yves.bollen@vu.nl ¤a Current address: Department of Chemistry and Pharmaceutical Sciences, VU University Amsterdam, Amsterdam, The Netherlands ¤b Current address: Departamento de Fisiolog´ıa Humana, Universidad de Castilla-La Mancha, Albacete, Spain ¤c Current address: Helmholtz Center for Infection Research, Braunschweig, Germany Introduction Twin-arginine transport (Tat) systems are present in plant chloroplasts and many prokaryotes. The Escherichia coli Tat system was first identified as a transporter of extracellular redox enzymes that require cofactor insertion and assembly in the cytoplasm prior to transport [1]. Later, a broader range of proteins was added to the list of E. coli Tat substrates. The E. coli Tat translocon consists of the proteins TatA/TatE, TatB, and TatC [2]. There is currently no explicit evidence for additional proteins other than TatA/EBC essential for transport. However, there are instances where cytoplasmic chaperones play a role along the Tat pathway, presumably assisting the folding of substrates (for a review see [3]). Recent evidence demonstrates that E. coli DnaK is directly involved in the Tat pathway [4,5] but the involvement of other general E. coli chaperones, e.g. GroEL and SlyD, remains obscure owing to their largely overlapping functions. In addition to general chaperones, a class of substrate-specific redox enzyme maturation proteins (REMPs) has been identified (reviewed in [6]). REMPs assist in the intracellular incorporation of complex cofactors into extracellular redox enzymes prior to Tat- dependent translocation. Usually, each REMP recognizes a single specific substrate protein. Several cognate REMP-substrate pairs have been identified. The two best-characterized REMPs are DmsD and TorD. TorD binds to trimethylamine N-oxide reductase (TorA) and assists in the incorporation of the molybdo-bis(molyb- dopterin guanine dinucleotide) cofactor. DmsD binds to dimethyl- sulfoxide reductase (DmsA) as well as to two homologous proteins YnfE and YnfF, and keeps these proteins in a conformation that allows incorporation of the same molybdo-bis(molybdopterin guanine dinucleotide) cofactor by other proteins. Similar to substrates of the ubiquitous general secretory (Sec) pathway, Tat substrates bear a transport-essential N-terminal signal sequence that consists of a positively charged N-region, a hydrophobic H-region and a short polar C-region. However Tat signal sequences bear several additional features [7]: the N-region is usually longer than is the case for Sec substrates; a consensus S/ TRRxFLK motif containing the highly conserved name-giving twin-arginine pair is found at the boundary between N- and H- regions; the H-region is less hydrophobic than is the case for Sec signal peptides; and finally the C-region usually contains at least one positively charged amino acid residue, sometimes referred to as the ‘‘Sec-avoidance motif’’ [8]. PLoS ONE | www.plosone.org 1 March 2012 | Volume 7 | Issue 3 | e34159