Specific cardiolipin-SecY interactions are required for proton-motive force stimulation of protein secretion

The transport of proteins across or into membranes is a vital biological process, achieved in every cell by the conserved Sec machinery. In bacteria, SecYEG combines with the SecA motor protein for secretion of preproteins across the plasma membrane, powered by ATP hydrolysis and the transmembrane proton-motive force (PMF). The activities of SecYEG and SecA are modulated by membrane lipids, particularly cardiolipin (CL), a specialized phospholipid known to associate with a range of energy-transducing machines. Here, we identify two specific CL binding sites on the Thermotoga maritima SecA-SecYEG complex, through application of coarse-grained molecular dynamics simulations. We validate the computational data and demonstrate the conserved nature of the binding sites using in vitro mutagenesis, native mass spectrometry, biochemical analysis, and fluorescence spectroscopy of Escherichia coli SecYEG. The results show that the two sites account for the preponderance of functional CL binding to SecYEG, and mediate its roles in ATPase and protein transport activity. In addition, we demonstrate an important role for CL in the conferral of PMF stimulation of protein transport. The apparent transient nature of the CL interaction might facilitate proton exchange with the Sec machinery, and thereby stimulate protein transport, by a hitherto unexplored mechanism. This study demonstrates the power of coupling the high predictive ability of coarse-grained simulation with experimental analyses, toward investigation of both the nature and functional implications of protein-lipid interactions.ACKNOWLEDGMENTS: We thank Audrey Solgadi for mass spectrometry analysis at L’Institut Paris Saclay d’Innovation Thérapeutique (IPSIT) and Mikhail Bogdanov for fruitful discussions. This work was funded by the Biotechnology and Biological Sciences Research Council (Grants BB/M003604/1, BB/I008675/1, and BB/N015126/1) and the Wellcome Trust (Grants 104632 and 109854/Z/15/Z). The mass spectrometry work was supported by the Centre National de la Recherche Scientifique, INSERM (Grant “DYNAMO,” ANR-11- LABEX-0011-01 to M.C.) and the Région Ile de France for cofunding the Le Service d’Analyse des Médicaments et Métabolites Mass Spectrometry Facility at IPSIT. E.P. is the recipient of an Imperial College London Institute of Chemical Biology Engineering and Physical Sciences Research Council (EPSRC) Centre for Doctoral Training studentship. This work was carried out using the computational facilities of the Advanced Computing Research Centre, University of Bristol. Additional simulations were carried out using Edinburgh ParallelComputing Centre’s Cirrus and the ARCHER UK National Supercomputing Service (www.archer.ac.uk), provided by HECBioSim, supported by the EPSRC