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

Abstract

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 pre-proteins across the plasma membrane, powered by ATP hydrolysis and the trans-membrane proton-motive-force (PMF). The activities of SecYEG and SecA are modulated by membrane lipids, particularly by cardiolipin, a specialised phospholipid known to associate with a range of energy-transducing machines. Here, we identify two specific cardiolipin 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 and biochemical analysis of Escherichia coli SecYEG. The results show that the two sites account for the preponderance of functional cardiolipin binding to SecYEG, and mediate its roles in ATPase and protein transport activity.In addition, we demonstrate an important role for cardiolipin in the conferral of PMFstimulation 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 an as yet unknown mechanism. This study demonstrates the power of coupling the high predictive ability of coarse-grained simulation with experimental analyses, towards investigation of both the nature and functional implications of protein-lipid interactions. Significance StatementMany proteins are located in lipid membranes surrounding cells and cellular organelles. The membrane can impart important structural and functional effects on the protein, making understanding of this interaction critical. Here, we apply computational simulation to the identification of conserved lipid binding sites on an important highly conserved bacterial membrane protein, the Sec translocase (SecA-SecYEG), which uses ATP and the proton motive force (PMF) to secrete proteins across the bacterial plasma membrane. We experimentally validate and reveal the conserved nature of these binding sites, and use functional analyses to investigate the biological significance of this interaction. We demonstrate that these interactions are specific, transient, and critical for both ATP-and PMF-driven protein secretion.

Interactions of a bacterial RND transporter with a transmembrane small protein in a lipid environment

“…Cardiolipin thus impacts processes ranging from electron transport to antimicrobial resistance by affecting protein localization, enhancing protein stability, mediating interactions between monomer units, and transmitting conformation changes between subunits (Dudek, 2017). Additionally, recent work has found that cardiolipin plays a role in proton motif force stimulation and modulation of ATPase activity in SecYEG (Corey et al, 2018). Cardiolipin promotes the distribution of the osmosensory transporter ProP to the cellular poles in E. coli (Romantsov et al, 2007).…”

Rate-limiting transport of positive charges through the Sec-machinery is integral to the mechanism of protein transport

“…When a proton is removed from the pre-protein at the cytosolic face of SecY it must be dispersed rapidly, or a sudden the drop in local pH would immediately reprotonate the lysine. We have recently identified two specific binding sites for cardiolipin (CL) near to the pKa-perturbed area, which are required for PMF stimulation of transport 35 (Fig. S3).…”

“…S3). CL is thought to buffer pH locally 36 , and simulations suggest that it is bound and released from SecYEG-SecA on the µs timescale 35 . Therefore, it seems plausible that protons extracted from lysine are transferred to CL for rapid dissipation (Fig.…”

“…Transport reaction components were produced as described previously 27 . To make inverted membrane vesicles (IMVs) at different pHs, a standard protocol was followed 35 coli strain BL21(DE3), which produces a PMF in the presence of ATP, or HB1(DE3), which lack ATP synthase, and therefore does not 43 .…”

“…Genes encoding translocation substrates were purchased from GeneArt (Thermo Fisher Scientific), cloned between the NcoI and HindIII sites of pBAD/Myc-HisC, then expressed and purified as described 35 . A complete list of protein sequences is shown in Table S1.…”