Molecular Dynamic Studies on the Interaction of a TatA Oligomer with Tat Translocon Substrates
The Tat Translocon directly utilizes the Proton Motive Force to transport folded proteins from the n-side to the p-side of energized membranes, targeting the thylakoid lumen of chloroplasts and the periplasmic space of Bacteria and Archaea. In most organisms the Translocon consists of three subunits, TatA, TatB and TatC exhibiting a stoichiometry of [~]20-50/1/1. While TatB/TatC recognize the canonical twin-arginine motif-containing signal sequence of substrate proteins, TatA has been hypothesized to interact with TatB/TatC and translocon substrates facilitating their transport across the membrane. TatA from E.coli contains a short transmembrane helix near the N-terminus, a longer amphipathic helix and a relatively large unstructured C-terminal domain. While the transmembrane and amphipathic helixes are required for Translocon activity, the C-terminal domain is, in large measure, dispensable. TatA has been hypothesized to form higher-order oligomers in the biological membranes. In this communication we have used 1000 ns-long course-grained molecular dynamic simulations to examine the interactions between a membrane-associated E. coli TatA nonamer, alone, and in association with two Tat Translocon substrate proteins, either OEE17 or TorA. In all simulations, either in the presence or absence of substrate, the TatA nonamer markedly thinned the lipid bilayer which may facilitate substrate translocation. The pore of the nonamer was occupied by a phospholipid layer consisting of [~]6 phospholipids in the absence of substrates and [~]11 phospholipids in their presence. Structurally, the amphipathic helix of TatA were observed to exhibit significant conformational flexibility which appears to facilitate TatA-substrate interactions. In the absence of substrate the TatA nonamer was unstable with its radial architecture collapsing in 200-300 ns. In the presence of substrate, however, the radial geometry of the nonamer persists for at least 1000 ns. Interestingly, in the presence of the smaller substrate OEE17, fewer TatA monomers are retained in a radial geometry then observed in the presence of the larger substrate TorA indicating that the molecularity of the TatA oligomer can adjust to the size of the substrate. Specific hydrophilic residues of the TatA amphipathic helixes were found to interact with both substrate molecules, and these form quite stable charge-pair or hydrogen-bonding interactions. While the substrate proteins were initially placed adjacent to the amphipathic helixes of the nonamer, during the simulation trajectories the substrates moved to a more central position adjacent to, and partially entering, the oligomer pore. Concomitantly, the oligomer was observed to lose phospholipids. These latter observations may constitute a glimpse of the initial stages of protein translocation.