Structure and dynamics of plant TatA in micelles and lipid bilayers studied by solution NMR

Abstract

The twin-arginine translocase (Tat) transports folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of plant chloroplasts. In Gram-negative bacteria and chloroplasts, the translocon consists of three subunits, TatA, TatB, and TatC, of which TatA is responsible for the actual membrane translocation of the substrate. Herein we report on the structure, dynamics, and lipid interactions of a fully functional C-terminally truncated 'core TatA' from Arabidopsis thaliana using solution-state NMR. Our results show that TatA consists of a short N-terminal transmembrane helix (TMH), a short connecting linker (hinge) and a long region with propensity to form an amphiphilic helix (APH). The dynamics of TatA were characterized using N relaxation NMR in combination with model-free analysis. The TMH has order parameters characteristic of a well-structured helix, the hinge is somewhat less rigid, while the APH has lower order parameters indicating structural flexibility. The TMH is short with a surprisingly low protection from solvent, and only the first part of the APH is protected to some extent. In order to uncover possible differences in TatA's structure and dynamics in detergent compared to in a lipid bilayer, fast-tumbling bicelles and large unilamellar vesicles were used. Results indicate that the helicity of TatA increases in both the TMH and APH in the presence of lipids, and that the N-terminal part of the TMH is significantly more rigid. The results indicate that plant TatA has a significant structural plasticity and a capability to adapt to local environments.

Thylakoid‐integrated recombinant Hcf106 participates in the chloroplast twin arginine transport system

“…Hcf106 is structurally similar to Tha4 in that both contain an amino terminal transmembrane domain (TMD), followed by a hinge region, an amphipathic α‐helix (APH), and a loosely structured carboxyl terminus (C‐tail). Recently, the structures of Tha4 and Hcf106 bacterial homologs, TatA and TatB, have been solved which agree with previous predictions (Hu, Zhao, Li, Xia, & Jin, ; Zhang, Wang, Hu, & Jin, ) and even more recently the structure to Tha4 from Arabidopsis thaliana (Pettersson et al., ). Despite the sequence similarity with Tha4, the two proteins are not interchangeable and thus appear to have distinct functions (Dabney‐Smith, Mori, & Cline, ).…”

Functional analysis of the polar amino acid in the TatA transmembrane helix

“…Such a hinge forming function at the N-terminus of the TatA TMH is consistent with our hypothesis that a transition to an even shorter TMH might be beneficial for the Tat mechanism. In addition, according to the TatA NMR structure (Hu et al, 2010;Pettersson et al, 2018), this 4-residue motif presents in the first coil of the TMH. Ser6 and Gln8 orientate oppositely than Ile6 and Trp7, suggesting a weak local amphipathic topology.…”

“…The structures of the three individual Tat subunits have been determined (Hu et al, 2010; Pettersson et al, 2018; Rollauer et al, 2012; Zhang et al, 2014). Both TatA and TatB possess a short transmembrane helix (TMH) followed by a hinge region, one or more amphipathic helices (APH), and then a relatively unstructured C-terminus.…”

Hydrophobic mismatch is a key factor in protein transport on the Tat pathway

“…Finally, even though TatB and TatC are present in a 1:1 stoichiometry, TatA joins the complex in variable stoichiometries (Leake et al, 2008). The structures of the three Tat subunits have been reported (Hu et al, 2010;Pettersson et al, 2018;Rollauer et al, 2012;Zhang et al, 2014b). TatA and TatB share an overall "L-shape" conformation composed of an N-terminal undefined short region located in the periplasm in bacteria, a remarkably short transmembrane alpha helix (TMH), a hinge region followed by one or more amphipathic helices (APH), and an unstructured C-terminal tail.…”