Membrane Alignment of the Pore-Forming Component TatA_d of the Twin-Arginine Translocase from Bacillus subtilis Resolved by Solid-State NMR Spectroscopy

Abstract

The twin-arginine translocase (Tat) provides protein export in bacteria and plant chloroplasts and is capable of transporting fully folded proteins across the membrane. We resolved the conformation and membrane alignment of the pore-forming subunit TatA(d) from Bacillus subtilis using solid-state NMR spectroscopy. The relevant structured part of the protein, TatA(2-45), contains a transmembrane segment (TMS) and an amphiphilic helix (APH). It was reconstituted in planar bicelles, which represent the lipid environment of a bacterial membrane. The SAMMY solid-state NMR experiment was used to correlate (15)N chemical shifts and (1)H-(15)N dipolar couplings in the backbone and side chains of the (15)N-labeled protein. The observed wheel-like patterns ("PISA wheels") in the resulting 2-dimensional spectra confirm the α-helical character of the two segments and reveal their alignment in the lipid bilayer. Helix tilt angles (τ(TMS) = 13°, τ(APH) = 64°) were obtained from uniformly labeled protein, and azimuthal rotations (ρ(Val15) = 235°, ρ(Ile29) = 25°) were obtained from selective labels. These constraints define two distinct families of allowed structures for TatA in the membrane-bound state. The manifold of solutions could be narrowed down to a unique structure by using input from a liquid-state NMR study of TatA in detergent micelles, as recently described [Hu, Y.; Zhao, E.; Li, H.; Xia, B.; Jin, C. J. Am. Chem. Soc. 2010, DOI: 10.1021/ja1053785]. Interestingly, the APH showed an unexpectedly slanted alignment in the protein, different from that of the isolated APH peptide. This finding implies that the amphiphilic region of TatA is not just a flexible attachment to the transmembrane anchor but might be able to form intra- or even intermolecular salt-bridges, which could play a key role in pore assembly.

Salt Sensitivity of Minimal Twin Arginine Translocases

“…Similarly, the apparent NaCl requirement for optimal activity of TatAyCy translocases containing the F19A-mutated TatAy protein could reflect the need for particular electrostatic interactions during TatAyCy-dependent protein translocation. Our present data would thus represent the very first experimental support for the hypothesis of Walther et al (56) that the amphiphilic region of TatA could form intra-or even intermolecular salt bridges with potentially important roles in pore assembly (56). It is presently not clear why some wild-type translocases (such as TatAC of B. cereus and S. aureus) are salt-sensitive, whereas other wild-type translocases (such as TatAyCy of B. subtilis and TatAC of L. monocytogenes) do not share this property.…”

“…1C and Refs. [55][56][57][58]. Furthermore, functional analyses suggested that the conserved hinge region connecting both helices might be of structural importance for TatAd (50).…”

The TatA component of the twin-arginine translocation system locally weakens the cytoplasmic membrane of Escherichia coli upon protein substrate binding

“…NMR studies (32) and biochemical accessibility studies (33) already indicated that the APH of TatA can adopt a tilted orientation at the membrane surface. The APH could directly contribute to membrane thickness at the membrane-anchor sites.…”

“…Accessibility of this segment in any orientation to the aqueous surface in the course of substrateinduced conformational changes should thus become detectable. Dyes at positions 22,23,26,27,28,32,33,34,35,42, and 45 cover the region from the hinge to the APH and an adjacent conserved negatively charged stretch. Position 51 served as positive control for quenching, as it is already positioned in an exposed region that is not anymore required for functionality (25).…”

Structure of TatA Paralog, TatE, Suggests a Structurally Homogeneous Form of Tat Protein Translocase That Transports Folded Proteins of Differing Diameter

“…1B. In general, the structure of TatE resembles those of E. coli TatA and Bacillus subtilis TatAd, which have been studied by NMR (30,31).…”