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

2010; American Chemical Society; Volume: 132; Issue: 45 Linguagem: Inglês

10.1021/ja106963s

1943-2984

Torsten H. Walther, Stephan L. Grage, Nadine Roth, Anne S. Ulrich,

Protein Structure and Dynamics

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 TatAd from Bacillus subtilis using solid-state NMR spectroscopy. The relevant structured part of the protein, TatA2−45, 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 15N chemical shifts and 1H−15N dipolar couplings in the backbone and side chains of the 15N-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 (τTMS = 13°, τAPH = 64°) were obtained from uniformly labeled protein, and azimuthal rotations (ρVal15 = 235°, ρIle29 = 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.