Structural Investigations of an Amphipathic Region of the Twin-Arginine Translocase Tata Subunit

Abstract

The twin-arginine translocase (Tat) system is used by many bacteria and plants to move folded proteins across the cytoplasmic membrane. Tat substrates contain a signature S/TRRxFLK twin-arginine motif in their N-terminal sequence. In most bacteria, the translocon consists of the TatABC subunits where TatA is the postulated pore subunit through homo-oligomerization with other TatA protomers, whereas TatBC is the substrate-receptor complex. The predicted structure of TatA includes a transmembrane helix, an amphipathic helix and a potentially unstructured C-terminal region. Biochemical and structural investigations were targeted at a peptide which represents the amphipathic region consisting of residues 22 to 44 of TatA (TatAH2). The dual topology of the region corresponding to TatAH2 in TatA was previously shown to be dependent on the membrane potential (Chan et al. 2007 Biochemistry 46: 7396-404), and thus warranted further investigations on its role for protein translocation. NMR and CD spectroscopy of TatAH2 show that it adopts helical structure in a membrane mimetic environment, in comparison to the random coil structure in aqueous solution. Microcalorimetry studies also show that it interacts with DPPG lipid vesicles to affect the phase transition temperatures. The solution NMR structure of TatAH2 shows conformation flexibility of the peptide around the acidic Asp31 at the center of the helix, a residue potentially important for the function of the TatA pore. The C-terminal half (residue 32 onwards) is α-helical, whereas the N-terminal half (23 to 30) has helical-like structure, suggesting that TatAH2 does not form a ‘typical’α-helix.