Solution structure of the TatB component of the twin-arginine translocation system

Abstract

The twin-arginine protein transport (Tat) system translocates fully folded proteins across lipid membranes. In Escherichia coli, the Tat system comprises three essential components: TatA, TatB and TatC. The protein translocation process is proposed to initiate by signal peptide recognition and substrate binding to the TatBC complex. Upon formation of the TatBC-substrate protein complex, the TatA subunits are recruited and form the protein translocation pore. Experimental evidences suggest that TatB forms a tight complex with TatC at 1:1 molar ratio and the TatBC complex contains multiple copies of both proteins. Cross-linking experiments demonstrate that TatB functions in tetrameric units and interacts with both TatC and substrate proteins. However, structural information of the TatB protein is still lacking, and its functional mechanism remains elusive. Herein, we report the solution structure of TatB in DPC micelles determined by Nuclear Magnetic Resonance (NMR) spectroscopy. Overall, the structure shows an extended 'L-shape' conformation comprising four helices: a transmembrane helix (TMH) α1, an amphipathic helix (APH) α2, and two solvent exposed helices α3 and α4. The packing of TMH and APH is relatively rigid, whereas helices α3 and α4 display notably higher mobility. The observed floppiness of helices α3 and α4 allows TatB to sample a large conformational space, thus providing high structural plasticity to interact with substrate proteins of different sizes and shapes.

Plant mitochondria contain the protein translocase subunits TatB and TatC

“…EcTatB contains four alpha helices that adopt an L-shape conformation ( Fig. 3B) (Zhang et al, 2014c). The model we obtained for At5g43680 has an almost identical structure containing four alpha helices in an L-shape conformation, further supporting the phylogenetic analysis that it is a TatB-like protein.…”

“…This was carried out using Phyre2 in the intensive mode, which returned a model fitting the identified structure of EcTatB (Fig. 3B) (Zhang et al, 2014c;Kelley et al, 2015). The model is based on the first 104 amino acids of EcTatB and the first 125 amino acids of At5g43680 as the Ctermini of both proteins are thought to be unstructured.…”

“…This utilizes the known structures of proteins to search genomes for similar proteins. So we used the known structures of the E. coli TatA (PDB: 2NM7) (Zhang et al, 2014b) and TatB (2MI2) (Zhang et al, 2014c) (hereafter EcTatA and EcTatB) to search the Arabidopsis nuclear genome (Table S1). Whereas both the known cpTatA and cpTatB proteins were identified with high confidence scores (Phyre2 confidence scores are a representation of the probability that the match between the sequence and the template is a true homology) a third unknown protein was also identified with similar confidence scores with the locus At5g43680 (Table S1).…”

Investigating the role of GXXXG motifs in helical folding and self-association of plasticins, Gly/Leu-rich antimicrobial peptides

“…However, this technique may be inefficient to detect the formation of transient oligomers as such weak non-covalent interactions might be disrupted by SDS detergent [53]. To test this hypothesis, peptide solutions were also preincubated with glutaraldehyde as a cross-linking agent to stabilize putative formed oligomers through covalent bonding [53,54]. Under cross-linking conditions, the bands corresponding to monomeric states remain prominent in both micelles and lipids environments (Fig.…”

Mechanistic Aspects of Folded Protein Transport by the Twin Arginine Translocase (Tat)

“…Solution and solid-state NMR structures of bacterial TatB (39) and TatA (40 -42) show that the TM and APH bend at the hinge to form an L-shaped molecule. Both of the E. coli proteins have a kink in their APH domain, with the TatB APH showing a more acute change in direction (39). TatB also has a longer C-tail than the TatA C-tail, both of which can be deleted without eliminating function (43,44).…”