The glove-like structure of the conserved membrane protein TatC provides insight into signal sequence recognition in twin-arginine translocation

Abstract

In bacteria, two signal-sequence-dependent secretion pathways translocate proteins across the cytoplasmic membrane. Although the mechanism of the ubiquitous general secretory pathway is becoming well understood, that of the twin-arginine translocation pathway, responsible for translocation of folded proteins across the bilayer, is more mysterious. TatC, the largest and most conserved of three integral membrane components, provides the initial binding site of the signal sequence prior to pore assembly. Here, we present two crystal structures of TatC from the thermophilic bacteria Aquifex aeolicus at 4.0 Å and 6.8 Å resolution. The membrane architecture of TatC includes a glove-shaped structure with a lipid-exposed pocket predicted by molecular dynamics to distort the membrane. Correlating the biochemical literature to these results suggests that the signal sequence binds in this pocket, leading to structural changes that facilitate higher order assemblies.

Figure 1. Overview of the structure of AaTatC

(A) Cartoon diagram of AaDHPC viewed from in the plane of the membrane color-ramped from N- to C-terminus (blue to red) from the front and rotated 90°. TMs and loops are numbered (1–6) according to the text. The amphipathic helix in Per1 is labeled as 1A. The bilayer is represented in the back as a rectangle with the most hydrophobic portions in gray and the hydrophilic head groups in blue. Dimensions are shown for the whole protein and the narrowest point in the glove. (B) As in A viewed from the cytoplasm. (C–E) Front molecular surface representations: (C) colored as in A; (D) electrostatic surface potential from negative −6 kB/e (red) to positive +6 kB/e (blue); (E) percent conservation based on an alignment of TatC Pfam seed sequences from 10 (white) to 90% (red). See also Figure S1. (F) Cytoplasmic views of E. (G) Sequence alignment of TatC homologs. The species are E. coli, A. aeolicus, Campylobacter jejuni, Thermus thermophilus and Staphylococcus aureus. Alignment and residue coloring are based on ClustalX output (Larkin et al., 2007). Secondary structure features are highlighted above the sequence with helices (rectangles) colored as in A. Sequence numbering is below the alignment for E. coli and A. aeolicus. Residues that are disordered in the crystal structure have red numbering. Residues mutated in the AaDHPC are underlined in black. Residues highlighted in Fig. 3 are shown above the sequence with boxes colored according to the type of mutation (see labeling below where Xlink indicates a signal sequence cross-link and SS Supp are signal sequence suppressor mutants). X indicates residues involved in crosslinks while other letters indicate specific mutations. The dashed line marks a salt bridge between K190 and E221.

Figure 2. Molecular dynamics simulation of TatC in the lipid bilayer

Panels A – D are oriented from the front and colored as in Fig. 1A. (A) A sample frame from the molecular dynamics simulation. The full representative frame 71 at 35.5ns with the lowest protein RMSD to all other frames. Shown are protein as a ribbons cartoon similar to Fig. 1A with waters (6856 total) and lipids as sticks (107). (B) A coil diagram of AaDHPC color-ramped from N- to C-terminus. The thickness of the coil is based on the average RMSD at each position across the entire MD simulation. Thicker regions have the most fluctuation. (C) Comparison of AaDHPC (‘xtal’ lightened relative to Fig. 1A) aligned by TM3 and TM4 to Frame 71 of the MD simulation (colored as in Fig. 1A). The rotation of TM5 is highlighted by an arrow. (D) The crystal contacts in AaTatC. The central protein is colored as in Fig. 1A. Symmetry related proteins are similarly colored except lighter. The orientations of TM1 and TM5 are highlighted by arrows pointing from the N- to C-terminus. See also Figure S2. (E) Side view of the MD simulation frame 71 highlighting the waters. Hydrogens are removed for clarity. Protein is shown as a Cα ribbon, waters as spheres and lipids as lines. The orange arrow indicates AaE165 and the penetration of water into the bilayer. The red arrow indicates the hydration of Per3. The blue arrow highlights the lipid filled hole at the back of the protein.

Figure 3. Residues correlated to TatC function based on mutagenesis or crosslinking

(A) Ribbons diagram of AaDHPC colored similar to Fig. 1A with discussed mutants shown as sticks. Residues are colored based on identified function with A. aeolicus numbering: signal sequence crosslink (orange), signal sequence suppression mutants (red), TatC to TatC crosslink (blue), TatC to TatA or TatB crosslink (cyan) and mutants that inactivate TatC (green) (Barrett et al., 2005; Buchanan et al., 2002; Holzapfel et al., 2007; Kneuper et al., 2012; Kreutzenbeck et al., 2007; Lausberg et al., 2012; Punginelli et al., 2007; Strauch and Georgiou, 2007; Zoufaly et al., 2012). Dashed boxes indicate regions highlighted in C and D. (B) View from the cytoplasm as in A. Labeling includes A. aeolicus numbers with E. coli numbering and mutations in parenthesis. C and D have similar schemes. (C) Per3 in sticks highlighting the conserved hydrogen bond network stabilized by the conserved D205. (D) Sticks view of the hydrogen bond stabilizing the kinks in TM3 and 1A facilitated by P42 and Y119.

Figure 4. The AaTatC dimer

(A) Molecular weight measured by MALLS. UV traces from sizing column on left axis as thick lines. Refractive index on the right axis is shown as dashed lines. AaTatC (dimer MW 54 kDa) is in black, the control BSA (MW 66 kDa) is shown in grey. (B) Representative dimer model viewed from the cytoplasm colored as in Fig. 1A (Model name 525 in Table S1 and Fig. S3). In this case, an MD model was used in calculating the interface. See also Figure S3.

Figure 5. A model for signal sequence binding by TatC

(A) AaDHPC as in Fig. 1A with an idealized helical SufI signal peptide including the first 12 amino acids (cylinder) modeled into the pocket of AaTatC with the C-terminus continuing as a dashed line. Residues identified as signal sequence crosslinks are shown as sticks (orange). L14, which crosslinks to some signal sequences, shown as sticks (magenta). The position of E165 is indicated. (B) Representative predicted Aquifex aeolicus signal sequences aligned based on the arginine pair colored as in Fig. 1H. The consensus sequence is shown above in red. The residues used to model the signal sequence are highlighted by a black box. The alignment ends at the position of the predicted signal sequence cleavage site for SufI indicated by a scissors. (C) Similar to A viewed from the cytoplasm with the MD frame 71 aligned as in Fig. 2C. The arginines in the consensus are highlighted and the lysine/AaE165 interaction is highlighted by a *. Arrows show predicted movement of TM1 and TM5. (D) A cartoon model for TatC recognition of a signal sequence. The hydrophilic N-terminus of the signal sequence initially binds the membrane. The RR pair is recognized by TatC, positioning the signal sequence near the pocket. The signal sequence would enter into the membrane forming a tight interface stabilizing the pocket. This would create a looped orientation priming the substrate for translocation. The cleavage site is indicated by a scissors.