A Potential Late Stage Intermediate of Twin-Arginine Dependent Protein Translocation in Escherichia coli

Abstract

The twin-arginine translocation (Tat) system transports folded proteins across membranes of prokaryotes, plant plastids, and some mitochondria. According to blue-native polyacrylamide gel electrophoresis after solubilization with digitonin, distinct interactions between the components TatA, TatB, and TatC result in two major TatBC-containing complexes in Escherichia coli that can bind protein substrates. We now report the first detection of a TatABC complex that likely represents the state at which transport occurs. This complex was initially found when the photo cross-linking amino acid p -benzoyl- l -phenylalanine (Bpa) was introduced at position I50 on the periplasmic side of the first trans-membrane domain of TatC. Cross-linking of TatC I50Bpa resulted in TatC-TatC-cross-links, indicating a close proximity to neighboring TatC in the complex. However, the new complex was not caused by cross-links but rather by non-covalent side chain interactions, as it was also detectable without UV-cross-linking or with an I50Y exchange. The new complex did not contain any detectable substrate. It was slightly upshifted relative to previously reported substrate-containing TatABC complexes. In the absence of TatA, an inactive TatBC I50Bpa complex was formed of the size of wild-type substrate-containing TatABC complexes, suggesting that TatB occupies TatA-binding sites at TatC I50Bpa . When substrate binding was abolished by point mutations, this TatBC I50Bpa complex shifted analogously to active TatABC I50Bpa complexes, indicating that a defect substrate-binding site further enhances TatB association to TatA-binding sites. Only TatA could shift the complex with an intact substrate-binding site, which explains the TatA requirement for substrate transport by TatABC systems.

TatA and TatB generate a hydrophobic mismatch that is important for function and assembly of the Tat translocon in Escherichia coli

“…In case of TatB, our data show that all length variations affect TatBC complex stability (Figure 4), and activity reductions are therefore likely due to weaker TatBC interactions. It is interesting that specific variations of the TMH in TatB can have selective effects on the different complexes, which might relate to conformational equilibria of the Tat complexes, such as previously described for mutations in TatC (Geise et al, 2019). Observations of residual activity with extended or shortened hydrophobic stretches in TatB are suggestive for significant flexibility of interactions within TatBC complexes, which likely are not rigid but rather dynamic oligomers.…”

“…Briefly, the MT3-tag (including the linker) was amplified from the pMAL-c2x-MT3 (Diestra et al, 2009) using primers MT-l-F and MT-R, cut with BglII/BssHII, and ligated into the backbone of the plasmid pAH120-Ptat- tatABC -strep cut with BamHI/BssHII. This resulted in pAH120-Ptat -tatABC -MT3, which was then cut with NdeI/BamHI and ligated into the corresponding sites of pABS- tatABC -H6 (Geise et al, 2019), resulting in pABS- tatABC -MT3. To construct pABS- tatA -MT3, tatA was amplified from pABS- tatABC -MT3 using primers pABS- tatA -MT3-R and pABS- tatABC -SalI-F, the fragment was cut with SalI/SacI and ligated into the plasmid backbone obtained by restriction of pABS- tatABC -H6 with the same enzymes.…”

“…It was therefore possible that functional effects of length variations in the hydrophobic TMH of TatA or TatB are simply due to disturbed interactions with TatC. To examine this aspect, we analyzed the stability of the known TatCcontaining complexes (Geise et al, 2019). Membrane fractions were solubilized by digitonin and analyzed by BN-PAGE/Western blotting to detect Tat complexes TC1 and TC2 (Figure 4).…”

“…A subpopulation of TatA protomers interacts with TatBC in a way that is not affected by mutations in TatC that abolish substrate-binding (Behrendt & Brüser, 2014), and resting-state-contacts of TatA to TatC have been identified (Aldridge et al, 2014). It is clear that TatA assemblies undergo substrate-induced conformational changes that relate to Tat transport (Alcock et al, 2016; Aldridge et al, 2012; Hou et al, 2018), and that the TatABC components rearrange during the translocation cycle (Geise et al, 2019; Habersetzer et al, 2017).…”

“…A subpopulation of TatA protomers interacts with TatBC in a way that is not affected by mutations in TatC that abolish substrate-binding [15], and resting-state-contacts of TatA to TatC have been identified [24]. TatA assemblies undergo substrate-induced conformational changes that relate to Tat transport [25,26], and the TatABC components rearrange during the translocation cycle [27,28]. in the transmembrane helix of TatA and TatB (yellow), the preceding polar position (bluish purple), and the amphipathic helix (turquoise).…”