Proofreading of substrate structure by the Twin-Arginine Translocase is highly dependent on substrate conformational flexibility but surprisingly tolerant of surface charge and hydrophobicity changes

Abstract

The Tat system transports folded proteins across the bacterial plasma membrane, and in Escherichia coli preferentially transports correctly-folded proteins. Little is known of the mechanism by which Tat proofreads a substrate's conformational state, and in this study we have addressed this question using a heterologous single-chain variable fragment (scFv) with a defined structure. We introduced mutations to surface residues while leaving the folded structure intact, and also tested the importance of conformational flexibility. We show that while the scFv is stably folded and active in the reduced form, formation of the 2 intra-domain disulphide bonds enhances Tat-dependent export 10-fold, indicating Tat senses the conformational flexibility and preferentially exports the more rigid structure. We further show that a 26-residue unstructured tail at the C-terminus blocks export, suggesting that even this short sequence can be sensed by the proofreading system. In contrast, the Tat system can tolerate significant changes in charge or hydrophobicity on the scFv surface; substitution of uncharged residues by up to 3 Lys-Glu pairs has little effect, as has the introduction of up to 5 Lys or Glu residues in a confined domain, or the introduction of a patch of 4 to 6 Leu residues in a hydrophilic region. We propose that the proofreading system has evolved to sense conformational flexibility and detect even very transiently-exposed internal regions, or the presence of unfolded peptide sections. In contrast, it tolerates major changes in surface charge or hydrophobicity.

Probing the quality control mechanism of the Escherichia coli twin-arginine translocase with folding variants of a de novo–designed heme protein

“…It has been reported that the length of the unstructured FG repeat polypeptide dramatically affects Tat export, with longer regions abolishing Tat export altogether ( 17 ). Conversely, Jones et al ( 18 ) recently reported that the Tat system was surprisingly tolerant of hydrophobic patches on the surface of structured single-chain variable fragment proteins, and export efficiency was increased with greater structural rigidity. Chaperones may also prevent export of a protein until cofactor insertion has taken place ( 19 – 21 ), and mutants incapable of cofactor binding are rapidly degraded once in contact with the Tat machinery ( 22 ).…”

A Robust Fractionation Method for Protein Subcellular Localization Studies in Escherichia Coli

“…Three periplasmic extraction methods, cold-osmotic shock, cold-osmotic shock with MgCl 2 and EDTA/lysozyme/cold-osmotic [30]; and two cell disruption techniques, sonication [30] and freezethaw cycles, were compared in combinations to assess fraction purity (Table4). Protein localization and cross-contamination was monitored by Western blot of overexpressed proteins and selected markers.…”

“…The periplasm was extracted by EDTA/lysozyme/cold osmotic shock and the cells were lysed by sonication as described previously [30]. …”

“…Alkaline phosphatase (PhoA) variants were used as reporter proteins to demonstrate either cytoplasmic or periplasmic cellular localization. The former lacks a signal peptide whereas in the latter, PhoA was fused to either a Sec-specific [28,29] or Tat-specific signal peptide [19,21,26,28,30] for export. PhoA is naturally exported by the Sec pathway [28], but has also been shown to export into the periplasm as a fully folded protein via the Tat pathway when fused to a TorA signal peptide [26,28].…”

The Twin-Arginine Pathway for Protein Secretion

“…Requirements for conformational stability were studied using non-native Tat substrates, like PhoA and antibody fragments. These are only exported in oxidising conditions where disulphide bonds form (38,148,149,152). Nevertheless, human growth hormone (hGH) and interferon α2b are also exported via Tat without disulphide bonds formed (143), suggesting that they assume near-native states as shown for E. coli CueO, which is exported without its copper cofactor bound (144).…”