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.
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.
School of Biosciences, University of Kent, Canterbury CT2 7NJ, United Kingdom.
Alexander S
AS
Jones
Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom.
James I
JI
Austerberry
Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom.
Rana
R
Dajani
Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom.
Jim
J
Warwicker
School of Chemical Engineering and Analytical Science, University of Manchester, Manchester M13 9PL, United Kingdom.
Robin
R
Curtis
Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom.
Jeremy P
JP
Derrick
School of Biosciences, University of Kent, Canterbury CT2 7NJ, United Kingdom. Electronic address: c.robinson-504@kent.ac.uk.
Colin
C
Robinson
D020816:Amino Acid Motifs; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D054730:Protein Interaction Domains and Motifs; D021382:Protein Sorting Signals; D017433:Protein Structure, Secondary; D021381:Protein Transport; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055672:Static Electricity; D013379:Substrate Specificity; D019076:Transgenes
D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D021382:Protein Sorting Signals; D057127:Single-Chain Antibodies; C408585:twin-arginine translocase complex, E coli
Protein translocation; Signal peptide; Tat; Twin-Arginine Translocase