Coordinating assembly and export of complex bacterial proteins.
ABSTRACT The Escherichia coli twin-arginine protein transport (Tat) system is a molecular machine dedicated to the translocation of fully folded substrate proteins across the energy-transducing inner membrane. Complex cofactor-containing Tat substrates, such as the model (NiFe) hydrogenase-2 and trimethylamine N-oxide reductase (TorA) systems, acquire their redox cofactors prior to export from the cell and require to be correctly assembled before transport can proceed. It is likely, therefore, that cellular mechanisms exist to prevent premature export of immature substrates. Using a combination of genetic and biochemical approaches including gene knockouts, signal peptide swapping, complementation, and site-directed mutagenesis, we highlight here this crucial 'proofreading' or 'quality control' activity in operation during assembly of complex endogenous Tat substrates. Our experiments successfully uncouple the Tat transport and cofactor-insertion activities of the TorA-specific chaperone TorD and demonstrate unequivocally that TorD recognises the TorA twin-arginine signal peptide. It is proposed that some Tat signal peptides operate in tandem with cognate binding chaperones to orchestrate the assembly and transport of complex enzymes.
Full-textDOI: · Available from: Alexandra Dubini, May 28, 2015
SourceAvailable from: Tara Winstone[Show abstract] [Hide abstract]
ABSTRACT: The system specific chaperone DmsD interacts with the twin-arginine leader peptide of its substrate; DmsA, allowing for proper folding and assembly of the DmsA catalytic subunit of DMSO (dimethylsulfoxide) reductase prior to translocation by the twin-arginine translocase. DmsD residues important for binding the complete 45 amino acid sequence of the DmsA leader (DmsAL) peptide were previously identified and found to cluster in a pocket of the DmsD structure. In the present study, we have utilized isothermal titration calorimetry (ITC) to determine the binding constant and thermodynamic parameters between 15 single substitution DmsD variant proteins and a synthetic DmsAL peptide made up of 27 amino acids (DmsAL15-41). The stoichiometry values were determined with ITC and the multimeric composition of the DmsD variants in the absence and presence of peptide were characterized with size exclusion chromatography and native-PAGE. Up to a four-fold change in affinity was observed for DmsD variant proteins relative to wild type DmsD and variation in entropic contribution to binding divided the binding site into two clusters: residues with either more or less favorable entropy. Substitution of hydrophobic residues along one helix face (helix 5) or prolines found on adjacent loops caused reduced binding affinity due to increased entropic cost and suggest that the twin-arginine motif of the DmsAL peptide binds to a preformed site on DmsD. Most DmsD variants were more than 90% monomeric in solution and bound a single peptide per protein molecule. The DmsD variant with the largest dimer population, showed increased affinity and induced the formation of tetramers in the presence of peptide, suggesting that dimeric or an alternatively folded form of DmsD may play an as yet undefined role in binding.Biochemistry 02/2015; 54(11). DOI:10.1021/bi500891d · 3.19 Impact Factor
[Show abstract] [Hide abstract]
ABSTRACT: Pathogenic bacteria adapt to their environment and manipulate the biochemistry of hosts by secretion of effector molecules. Serratia marcescens is an opportunistic pathogen associated with healthcare-acquired infections and is a prolific secretor of proteins, including three chitinases (ChiA, ChiB, and ChiC) and a chitin binding protein (Cbp21). In this work, genetic, biochemical, and proteomic approaches identified genes that were required for secretion of all three chitinases and Cbp21. A genetic screen identified a holin-like protein (ChiW) and a putative l-alanyl-d-glutamate endopeptidase (ChiX), and subsequent biochemical analyses established that both were required for nonlytic secretion of the entire chitinolytic machinery, with chitinase secretion being blocked at a late stage in the mutants. In addition, live-cell imaging experiments demonstrated bimodal and coordinated expression of chiX and chiA and revealed that cells expressing chiA remained viable. It is proposed that ChiW and ChiX operate in tandem as components of a protein secretion system used by gram-negative bacteria. © 2014 Hamilton et al.The Journal of Cell Biology 12/2014; 207(5):615-26. DOI:10.1083/jcb.201404127 · 9.69 Impact Factor