Thermodynamic Characterization of the DmsD Binding Site for the DmsA Twin-Arginine Motif

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 dimethyl sulfoxide 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 this study, we have utilized isothermal titration calorimetry (ITC) to determine the dissociation constant and thermodynamic parameters of 15 single-substitution DmsD variant proteins and a synthetic DmsAL peptide consisting of 27 amino acids (DmsAL₁₅₋₄₁). The stoichiometry values were determined via ITC, and the multimeric compositions of the DmsD variants in the absence and presence of peptide were characterized via size exclusion chromatography and native polyacrylamide gel electrophoresis. An up to 4-fold change in affinity was observed for DmsD variant proteins relative to that of wild-type DmsD, and variation of the 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 because of the increased entropic cost, which suggests 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 DmsD or an alternatively folded form of DmsD may play an as yet undefined role in binding.

Biosynthesis of selenate reductase in Salmonella enterica: critical roles for the signal peptide and DmsD

“…A key residue for efficient binding of the E. coli DmsA signal peptide by E. coli DmsD is W87, which has a direct equivalent in S. enterica DmsD and is distinct from W91 identified here (Winstone & Turner, 2015). While the physiological function of E. coli DmsD W87S and W87Y variants has not been reported, the identification of an S. enterica variant in this region by a completely random screen reinforces the critically important nature of this part of DmsD to its function.…”

“…Note, however, that, for Archaeoglobus fulgidus TtrD, which is related to DmsD, and E. coli NapD, which is from a different protein family to DmsD (Turner et al , 2004), the twin-arginine motif, or residues close to it, was found to be part of the chaperone binding epitope (Coulthurst et al , 2012; Grahl et al , 2012). There is also some biochemical evidence that E. coli DmsD does interact with the twin-arginine motif to some extent (Winstone & Turner, 2015), while the hydrophobic h-region contains the major drivers for specificity (Shanmugham et al , 2012; Winstone et al , 2013). …”

“…The E. coli DmsD protein has been extensively mutagenized and peptide binding of the variant proteins analysed in vitro (Chan et al , 2008; Winstone & Turner, 2015). A key residue for efficient binding of the E. coli DmsA signal peptide by E. coli DmsD is W87, which has a direct equivalent in S. enterica DmsD and is distinct from W91 identified here (Winstone & Turner, 2015).…”

Liquid-Liquid Phase Transition Drives Intra-chloroplast Cargo Sorting

“…In addition, this induced phase separation of the STT complex was sharply concentration-dependent and occurred when the concentration of STT and LTP reaches 2.5 mM (Figure 4A). Previous studies together with our data have shown that Tat translocon components including TorD, NapD, DmsD, cpTatC/Hcf106, and STTs may bind the Tat substrate with a 1:1 stoichiometry (Maillard et al, 2007;Ma and Cline, 2013;Dow et al, 2013;Winstone and Turner, 2015). Considering that the concentration of substrate transported in vivo may be lower than that of the STTs, we also checked whether low concentrations of cpTat substrate could induce the phase separation of STTs.…”

Influence of the TorD signal peptide chaperone on Tat-dependent protein translocation

“…At least 8 proteins exported from the E. coli cytoplasm by the Tat machinery have signal peptides that bind to a REMP (17,42). While signal peptide binding affinities for REMPs are in the low micromolar to high nanomolar range (43)(44)(45)(46), these are potentially significantly modulated by high affinity REMP interactions with the folded mature domain of the cognate protein (47,48). How the signal peptide is able to efficiently locate the signal peptide binding site on the Tat translocon in the presence of REMPs with similar affinities remains unresolved, though a GTP binding site suggests that affinities could be modulated by nucleotide hydrolysis (20,45,49).…”