The Hydrophobic Core of Twin-Arginine Signal Sequences Orchestrates Specific Binding to Tat-Pathway Related Chaperones

Abstract

Redox enzyme maturation proteins (REMPs) bind pre-proteins destined for translocation across the bacterial cytoplasmic membrane via the twin-arginine translocation system and enable the enzymatic incorporation of complex cofactors. Most REMPs recognize one specific pre-protein. The recognition site usually resides in the N-terminal signal sequence. REMP binding protects signal peptides against degradation by proteases. REMPs are also believed to prevent binding of immature pre-proteins to the translocon. The main aim of this work was to better understand the interaction between REMPs and substrate signal sequences. Two REMPs were investigated: DmsD (specific for dimethylsulfoxide reductase, DmsA) and TorD (specific for trimethylamine N-oxide reductase, TorA). Green fluorescent protein (GFP) was genetically fused behind the signal sequences of TorA and DmsA. This ensures native behavior of the respective signal sequence and excludes any effects mediated by the mature domain of the pre-protein. Surface plasmon resonance analysis revealed that these chimeric pre-proteins specifically bind to the cognate REMP. Furthermore, the region of the signal sequence that is responsible for specific binding to the corresponding REMP was identified by creating region-swapped chimeric signal sequences, containing parts of both the TorA and DmsA signal sequences. Surprisingly, specificity is not encoded in the highly variable positively charged N-terminal region of the signal sequence, but in the more similar hydrophobic C-terminal parts. Interestingly, binding of DmsD to its model substrate reduced membrane binding of the pre-protein. This property could link REMP-signal peptide binding to its reported proofreading function.

Nitrate and periplasmic nitrate reductases

“…267 While the purpose of the NapA-D interaction is predicted to be comparable in all organisms, the chemical basis of the interaction is species-specific, thus may be considered as a redox enzyme maturation protein (REMP). 268,269 NapD lacks a conserved NapA binding motif.…”

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

“…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). …”

“…Although the E. coli DmsD protein can form homo-oligomers in vitro (Sarfo et al , 2004), the monomeric from has been structurally characterized (Ramasamy & Clemons, 2009; Stevens et al , 2009, 2012). The E. coli DmsD protein is thought to bind to the h-region of the DmsA Tat signal peptide (Shanmugham et al , 2012; Winstone et al , 2013), but a precise binding epitope has not yet been established experimentally.…”

Assembly pathway of a bacterial complex iron sulfur molybdoenzyme

“…Rather, they 'escort' their CISM substrates though the entire maturation process as implied by their interactome (30) (Figure 1). Accordingly, many potential roles for REMPs have been proposed, including functioning as: (i) foldases to ensure correct secondary and tertiary structure (25,31); (ii) unfoldases to correct folding mistakes (25); (iii) avoidance chaperones to prevent incorrect membrane targeting during folding and assembly (32); (iv) cofactor-assembly chaperones to maintain apoenzymes in a cofactor-binding competent conformation (30,31); (v) cofactor-binding proteins, which bind the cofactor prior to its transfer to the apoenzyme (30,33); (vi) targeting proteins directing substrates to specific cellular locations (34,35); (vii) escort chaperones to promote transmembrane transport of enzyme complexes (30,31,34); (viii) proofreading chaperones to suppress transport until essential prior steps in the assembly process are complete (33,36,37); and (ix) protease protection chaperones to prevent degradation during assembly (38). Yet despite these proposed roles, a detailed path of actions for REMPs has yet to be defined.…”