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The hydrophobic core of twin-arginine signal sequences orchestrates specific binding to Tat-pathway related chaperones.

Shanmugham A, Bakayan A, Völler P, Grosveld J, Lill H, Bollen YJ - PLoS ONE (2012)

Bottom Line: This ensures native behavior of the respective signal sequence and excludes any effects mediated by the mature domain of the pre-protein.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.This property could link REMP-signal peptide binding to its reported proofreading function.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cell Biology, VU University Amsterdam, Amsterdam, The Netherlands.

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.

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Related in: MedlinePlus

The signal sequence ensures specific REMP binding.Surface plasmon resonance sensorgrams for the injection of various proteins over immobilized TorD (A) and DmsD (B) are shown. The model pre-proteins ssDmsA-GFP (black lines), ssTorA-GFP (red lines) and signal sequence-free GFP (blue lines) were injected for 60 seconds at a concentration of 200 nM, a flow rate of 50 µl/min, and a temperature of 25°C.
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pone-0034159-g001: The signal sequence ensures specific REMP binding.Surface plasmon resonance sensorgrams for the injection of various proteins over immobilized TorD (A) and DmsD (B) are shown. The model pre-proteins ssDmsA-GFP (black lines), ssTorA-GFP (red lines) and signal sequence-free GFP (blue lines) were injected for 60 seconds at a concentration of 200 nM, a flow rate of 50 µl/min, and a temperature of 25°C.

Mentions: Binding of the Tat signal peptides to REMPs was investigated by SPR. His-tagged DmsD and TorD were immobilized in separate lanes on the surface of a Ni-NTA chip. The signal sequence of DmsA fused to GFP (ssDmsA-GFP) and the signal sequence of TorA fused to GFP (ssTorA-GFP) were separately injected over this surface for a time period of 60 s. The observed SPR responses (Figure 1) imply that ssDmsA-GFP binds to the DmsD covered surface, but not to the TorD surface. Similarly ssTorA-GFP shows specific binding to TorD. GFP by itself did not bind to either TorD or DmsD (Figure 1); the step-like SPR responses observed are a direct result of the higher refractive index of the protein solution compared to the protein-free buffer. The specific interactions between the REMPs and their cognate GFP-fusion pre-proteins thus are apparently governed by the respective Tat signal peptide.


The hydrophobic core of twin-arginine signal sequences orchestrates specific binding to Tat-pathway related chaperones.

Shanmugham A, Bakayan A, Völler P, Grosveld J, Lill H, Bollen YJ - PLoS ONE (2012)

The signal sequence ensures specific REMP binding.Surface plasmon resonance sensorgrams for the injection of various proteins over immobilized TorD (A) and DmsD (B) are shown. The model pre-proteins ssDmsA-GFP (black lines), ssTorA-GFP (red lines) and signal sequence-free GFP (blue lines) were injected for 60 seconds at a concentration of 200 nM, a flow rate of 50 µl/min, and a temperature of 25°C.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3316669&req=5

pone-0034159-g001: The signal sequence ensures specific REMP binding.Surface plasmon resonance sensorgrams for the injection of various proteins over immobilized TorD (A) and DmsD (B) are shown. The model pre-proteins ssDmsA-GFP (black lines), ssTorA-GFP (red lines) and signal sequence-free GFP (blue lines) were injected for 60 seconds at a concentration of 200 nM, a flow rate of 50 µl/min, and a temperature of 25°C.
Mentions: Binding of the Tat signal peptides to REMPs was investigated by SPR. His-tagged DmsD and TorD were immobilized in separate lanes on the surface of a Ni-NTA chip. The signal sequence of DmsA fused to GFP (ssDmsA-GFP) and the signal sequence of TorA fused to GFP (ssTorA-GFP) were separately injected over this surface for a time period of 60 s. The observed SPR responses (Figure 1) imply that ssDmsA-GFP binds to the DmsD covered surface, but not to the TorD surface. Similarly ssTorA-GFP shows specific binding to TorD. GFP by itself did not bind to either TorD or DmsD (Figure 1); the step-like SPR responses observed are a direct result of the higher refractive index of the protein solution compared to the protein-free buffer. The specific interactions between the REMPs and their cognate GFP-fusion pre-proteins thus are apparently governed by the respective Tat signal peptide.

Bottom Line: This ensures native behavior of the respective signal sequence and excludes any effects mediated by the mature domain of the pre-protein.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.This property could link REMP-signal peptide binding to its reported proofreading function.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cell Biology, VU University Amsterdam, Amsterdam, The Netherlands.

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.

Show MeSH
Related in: MedlinePlus