DmsD, a Tat system specific chaperone, interacts with other general chaperones and proteins involved in the molybdenum cofactor biosynthesis

Abstract

Many bacterial oxidoreductases depend on the Tat translocase for correct cell localization. Substrates for the Tat translocase possess twin-arginine leaders. System specific chaperones or redox enzyme maturation proteins (REMPs) are a group of proteins implicated in oxidoreductase maturation. DmsD is a REMP discovered in Escherichia coli, which interacts with the twin-arginine leader sequence of DmsA, the catalytic subunit of DMSO reductase. In this study, we identified several potential interacting partners of DmsD by using several in vitro protein-protein interaction screening approaches, including affinity chromatography, co-precipitation, and cross-linking. Candidate hits from these in vitro findings were analyzed by in vivo methods of bacterial two-hybrid (BACTH) and bimolecular fluorescence complementation (BiFC). From these data, DmsD was confirmed to interact with the general molecular chaperones DnaK, DnaJ, GrpE, GroEL, Tig and Ef-Tu. In addition, DmsD was also found to interact with proteins involved in the molybdenum cofactor biosynthesis pathway. Our data suggests that DmsD may play a role as a "node" in escorting its substrate through a cascade of chaperone assisted protein-folding maturation events.

Fig. 1

Representative findings from affinity chromatography approaches. (A) Co-purifying proteins with DmsD preparation (lane 1; lane 2. control protein preparation). Proteins with asterisk confirmed to interact with DmsD by far-western (not shown). (B) Potential His₆-T₇-DmsD-interacting species from anaerobic solubilized membrane prey fractions. Eluted fractions from Ni²⁺-NTA resin exposed to CHAPS-solubilized E. coli membrane prey fractions. Lane 1, His₆-T₇-DmsD-immobilized resin; lane 2, His₆-T₇-TehB control protein immobilized resin. (C) Co-purification experiments demonstrating potential His₆-T₇-DmsD-interacting species from anaerobic expressed DmsD soluble fractions. Lane 1, MC4100; lane 2, MC4100/pTDMS28. (D) Co-purification experiments demonstrating potential His₆-T₇-DmsD-interacting species from anaerobic solubilized membranes. Putative interacting proteins are indicated with asterisks. Arrows denote the His₆-T₇-DmsD or control protein. Molecular mass markers (in kDa) are indicated to the left.

Fig. 2

Representative interaction experiments using Sulfo-SBED cross-linker. Sulfo-SBED reacted purified His₆-T₇-DmsD was incubated with wild-type soluble prey protein fraction (~1.6 μg bait to prey extract protein ratio) and exposed to UV to permit cross-linking. Parallel experiments performed with non-reacted control. Cross-linked samples were cleaved with DTT to transfer the biotin label to the interacting partner and purified on an avidin column. After incubation the various soluble cell extract samples were run on a 12% SDS-PAGE gel and electroblotted. Following blocking in 10% milk/TBS/0.2% azide, the blot was initially probed with 100 ng/mL Streptavidin–HRP (Novagen) prior to development with Opti-4CN HRP developer reagent (Bio-Rad). Therefore bands in the figure contain biotin. Lane 1, Sulfo-SBED cross-linker reacted His₆-T₇-DmsD with soluble cell extract; lane 2, His₆-T₇-DmsD with no cross-linker exposed to cell extract; lane 3, no His₆-T₇-DmsD added to the cell extract. Molecular masses (in kDa) are indicated to the left. Non cross-linked biotin labeled DmsD bands are highlighted with an arrow indicating both monomer and dimer forms (confirmed by anti-DmsD antibody). Additional bands found in control lanes 2 and 3 are naturally occurring biotinylated or biotin-binding proteins.

Fig. 3

DmsD interactions revealed by bacterial two-hybrid approach. Functional complementation of the two CyaA domains brought together by the interaction of DmsD and a given interacting partner result in cAMP synthesis and the subsequent induction of the Lac operon that is evaluated by the β-galactosidase activity. Miller unit activities represent the relative levels of hybrid protein–protein interaction. The background was established using the control system of pDmsDT25 interacting with pUT18-Zip, which is not expected to have any interaction. A: Interactions with general chaperones. For comparison DmsD interaction with its DmsA leader substrate is also shown. To show relative amounts the Tig and DmsA interactions are shown at 1/3 (×3) and 1/5 (×5) their values obtained. B: Interactions with proteins involved in the molybdopterin cofactor biosynthesis. Data was subjected to the T-Test and were found to be significantly different than background control for all samples except for MobA.

Fig. 4

DmsD interactions revealed by the biomolecular fluorescence complementation assay. The specific intensity is the relative fluorescence from the reconstituted YFP protein ratio to the cell density. The negative control for these experiments is the data obtained from transformants containing both pDmsDYcK and pYnK which is based on the same parent plasmid for the other constructs used in the BiFC assay but only harbours the gene encoding the N-terminal moiety of YFP. A: Interactions with general chaperones. B: Interactions with proteins involved in the molybdopterin cofactor biosynthesis. For comparison DmsD interaction with its DmsA leader substrate is also shown in panel B. Shaded bars represent data obtained using a Δtat strain, whereas open bars are data for WT E. coli. Data was subjected to the T-Test and were found to be significantly different than background control for all samples except interaction with DnaJ in WT host.

Fig. 5

Interaction web for DmsD. Interactions are cartooned showing in solid line interactions described by the Bacterial Protein interaction database (Bacteriome.org). Dashed lines indicate strong interactions demonstrated by this study and previous work from our group. Dotted lines are weaker or more transient interactions suggested by this study. The interaction network of the molybdopterin cofactor biosynthesis is shown, however, for clearer viewing interactomes of the chaperones are not included.