Journal: Nature Communications

Article Title: Structure and mechanism of the K + /H + exchanger KefC

doi: 10.1038/s41467-024-49082-7

Figure Lengend Snippet: a Cartoon representation of the cytosolic region of the KefC homodimer shown from the membrane plane. Core domains are shown in blue, dimerization domain in sand, linker helix in light purple, and the C-terminal regulatory RCK domains (pink and red). A stretch of four arginine residues in the amphipathic helix at the end of TM7are shown as cyan sticks. Each RCK domain binds AMP in the same coordination as shown as a zoomed in view for one of the bound AMP molecules. The AMP is shown as cyan sticks, and residues coordinating AMP as pink sticks. Dashed lines represent hydrogen bond distances at ~2.7–3.5 Å. b Two interaction sites between parallel domain-swapped helices in the RCK domain and the KefC transporter module are shown. Side-chains are labeled and shown as sticks with dashed lines representing hydrogen bond interactions. c Interactions involved in binding of GSH (shown in sand sticks). GSH molecules binds at the interface of dimeric RCK domain. Interacting residues from individual protomers are shown in pink and red sticks, respectively. d Electrostatic surface representation of the RCK dimer as viewed from the cytoplasm with bound AMP (cyan) and GSH (sand) shown in stick.

Article Snippet: Membranes of KefC WT*-GFP were solublized in buffer containing 20 mM Tris pH 7.5, 300 mM KCl, 1 mM AMP (A2252 Sigma Aldrich) or 1 mM CMP (C1131 Sigma Aldrich), or 1 mM cyclic di-AMP (17753 Cayman chemicals) for 1 h. Non-solubilised membrane material was pelleted by ultracentrifugation at 195,000 × g for 45 min at 4 °C.

Techniques: Membrane, Labeling, Binding Assay

Journal: Nature Communications

Article Title: Structure and mechanism of the K + /H + exchanger KefC

doi: 10.1038/s41467-024-49082-7

Figure Lengend Snippet: a Left: representative FSEC traces for solubilized membranes of KefC WT* in the presence (black) and absence of AMP (orange) at 4 °C. Right: normalized FSEC traces of solubilized membranes of KefC WT* with AMP (black) and KefC WT* incubated with alkaline phosphatase (orange). Removal of AMP in both cases destabilizes KefC dimers. b Fraction of dimeric protein retained in DDM solubilized membranes of KefC and RCK-transporter module interface mutants after heating at 60 °C in the presence of AMP. Error bars indicate mean values ± sd of n = 3 independent experiments. The source data are provided as a file. c Representative FSEC traces for KefC WT*, K81A, N135A, R146A, H259A, R401A, E465A, and R543A at 4 °C (black) and after heating at 60 °C for 10 min (orange) in buffer containing AMP. d Schematic of the KefC activation mechanism. Under resting conditions, AMP and GSH bind to the homodimer assembly of the RCK domains and the unique C-terminal helices (α7) interact with the transporter module inhibiting transport, as shown in the cryo-EM structure here. Upon displacement of GSH by glutathione adducts the RCK domains likely detach from the transporter module and interacts with KefF, which is predicted here based on the AlphaFold KefC model. Detachment of the RCK domains allows the KefC transporter to operate according to ion-concentration gradients and the pKa of the ion-binding site, which is specific for K + co-ordination ( K d ~ 10 mM). Figure 4d was created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en .

Article Snippet: Membranes of KefC WT*-GFP were solublized in buffer containing 20 mM Tris pH 7.5, 300 mM KCl, 1 mM AMP (A2252 Sigma Aldrich) or 1 mM CMP (C1131 Sigma Aldrich), or 1 mM cyclic di-AMP (17753 Cayman chemicals) for 1 h. Non-solubilised membrane material was pelleted by ultracentrifugation at 195,000 × g for 45 min at 4 °C.

Techniques: Incubation, Activation Assay, Cryo-EM Sample Prep, Concentration Assay, Binding Assay

Journal: Nature Communications

Article Title: Structures of the DarR transcription regulator reveal unique modes of second messenger and DNA binding

doi: 10.1038/s41467-023-42823-0

Figure Lengend Snippet: a Fluorescence polarization (FP) binding isotherms of WT Rhodococcus sp. USK13 DarR (red, open circles) and Rhodococcus sp. USK13 DarR(W177A-Q184A-L192A) (blue, open squares), respectively, to F-c-di-AMP. The x and y axes are concentration of Rhodococcus sp. USK13 DarR in μM and millipolarization units (mP), respectively. WT Rhodococcus sp. USK13 DarR bound with a K d of 21.9 ± 2.7 μM, while the mutant showed no detectable binding. Data points represent mean values ± SD with the error bars centered at the mean. The error in overall K d was determined as the SD between the calculated K d s for three runs. b Overall structure of the WT Rhodococcus sp. USK13 DarR-c-di-AMP complex. One subunit is colored magenta and the other green. Electron density (mF o -DF c ) calculated after omitting the c-di-AMP (blue mesh) is included and contoured at 2.9 σ. Helices that contain residues contributing to c-di-AMP binding are labeled. c c-di-AMP mediated bridging between DarR dimers observed in the crystal packing. d and e Close-up of the two nucleotide binding interactions at each of the DarR dimer interfaces with the omit electron density included (blue mesh) and contoured at 2.9 σ. One DarR subunit is colored green and the other, magenta. Residues that participate in nucleotide binding are shown as sticks and labeled.

Article Snippet: To measure c-di-AMP binding to DarR or DarR mutants, 2′-O-(6-[Fluoresceinyl]aminohexylcarbamoyl)-cyclic diadenosine monophosphate (2′-Fluo-AHC-c-di-AMP) (BioLog), was used as a fluoresceinated reporter ligand.

Techniques: Fluorescence, Binding Assay, Concentration Assay, Mutagenesis, Labeling

Journal: Nature Communications

Article Title: Structures of the DarR transcription regulator reveal unique modes of second messenger and DNA binding

doi: 10.1038/s41467-023-42823-0

Figure Lengend Snippet: a FP isotherms showing binding of WT Rhodococcus sp. USK13 DarR (red, open circles) and Rhodococcus sp. USK13 DarR(W177A-Q184A-L192A) (blue, open squares), respectively, to F-cAMP. The x and y axes are concentration of Rhodococcus sp. USK13 DarR in μM and mP, respectively. WT Rhodococcus sp. USK13 DarR bound with a K d of 28 ± 3 μM, while the mutant showed no binding. Data points represent mean values ± SD with the error bars centered at the mean. The error in K d was determined as the SD between the calculated K d s for three technical replicates. b Structure of the WT Rhodococcus sp. USK13 DarR-cAMP complex. One subunit is magenta and the other green. Sigma-A weighted omit electron density (mF o -DF c ) is shown as a blue mesh and contoured at 3.3 σ. c Close up of the cAMP binding-DarR interactions with omit electron density. One DarR subunit is colored green and the other, magenta. Residues that interact with cAMP are shown as sticks and labeled. d Overlay of DarR-cAMP (yellow) and the two DarR-c-di-AMP bound structures (red and slate).

Article Snippet: To measure c-di-AMP binding to DarR or DarR mutants, 2′-O-(6-[Fluoresceinyl]aminohexylcarbamoyl)-cyclic diadenosine monophosphate (2′-Fluo-AHC-c-di-AMP) (BioLog), was used as a fluoresceinated reporter ligand.

Techniques: Binding Assay, Concentration Assay, Mutagenesis, Labeling

Journal: Nature Communications

Article Title: Structures of the DarR transcription regulator reveal unique modes of second messenger and DNA binding

doi: 10.1038/s41467-023-42823-0

Figure Lengend Snippet: a Close-up of the interactions between the two centrally bound DarR subunits of the DarR dimer-of-dimers-DNA complex. b FP isotherms of WT Rhodococcus sp. USK13 DarR (red, open circles) and Rhodococcus sp. USK13 DarR(A119E-L120R) (blue, open squares), respectively, binding to the fluoresceinated 20 bp operator site. The x and y axis are concentration of Rhodococcus sp. USK13 DarR in nM and mP, respectively. Data points represent mean values ± SD with the error bars centered at the mean. The error in overall K d was determined as the SD between the calculated K d s for three runs. The error in K d was determined as the SD between calculated K d s for three experiments. c SEC analyses comparing elution of WT Rhodococcus sp. USK13 DarR and Rhodococcus sp. USK13 DarR(119E-L120R)-DNA complex. The x and y axes are LogMW and elution parameter (Kav), respectively. Kav calculated by Kav = (elution volume for the standard – void volume)/(column volume – void volume). Apo DarR eluted (blue oval) at a calculated molecular weight (MW) of 50 kDa, consistent with a dimer, while the DarR(A119E-L120R)-DNA complex (red oval) eluted at a MW of 60 kDa consistent with a DarR dimer-DNA complex. The standards used for calculation of the standard curve are the same as in Fig. .

Article Snippet: To measure c-di-AMP binding to DarR or DarR mutants, 2′-O-(6-[Fluoresceinyl]aminohexylcarbamoyl)-cyclic diadenosine monophosphate (2′-Fluo-AHC-c-di-AMP) (BioLog), was used as a fluoresceinated reporter ligand.

Techniques: Binding Assay, Concentration Assay, Molecular Weight

Journal: Nature Communications

Article Title: Structures of the DarR transcription regulator reveal unique modes of second messenger and DNA binding

doi: 10.1038/s41467-023-42823-0

Figure Lengend Snippet: a Ribbon diagram showing DarR-DNA contacts. b DNA schematic showing contacts to the phosphates and bases. c Ribbon diagram showing the recognition helices of the DarR subunits bound to the DNA major groove and the locations and contacts of base interacting residues, Lys44 and Gly45. d FP isotherms for WT Rhodococcus sp. USK13 DarR binding to fluoresceinated 20 bp operator (top strand, 5´-TAGATACTCCGGAGTATCTA-3´) (red open circles), T mutant 20 bp operator (5´-TAG A G A CTC CGG C GT C TC TA-3´) (green open diamonds), G mutant 20 bp operator (5´- TAG ATA A TC C T G A T T ATC TA-3´) (blue open squares), optimized 20 bp operator (5´-T T G C TA C TC CGG AGT AT CTA-3´) (crosses). The x and y axes are concentration of Rhodococcus sp. USK13 DarR in nM and normalized D millipolarization units (mP) ((A-A 0 )/(A max -A 0 )), respectively. A is change in mP reading, A 0 is the initial mP value before addition and A max is the maximal mP reading upon binding saturation. Normalization was done here to account for slightly different A max values obtained for the different DNA sites. Data points represent mean values ± SD with the error bars centered at the mean. The error in K d was determined as the SD between the calculated K d s for three technical replicate runs. e FP binding isotherms comparing the binding of WT Rhodococcus sp. USK13 DarR (red open circles), DarR(K44A) (green open diamonds) and DarR(G45V) (blue open squares) to the WT 20 bp operator. The x and y axes are concentration of Rhodococcus sp. USK13 DarR WT or mutant in nM and mP, respectively. Data points represent mean values ± SD with the error bars centered at the mean. The error in overall K d was determined as the SD between the calculated K d s for three runs. The error in K d was determined as the SD between the calculated K d s for the three technical replicate runs. Note, the DarR(K44A), DarR(G45V), T mutant and G mutant DNA data showed no saturable binding and hence were not fit but the points indicated with a straight line.

Article Snippet: To measure c-di-AMP binding to DarR or DarR mutants, 2′-O-(6-[Fluoresceinyl]aminohexylcarbamoyl)-cyclic diadenosine monophosphate (2′-Fluo-AHC-c-di-AMP) (BioLog), was used as a fluoresceinated reporter ligand.

Techniques: Binding Assay, Mutagenesis, Concentration Assay