Research ArticleSTRUCTURAL BIOLOGY

Gating by ionic strength and safety check by cyclic-di-AMP in the ABC transporter OpuA

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Science Advances  18 Nov 2020:
Vol. 6, no. 47, eabd7697
DOI: 10.1126/sciadv.abd7697
  • Fig. 1 Domain organization of OpuA and activity in nanodiscs.

    (A) Left, SDS–polyacrylamide gel electrophoresis (PAGE) analysis of OpuA in nanodiscs; right, domain organization of OpuA protomers. (B) Schematic of the coupled enzyme assay to monitor ATPase activity, as shown in (C) to (F). LDH, lactate dehydrogenase; PK, pyruvate kinase. For (C) to (F), the standard assay mixture was composed of 50 mM K-Hepes (pH 7.0), 450 mM KCl, 4 mM phosphoenolpyruvate, 600 μM NADH, 2.1 to 3.5 U of pyruvate kinase, and 3.2 to 4.9 U of lactate dehydrogenase. (C) ATPase activity of OpuA in nanodiscs in the presence of 10 mM ATP with (black circles) and without (blue triangles) 10 μM cyclic-di-AMP. (D) ATPase activity of OpuA in nanodiscs as a function of ionic strength (generated by addition of KCl) in the presence of 100 μM glycine betaine and 10 mM ATP with (black circles) and without (blue triangles) 10 μM cyclic-di-AMP. (E) ATPase activity of OpuA in nanodiscs as a function of the cyclic-di-AMP concentration in the presence of 100 μM glycine betaine and 10 mM ATP. (F) ATPase activity of OpuA as a function of the ATP concentration in the presence of 100 μM glycine betaine. We normalized the ATP hydrolysis activities, as described in Materials and Methods. The error bars represent the SD of three technical replicates. PEP, phosphoenolpyruvate; NADH, reduced form of nicotinamide adenine dinucleotide; NAD+, oxidized form of nicotinamide adenine dinucleotide.

  • Fig. 2 Conformational states of OpuA during the transport cycle.

    (A) Cryo-EM map of the apo inward-facing conformation of wild-type OpuA at high ionic strength [50 mM KPi (pH 7.0) and 200 mM KCl (pH 7.0)] in the absence of glycine betaine. The NBDs are highly flexible, exposing different opening angles, as emphasized by the gray arrow. The SBD and CBS domains are not resolved. (B) Cryo-EM map of the substrate-loaded occluded conformation of OpuA (E190Q) in the presence of 100 μM glycine betaine, high ionic strength [20 mM Hepes (pH 7.0) and 300 mM KCl], and 10 mM MgATP. The CBS domains are not resolved. (A and B) The color code of the individual domains of one protomer is the same as in Fig. 1, and membrane boundaries are indicated by black lines. (C) ATP (blue sticks with phosphates in orange) bound to the NBD in the occluded conformation. Walker A, walker B, and signature motifs are shown in yellow (WA), red (WB), and green (C motif), respectively (density around ATP is shown as mesh at 6σ). Glutamine-190 is shown as stick. (D) Superposition of the scaffold that is covalently linked to the TMD of OpuA of both the IF (yellow) and occluded (green) conformation. (E) Single-molecule fluorescence resonance energy transfer (smFRET) measurements by alternating laser excitation (ALEX) of Alexa555- and Alexa647-labeled OpuA (S24C) in 20 mM K-Hepes (pH 7.0) at high ionic strength (+300 mM KCl), low ionic strength, turnover conditions [300 mM KCl, 10 mM MgATP, and 1 mM glycine betaine (GB)], and turnover-inhibited conditions [300 mM KCl, 10 mM MgATP, 1 mM glycine betaine, and 100 μM cyclic-di-AMP]. The dashed line marks the peak of the FRET signal.

  • Fig. 3 Substrate delivery and translocation in OpuA.

    For a global indication of the location with respect to the full map, consult Fig. 2B. (A) Comparison of the x-ray structures of the soluble/isolated OpuA-SBD in an open [yellow; Protein Data Bank (PDB) ID: 3L6G] and a closed substrate-loaded conformation (green; PDB ID: 3L6H) to the docked conformation, as seen in the full-length occluded structure (blue). (B) Comparison of the substrate entry point in the TMD seen from the extracellular side, for the IF conformation (left) and the occluded conformation (right), with residues F142 and M233 presumably closing the substrate entry. (C) G seen from the membrane plane, showing F166 and M227 as well. Unassigned density found in the occluded space of the TMD of the occluded conformation is shown in red (density is shown as mesh at 6σ).

  • Fig. 4 Ionic strength sensor and ionic gating of OpuA.

    (A) Sequence alignment of part of the NBD with the putative ionic strength sensor. The top three sequences are OpuA homologs that are regulated by ionic strength; the bottom three sequences are nonregulated type 1 ABC importers. KRIK residues are shown in the blue box. Numbering is based on the OpuA sequence. UniProt IDs for the alignment are as follows: Ll-OpuAA, Q9KIF7; Bs-OpuAA, P46920; Lm-GbuA, Q9RR46; Ec-ProV, P14175; Ec-MetN, P30750; Cs-ArtN, Q8RCC2; and Ec-MalK, P68187. (B) HTH region of the NBD in the occluded conformation. The basic residues of KRIK are shown in stick representation in blue. (C) In vivo 14C-glycine betaine uptake by wild-type OpuA (blue triangles) and the KRIK to AAIA mutant (black circles) as a function of osmotic stress (sucrose addition to L. lactis cells), which increases the internal ionic strength. Error bars represent the SD of three independent experiments.

  • Fig. 5 Regulation of OpuA by cyclic-di-AMP and proposed mechanism.

    (A) Schematic representation of OpuA reconstituted in vesicles together with the ATP-regenerating system described in (32). (B) Uptake of glycine betaine by OpuA in the vesicles in the presence (black triangles) or absence (blue circles) of 200 μM cyclic-di-AMP. The ATP/ADP (adenosine diphosphate) ratio is kept constant (with ATP ~ 6.5 mM) by the co-reconstituted arginine breakdown pathway (32); error bars represent the SD of three independent experiments. (C) Binding site of cyclic-di-AMP in the substrate-bound cyclic-di-AMP–inhibited IF conformation of OpuA. Density around cyclic-di-AMP is shown as mesh at 6σ. We find one molecule of cyclic-di-AMP coordinated between both CBS domains. (D) Cryo-EM map of the substrate-bound inward-facing cyclic-di-AMP–inhibited conformation of OpuA in 20 mM K-Hepes (pH 7.0) at high ionic strength (+300 mM KCl) and in the presence of 10 μM cyclic-di-AMP, 10 μM MgAMP-PNP, and 100 μM glycine betaine. (E) Cryo-EM map of the IF cyclic-di-AMP–inhibited conformation of OpuA in 50 mM KPi (pH 7.0) at high ionic strength (+200 mM KCl) and in the presence of 10 μM cyclic-di-AMP.

  • Fig. 6 Mechanism of translocation and regulation of transport.

    Schematic of the proposed transport cycle of OpuA. At low ionic strength, OpuA is in an inactive state. Ionic strength (~0.2 M) breaks the interaction of the sensor (cationic residues of HTH) with the anionic membrane and activates OpuA (gray shaded area). The transport cycle starts with a flexible IF. The substrate is bound by the SBD (blue), which docks onto the IF conformation. The substrate is translocated into the hydrophobic occluded space inside the outwardly oriented TMD (green). ATP hydrolysis returns the transporter to the IF conformation, and the substrate is pushed into the intracellular environment. The SBD undocks, resetting the transport cycle (unshaded area). In the inhibition cycle, the CBS domains (red) of OpuA dimerize by the binding of cyclic-di-AMP (green). This state leads to the substrate-dependent futile hydrolysis of ATP, as shown in Fig. 1 (C and D), and inhibition of transport, as shown in Fig. 3D.

Supplementary Materials

  • Supplementary Materials

    Gating by ionic strength and safety check by cyclic-di-AMP in the ABC transporter OpuA

    Hendrik R. Sikkema, Marco van den Noort, Jan Rheinberger, Marijn de Boer, Sabrina T. Krepel, Gea K. Schuurman-Wolters, Cristina Paulino, Bert Poolman

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