The substrate-binding domains of the osmoregulatory ABC importer OpuA transiently interact

Abstract

Bacteria utilize various strategies to prevent internal dehydration during hypertonic stress. A common approach to countering the effects of the stress is to import compatible solutes such as glycine betaine, leading to simultaneous passive water fluxes following the osmotic gradient. OpuA from Lactococcus lactis is a type I ABC-importer that uses two substrate-binding domains (SBDs) to capture extracellular glycine betaine and deliver the substrate to the transmembrane domains for subsequent transport. OpuA senses osmotic stress via changes in the internal ionic strength and is furthermore regulated by the 2nd messenger cyclic-di-AMP. We now show, by means of solution-based single-molecule FRET and analysis with multi-parameter photon-by-photon hidden Markov modeling, that the SBDs transiently interact in an ionic strength-dependent manner. The smFRET data are in accordance with the apparent cooperativity in transport and supported by new cryo-EM data of OpuA. We propose that the physical interactions between SBDs and cooperativity in substrate delivery are part of the transport mechanism.

Keywords: ABC importer; interdomain dynamics; molecular biophysics; osmoregulatory transporter; single-molecule FRET; single-particle cryo-EM; structural biology; transient protein-protein interactons.

Figure 1.. Experimental approach for measuring interdomain dynamics in the SBDs of OpuA.

(A) A cryo-EM structure of OpuA (PDB: 7AHH). Mutations K521C and N414C are highlighted as grey spheres. (B) Size-exclusion chromatography profiles of OpuA-nanodiscs that were purified according to the previously described protocol (Sikkema et al., 2020) (blue) or according to the new protocol that is described here (yellow). The Latin numbers refer to the four different nanodisc species as is described in the first paragraph of the Results section. The other numbers refer to the elution fractions that were loaded on an SDS-PAA gel. (C) SDS-PAA gel with the size exclusion fractions of the blue line in (B). (D) SDS-PAA gel with the size-exclusion fractions of the yellow line in (B). (E) A schematic representation of how confocal, solution-based smFRET was used to study different states of the SBDs. (F) A representation of a fluorescent burst time trace, displaying the photon counts in the donor (green) and acceptor (red) detection channel over time (left). The zoom-in is a representation of a photon time trace, in which single photons are represented as lines and the most likely state path from the Viterbi algorithm in mpH2MM as a blue line.

Figure 1—figure supplement 1.. Enzyme-coupled ATPase assay of wildtype OpuA (circles, grey shading), OpuA-K521C (squares, blue shading) and OpuA-N414C (triangles, yellow shading).

A standard sample contains 50 mM HEPES-K pH 7.0, 450 mM KCl, 20 mM Mg-ATP, 100 µM glycine betaine, 4 mM phosphoenolpyruvate, 600 μM NADH, 2.1–3.5 U of pyruvate kinase plus 3.2–4.9 U of lactate dehydrogenase. Standard deviation over at least two measurements with different protein purifications and membrane reconstitutions, each consisting of three technical replicates is represented as shaded areas.

Figure 2.. The SBDs of OpuA sample two dynamic FRET states.

The proteins were analyzed in 50 mM HEPES-K pH 7.0, 600 mM KCl with the following additions: (A) OpuA-N414C without further additions. (B) OpuA-K521C without further additions. (C) OpuA-E190Q-K521C with 20 mM Mg-ATP plus 100 µM glycine betaine. (D) OpuA-K521C with 20 mM Mg-ATP. From top to bottom: (i) FRET histogram showing the corrected bursts that were selected after removing donor-only and acceptor-only bursts. (ii) 2D E-S histogram showing the same data as in (i). Black dots represent the average value of each state after mpH2MM and after application of the correction factors. (iii) Burst variance analysis of the same burst data as in (i). The standard deviation of FRET in each burst is plotted against its mean FRET. Black squares represent average values per FRET bin. The black dotted line shows the expected standard deviation in the absence of within-burst dynamics. (iv) E-S scatter plot of the corrected dwells. Dwells are colored on the basis of the assigned state of the chosen mpH2MM model. Black dots represent the average value of each state and the numbers at the arrows show transition rate constants (s^–1) between the two FRET states. (v) Plot of the ICL-values for each final model. The model used in the analysis is shown as a red star.

Figure 2—figure supplement 1.. Typical 2D E-S histograms of FRET bursts after γ-correction and correction for leakage and crosstalk.

Graphs represent data of OpuA-K521C in 50 mM HEPES-K pH 7.0, 600 mM KCl. Left graph shows the bursts after burst selection with a cut-off of minimally 35 photons per burst. Right graph shows the bursts after removal of donor-only and acceptor-only bursts. To filter out acceptor-only bursts, a threshold of minimally 15 photons after donor excitation was set. To filter out donor-only bursts, a threshold of minimally 15 photons was set. Similar graphs for all other OpuA variants and conditions can be found in the publicly available python notebooks (https://doi.org/10.34894/GSIEBW).

Figure 3.. Reduction in SBD docking efficiency does not affect the two dynamic FRET states.

(A) A cryo-EM structure of OpuA (PDB: 7AHH) highlighting Val-149 and the two most important residues in the vicinity of Val-149. Coloring of the domains is similar to that in Figure 1A. (B) Results of an enzyme-coupled ATPase assay for OpuA-WT (filled circles, grey) and OpuA-V149Q-K521C (open circles, yellow). Each sample contains 50 mM HEPES-K pH 7.0, 450 mM KCl, 10 mM Mg-ATP, 4 mM phosphoenolpyruvate, 600 μM NADH, 2.1–3.5 U of pyruvate kinase, and 3.2–4.9 U of lactate dehydrogenase. Standard deviation over at least two measurements with different protein purifications and membrane reconstitutions, each consisting of three technical replicates is represented as shaded areas. (C) Results of (B) represented as activity relative to the activity at 100 µM glycine betaine. (D) smFRET results for OpuA-V149Q-K521C in 50 mM HEPES-K pH 7.0, 600 mM KCl. From top to bottom: (i) FRET histogram showing the corrected bursts that were selected after removing donor-only and acceptor-only bursts. (ii) 2D E-S histogram showing the same data as in (i). Black dots depict the average value of each state after mpH2MM and after application of the correction factors. (iii) Burst variance analysis of the same burst data as in (i). The standard deviation of FRET in each burst is plotted against its mean FRET. Black squares represent average values per FRET bin. Black dotted line shows the expected standard deviation in the absence of within-burst dynamics. (iv) E-S scatter plot of the corrected dwells. Dwells are colored on the basis of the assigned state of the chosen mpH2MM model. Black dots represent the average value of each state and the numbers at the arrows show transition rate constants (s^–1) between the two FRET states. (v) Plot of the ICL-values for each final model. The model used in the analysis is shown as a red star.

Figure 3—figure supplement 1.. Enzyme-coupled ATPase assay for wildtype OpuA (squares, grey shading) and OpuA-V149Q-K521C (circles, yellow shading).

A standard sample contains 50 mM HEPES-K pH 7.0, 450 mM KCl, 20 mM Mg-ATP, 100 µM glycine betaine, 4 mM phosphoenolpyruvate, 600 μM NADH, 2.1–3.5 U of pyruvate kinase plus 3.2–4.9 U of lactate dehydrogenase. Standard deviation over at least two measurements with different protein purifications and membrane reconstitutions, each consisting of three technical replicates is represented as shaded areas.

Figure 4.. Shifts in FRET states under different conditions.

(A, B) FRET (E) histograms of OpuA-K521C (A) and OpuA-N414C (B) corresponding to the corrected dwells of the two FRET states after mpH2MM. Black lines show the mean E of the two states. (C) A schematic representation of the diffusion freedom in 2D of the SBDs, shown as blue semicircles. The radius is defined as the sum of the radius of an SBD and the length of the linker region in a fully extended conformation. The sequence of the linker region was defined based on the occluded OpuA structure (PDB: 7AHD) and spans the N- and C-terminal end of the SBD and anchoring helix, respectively. (D) Lowpass-filtered cryo-EM density maps of OpuA-WT in three different ionic strength conditions (50 mM, 100 mM and 200 mM KCl). The top-views of each condition are horizontally aligned (left) and super-positioned (right) for better comparison. The densities corresponding to the SBDs are highlighted in teal, blue and brown ellipses, respectively.

Figure 4—figure supplement 1.. Multiple sequence alignment of close homologs of OpuA from L.

lactis. The alignment was made by the Clustal Omega webserver using the default settings. The alignment was visualized and the JPRED secondary structure prediction were performed in Jalview (version 2.11.2.6). The black box indicates the linker region, based on OpuA from L. lactis. Jnetpred shows the consensus prediction of the different predictions in JPRED; α-helices are shown as red tubes and β-sheets as dark green arrows. JNETCONF is a confidence estimate of the jnetpred prediction. NCBI reference sequence IDs are: L. lactis, WP_003130445.1; L. taiwanensis, WP_205272268.1; L. allomyrinae, WP_120771492.1; L. garvieae, WP_004257235.1; L. hircilactis, WP_153496572.1; W. confusa, WP_199402959.1; W. muntiaci, WP_187387617.1;, W. ceti, WP_213409458.1; W. soli, WP_147152447.1; L. citreum, WP_040177303.1; W. halotolerans, WP_022790870.1; W. paramesenteroides, WP_150189650.1; L. plantarum, WP_068161132.1; L. fallax, WP_010007192.1; W. bombi, WP_092461275.1.

Figure 4—figure supplement 2.. Image processing of OpuA wild-type in MSP1E3D1 nanodiscs in 20 mM HEPES-K pH 7.0, 50 mM KCl.

(A) Representative elution profile from Superdex 200 increase 10/300 GL size-exclusion column. The collected fraction for cryo-EM sample preparation is indicated by dashed lines. (B) Representative micrograph at 1.3 μm defocus followed by detailed overview of the image processing leading to the map used. Abbreviations: gmodel t=crYOLO general model and K=number of classes. Scale bar, 10 nm.

Figure 4—figure supplement 3.. Image processing of OpuA wild-type in MSP1E3D1 nanodiscs in 20 mM HEPES-K pH 7.0, 100 mM KCl.

(A) Representative elution profile from Superdex 200 increase 10/300 GL size-exclusion column. The collected fraction for cryo-EM sample preparation is indicated by dashed lines. (B) Representative micrograph of the sample at 1.3 μm defocus, followed by detailed overview of the image processing leading to the map used. Abbreviations: model03=in-house trained crYOLO model generated by Sikkema et al., 2020, K=number of classes and T=tau_fudge. Scale bar, 10 nm.

Figure 5.. Schematic of possible states of the SBDs of OpuA.

(A) Both SBDs dock in a non-productive manner onto the TMDs. (B) The SBDs interact with each other in an upright orientation either back-to-back, front-to-front or front-to-back. (C) The SBDs interact sideways with each other.

Author response image 1.

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Author response image 3.