Inhibition effect of bisphosphonates on TmPPase.

A. Overall structure of the monomer TmPPase structure, consisting of hydrolytic centre, coupling funnel, ion gate and exit channel. B. Chemical structure of IDP and bisphosphonates and their inhibition activity against TmPPase. C. Activity of TmPPase modified with NEM (N-Ethylmaleimide) in the presence of MgCl (2.5 mM) and NaCl (20 mM) after incubation with Ca2+ (2 mM) and bisphosphonates (0.5 mM). Measurement was done in triplicate.

Structural asymmetry in the dimer active-site of TmPPase:ETD complex.

A. Top view of the superposition of chain A (cyan) and chain B (wheat) showing the relative movements (black arrow) of helices. B. Side view of the superposition of TMH5 and TMH6 in TmPPase:ETDs (chain A (cyan) and chain B (wheat)) and TmPPase:IDP (light blue; PDB: 5LZQ) showing the movement of loop5-6 and reorientation of ETDA, ETDB and IDP. The yellow dashed line shows the interaction of E2175.76 of loop5-6 in Chain A with ETD and IDP, and D2185.77 of loop5-6 in Chain B with ETDB.; Close-up view of IDP superposed with ETDA and ETDB. C. Stereo representation (wall-eyed view) of residues in the active site with ETD coordinated (dashed lines), Ca2+ (pink sphere) and nucleophilic water (red spheres) in a Mg2+ metal cage (green spheres). D. Stereo representation (wall-eyed view) of residues in the active site with ETDB coordinated (dashed lines), Ca2+ (pink sphere) and water (red spheres) in a Mg2+ metal cage (green spheres).

Comparison of the TmPPase:ZLD and TmPPase:IDP structures in the active site.

A. Top view of the superposition of TmPPase:ZLD (chain A, pink) and TmPPase: IDP (chain A, orange) (PDB: 5LZQ). Helices movements are indicated by a black arrow. B Cross-section view of the active site in TmPPase:ZLD. C. Cross-section side view of the active site in TmPPase:IDP. D. Top view of the superposition of TMH11, TMH12 and TMH15 in TmPPase:ZLD and TmPPase:IDP showed the movement of the hydrolytic centre and the orientation of ZLD and IDP. E. Stereo representation (wall-eyed view) showing the coordination of key residues in the active site with ZLD (dash line), and water (red sphere) in a Mg2+ metal cage (green spheres). F. Stereo representation (wall-eyed view) showing the coordination of key residues in the active site with IDP (dash line) in a Mg2+ metal cage (green spheres).

DEER distance distributions of TmPPase S525R1 and C599R1 under different conditions.

A. Symmetric structures (TmPPase:IDP (PDB: 5LZQ) and TmPPase:Ca (PDB: 4AV3) and asymmetric model (TmPPase:IDP(A)_Ca(B)) of TmPPase. The sites mutated and labelled with MTSSL are shown as spheres, with T211R1 (Cyan) and C599R1 (maroon) on the cytoplasmic side (top) and S525R1(maroon) on the periplasmic side (bottom) of the membrane. Distances between spin pairs are indicated as dashed lines, consistent with sphere colouring. DEER data of T211R1 is shown in supplementary Figure EV8. B and E. DEER raw data traces for S525R1 and C599R1, respectively. Each condition is labelled, and the raw data are colour-coded, with the background function indicated as solid black lines. C and F. DEER background-corrected time-domain traces for S525R1 and C599R1, respectively. The vertical black dashed line represents the minimum of the first oscillation in the apo state and aids visualisation to highlight the shifts in the oscillation minimum under different conditions. D and G. Distance distributions of S525R1 and C599R1, respectively. The in silico distance distribution corresponding to each spin pair modelled onto the asymmetric hybrid structure (TmPPase:IDP(A)_Ca(B)) is shown at the top as a solid black line, with the modal distance shown as a vertical dashed line. In silico predicted distance distributions for each condition, modelled using the solved structures (TmPPase:Ca, TmPPase:Ca:ETD (PDB 9G8K), TmPPase:ZLD (PDB 9G8J), and TmPPase:IDP) are presented as coloured dashed lines overlaying the experimental distributions. The shaded regions represent the 95% (2σ) confidence interval of the distributions, and the colour bars represent an assessment of the reliability of the distributions. The probability density within the green region indicates the mean distance, width, and peak shape are all reliable; the probability density within the yellow region indicates the mean distance and width are reliable; the probability density within the orange region indicates that the mean distance is reliable; the probability density within the red region indicates no quantitation is possible.

Transient currents of TmPPase Na+ pumping and ion gate of TmPPase structures.

A. Curve of Na+ pumping current triggered by 100 μM of K4PPi, 50 μM of IDP, 50 μM of ETD, 50 μM of ZLD, and 200 μM of K2HPO4. The vertical black dased line represents the addition of activating buffer and non-activating buffer. B-E. Ion gate of TmPPase:Ca (yellow); TmPPase:ETD (cyan); TmPPase:ZLD (green); TmPPase:IDP (purple). The black arrows show the movement of residues of D70316.46 and K70716.50.

Models based on DEER distance distributions for TmPPase S525R1 and C599R1.

Four DEER models showing major conformational ensembles of TmPPase in solution. Two monomers are colored purple and green, respectively. All TMHs are shown in brown; mobile loop5-6 is indicated by a black dashed line, while fixed loop5-6 and loop12-13 are indicated by a solid black line; The labelling sites are represented by maroon spheres. Ca2+ is shown as a magenta circle; IDP is shown as purple squares; ETD as cyan squares connected by a cyan stick; ZLD as an orange pentagon.

Inhibition of TmPPase by bisphosphonates.

All data are shown as mean ± SD with three replicates.

Top view of accessible cysteines for NEM modification.

A. Two exposed cysteines in both monomers of TmPPase:Ca (PDB: 4AV3; cyan). B. One exposed cysteine in both monomers of TmPPase:IDP (PDB: 5LZQ; wheat).

Electron density maps of ETD at the active sites.

A. mFo–Fc omit map with positive density of the ETDA shown in green mesh (Top left); Polder omit map of ETDA (Bottom left) shown in yellow mesh; 2mFo–Fc (light blue mesh) map of ETDA, ions and surrounding residues (Right). B. mFo–Fc omit map with positive density of the ETDB shown in green mesh (Top left); Polder omit map of ETDB (Bottom left) shown in yellow mesh; 2mFo–Fc (light blue mesh) map of ETDB, ions and surrounding residues (Right). C. mFo-Fc omit map with positive density and negative density of the exchanged ETDs shown in green and red mesh, respectively; 2mFo-Fc (light blue mesh) map of the exchanged ETDs. Ca2+ ion is shown in purple; Mg2+ ions are shown in green and water molecules are shown in red.

Electron density maps of ZLD at the active sites.

A. mFo– Fc omit map with positive density of the ZLDA shown in green mesh (Top left); Polder omit map of ZLDA (Bottom left) shown in yellow mesh; 2mFo–Fc (light blue mesh) map of ZLDA, ions and surrounding residues (Right). B. mFo– Fc omit map with positive density of the ZLDB shown in green mesh (Top left); Polder omit map of ZLDB (Bottom left) shown in yellow mesh; 2mFo–Fc (blue mesh) map of ZLDB, ions and surrounding residues (Right). C. mFo– Fc omit map with positive density of the ZLDC shown in green mesh (Top left); Polder omit map of ZLDC (Bottom left) shown in yellow mesh; 2mFo–Fc (blue mesh) map of ZLDC, ions and surrounding residues (Right). D. mFo– Fc omit map with positive density of the ZLDD shown in green mesh (Top left); Polder omit map of ZLDD (Bottom left) shown in yellow mesh; 2mFo–Fc (blue mesh) map of ZLDD, ions and surrounding residues (Right).

Electron density maps of loop5-6 in the TmPPase:ETD structure.

A. 2mFo–Fc (blue mesh) electron density map of loop5-6 at chain A. B. 2mFo–Fc (blue mesh) electron density map of loop5-6 at chain B.

Helix curvature comparison between chain A and chain B of the TmPPase:ETD structure.

Changes in helix curvature are shown in the hydrolytic side of TMH11, TMH12, and TMH15. The black bar shows the region in the hydrolytic centre side.

Ion gates of TmPPase structures.

A. Superposition of the ion gate of the four TmPPase structures (yellow: TmPPase:Ca; cyan: TmPPase:ETD; green: TmPPase:ZLD; purple: TmPPase:IDP; Na+ is shown as a purple sphere). The movement of TMH16 is shown as the black arrow. B-C. The 2mFo-Fc and mFo-Fc density map of ion gate in the TmPPase:ETD (cyan) and TmPPase:ZLD (green) structure.

DEER distance distributions of TmPPase T211R1 under different conditions.

A. Structure of the TmPPase dimer (PDB 5LZQ), with monomers A and B coloured cyan and purple, respectively. The sites that were mutated to cysteine and labelled with MTSSL are shown by maroon spheres, with T211R1 on the cytoplasmic (top) side of the membrane interface. B. DEER raw data traces for T211R1. Each condition measured is coloured according to the condition used. C. DEER background-corrected time-domain traces for T211R1. D. The overlap between the predicted distance distribution of T211R1 from the solved crystal structures (TmPPase:Ca, TmPPase:Ca:ETD, TmPPase:ZLD, and TmPPase:IDP), shown as dashed lines, with the resulting DEER distance distributions at the respective conditions. The grey-shaded regions represent the uncertainty in the distribution. The data were all processed in DeerAnalysis2022, with validation in the same way as described in the methods.

CW-EPR spectra of TmPPase T211R1, C599R1, and S525R1 under different conditions

The CW-EPR data were collected at X-band frequency (9.4 GHz) and room temperature (298 K) before the addition of deuterated ethylene glycol for snap freezing. A. The normalised CW-EPR data for TmPPase T211R1 in its apo form (red solid line), +ETD (maroon solid line) and +IDP (cyan solid line) added. The CW-EPR spectra are vertically offset to aid visualisation. B. The normalised CW-EPR data for TmPPase S525R1 for apo (red solid line), +Ca (II) (green solid line), +Ca/ETD (magenta solid line) and +ETD (maroon solid line) conditions. The CW-EPR spectra are vertically offset to aid visualisation. C. The normalised CW-EPR data for TmPPase C599R1 for all tested conditions. The features corresponding to immobile components in the spectra are indicated by grey dashed lines. The CW-EPR spectra are vertically offset to aid visualisation.

ComparativeDeerAnalyzer (CDA) data of TmPPase S525R1, C599R1 and T211R1.

A. The raw data for TmPPase S525R1, colour coded as in the main text. The grey solid lines correspond to three-dimensional homogeneous background functions, while the black dashed lines are the associated Tikhonov fits generated by the automated CDA software. The data are offset vertically to aid visualisation. B. The corresponding consensus distance distributions for TmPPase S525R1, generated by the automated CDA software. The shaded regions correspond to the 95% (2σ) confidence interval. The colour scheme is consistent with panel A. C. The raw data for TmPPase C599R1, colour coded as in the main text. The grey solid lines correspond to three-dimensional homogeneous background functions, while the black dashed lines are the associated Tikhonov fits generated by the automated CDA software. The data are offset vertically to aid visualisation. D. The corresponding consensus distance distributions for TmPPase C599R1, generated by the automated CDA software. The shaded regions correspond to the 95% (2σ) confidence interval. The colour scheme is consistent with panel C. E. The raw data for TmPPase T211R1, colour coded as in the main text. The grey solid lines correspond to three-dimensional homogeneous background functions, while the black dashed lines are the associated Tikhonov fits generated by the automated CDA software. The data are offset vertically to aid visualisation. F. The corresponding consensus distance distributions for TmPPase T211R1, generated by the automated CDA software. The shaded regions correspond to the 95% (2σ) confidence interval. The colour scheme is consistent with panel E. The data are offset vertically to aid visualisation.

X-ray data collection and refinement statistics.

Structural alignments between chains of different TmPPase structures