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 MgCl2 (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. Superposition of chain A (cyan) and chain B (wheat) showing the relative movements (black arrow) of helices. B. Superposition of TMH5 and TMH6 in TmPPase:ETDs and TmPPase:IDP (orange; PDB: 5LZQ) showing the movement of loop5-6 and reorientation of ETDA, ETDB and IDP. The dashed line shows the interaction of E2175.76 of loop5-6 in Chain A with ETDA and IDP, and D2185.77 of loop5-6 in Chain B with ETDB.; Close-up view of IDP superposed with ETDA and ETDB. C. Residues in the active site with ETDA coordinated (dashed lines), Ca2+ (pink sphere) and nucleophilic water (red spheres) in a Mg2+ metal cage (green spheres). D. Residues in 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. 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 view of the active site in TmPPase:IDP. D. 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. 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. 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 under different conditions.

A. Structure of the TmPPase dimer, with monomers A and B coloured cyan and purple, respectively. The sites mutated to cysteine and labelled with MTSSL are shown by maroon spheres, with T211R1 on the cytoplasmic (top) side and S525R1 on the periplasmic (bottom) side of the membrane. B. DEER raw data traces for S525R1. Each condition measured is coloured accordingly. C. DEER background corrected time-domain traces for S525R1. The black dashed line represents the base of the oscillation in the apo state and acts as a guide to highlight the small distance changes where the base of the oscillation comes before (shorter distance) or after (longer distance) the line. D. The overlap between the predicted distance distribution of S525R1 from the solved crystal structures (TmPPase:Ca, TmPPase:Ca:ETD, TmPPase:ZLD, and TmPPase:IDP), shown as coloured dashed lines, with the resulting DEER distance distributions at the respective conditions. The vertical black dashed line represents the mean distance for the apo state to highlight changes from this to shorter and longer distances. The grey-shaded regions represent the uncertainty in the distribution and the coloured bar above the x-axis represents the DeerAnalysis ‘traffic light’ system for assessing the reliability of the distribution presented. For the time window of this dataset, the distances within the regions highlighted in green and yellow indicate the reliability of the mean distance, peak shape, and width. For distance peaks within the orange region (> 5.0 nm), only the mean distance is considered reliable70. The data were all processed in DeerAnalysis, with validation in the same way as described in the methods.

Combinded model of Ca (monomer A) and IDP (monomer B) structures

A. Side and bottom views of the symmetric and asymmetric structures for IDP bound to TmPPase. The asymmetric model combines a monomer (A) of TmPPase_Ca and a monomer (B) of TmPPase_IDP. B. The overlap of their predicted distance distribution and resulting experimental DEER distance distribution for S525R1.

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. C-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 T211R1.

Five DEER models of PfPPase bound bisphosnates. TMH12, 13 and loop12-13 are shown in brown; TMH5, 6 and loop5-6 are shown in green. The labelling sites are represented by maroon spheres. Within the structures, Ca2+ is shown as a green circle; ETD by a purple square; ZLD by an orange pentagon and IDP as blue square.

Inhibition of TmPPase by bisphosphonates.

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

Accessible cystines for NEM modification.

A. Two exposed cysteines in both monomers of TmPPase:Ca (PDB: 4AV3; cyan). B. One exposed cystine 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 (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 (blue mesh) map of ETDB, ions and surrounding residues (Right). 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 (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 and S525R1 on the periplasmic (bottom) 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 DeerAnalysis, with validation in the same way as described in the methods.

CW EPR spectra of TmPPase T211R1 and S525R1 under different conditions.

The CW EPR data were collected at room temperature before the addition of ethylene glycol and therefore represent the rotational motion of the spin labels in comparison to each other. A. The normalized CW EPR data for T211R1 in its apo form, and with ETD and IDP added. B. The normalized CW EPR data for S525R1 for apo, +Ca(II), +Ca/ETD and +ETD conditions.

ComparativeDeerAnalyzer (CDA) data of TmPPase S525R1 and T211R1.

A. The background-corrected data and distance distributions for S525R1 generated by the automated CDA software. B. The background-corrected data and distance distributions for T211R1 generated by the automated CDA software.

X-ray data collection and refinement statistics.

Structural alignments between chains of different TmPPase structures