Figures and data in Mechanism of Ca2+ transport by ferroportin

Figures
Tables
Additional files

5 figures, 2 tables and 1 additional file

Figures

Figure 1 with 4 supplements

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Specific Ca²⁺ uniport by HsFpn.

(a) Ca²⁺ influx by HsFpn in proteoliposomes measured by fluorescence changes (F/F₀) of Fluo-4 (green). The presence of 11F9 Fab (purple) or hepcidin (orange) reduces the Ca²⁺ influx. (b) Comparison of initial rates of Ca²⁺ influx in (a). (c) Ca²⁺ uptake in HEK cells expressing HsFpn monitored by Fluo-4 loaded inside cells. Cells overexpressing Fpn show a significantly faster Ca²⁺ uptake compared to control cells transfected with an empty vector. (d) Comparison of initial rates of Ca²⁺ uptake in (c). (e) Ca²⁺ influx by HsFpn in proteoliposomes at different outside (o) pHs. The inside pH is maintained at 7.5. (f) Comparison of initial rates of Ca²⁺ transport at different outside pHs. (g) Ca²⁺ influx by HsFpn in proteoliposomes at different membrane potentials. Valinomycin was used to generate a membrane potential prior to the addition of Ca²⁺. (h) Comparison of initial rates of Ca²⁺ transport at different membrane potentials. In all the transport assays, 500 µM of Ca²⁺ was added at time zero. Statistical significances were analyzed with one-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparisons. In this article, all time-dependent fluorescence traces are shown as solid lines (mean) with shaded regions (standard deviation, SD) from at least three biological repeats. For all bar graphs, a scatter plot is overlaid on each bar. The height represents the mean of at least three measurements, and the error bar standard error of the mean (SEM). Statistical significances are indicated as follows: ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p<0.0001.

Figure 1—figure supplement 1

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Expression of HsFpn and validation of Ca²⁺ transport in proteoliposomes.

(a) SEC profile (left) and SDS-PAGE gel image (right) of purified HsFpn. (b) Calcimycin, a Ca²⁺ ionophore, collapses the Ca²⁺ gradient after 450 s of Ca²⁺ influx by Fpn in proteoliposomes. Schematics of Ca²⁺ transport by Fpn (pale green and light orange lobes) in proteoliposomes with (c) no Fab; (d) Fab (slate purple lobes) on the outside (o); (e) Fab on both the inside and outside (i/o). (f) Inhibition of Fpn-mediated Ca²⁺ influx in proteoliposomes by Fab added to different sides. (g) Comparison of initial rates of Ca²⁺ influx in (f). Unpaired t-test with Welch correction was used to analyze statistical significances. In (b) and (f), 500 µM of Ca²⁺ was added at time zero.

Figure 1—figure supplement 1—source data 1 Uncropped SDS-PAGE gel image of purified HsFpn. Black dashed rectangle indicates the region of interest.: https://cdn.elifesciences.org/articles/82947/elife-82947-fig1-figsupp1-data1-v2.zip
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Figure 1—figure supplement 2

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H⁺-independent Ca²⁺ uptake by HsFpn in HEK cells.

Intracellular pH change induced by addition of Co²⁺ (a) or Ca²⁺ (b) on the extracellular side monitored by a pH-sensitive dye (pHrodo Red). (c) Comparison of cytosolic pH changes induced by the Co²⁺ or Ca²⁺ uptake. The mean final fluorescence changes from the traces in (a) and (b) are plotted. Decreased relative fluorescence (ΔF/F₀) correlates to increased pH. Sidak’s multiple comparisons test was used following two-way ANOVA. (d) Ca²⁺ transport rates at different extracellular pHs. Two-way ANOVA: among different pHs, p=0.912; between empty and WT, p<0.0001; interaction, p=0.918.

Figure 1—figure supplement 3

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Effect of Na⁺, K⁺, and Cl^- on Ca²⁺ transport by HsFpn.

(a) Ca²⁺ influx in proteoliposomes with symmetrical NaCl, KCl, and K-Gluconate. 500 µM Ca²⁺ was added at time zero. (b) Comparison of initial rates of Ca²⁺ transport in different salts. Statistical significances were analyzed with one-way ANOVA.

Figure 1—figure supplement 4

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Dose-dependent Ca²⁺ transport by Fpn.

(a) Ca²⁺ transport into liposome with purified Fpn monitored by impermeable Fluo-4. Different concentrations of Ca²⁺ (labeled to the right of each trace) were added followed by the addition of valinomycin (val) to clamp membrane potential. The time points for the addition of Ca²⁺ or valinomycin are indicated by vertical dashed lines. For the ‘No protein’ control, 500 µM Ca²⁺ was used. The inset shows the schematic of the proteoliposomes with a 100:1 inside:outside K⁺ gradient. (b) Initial rates of fluorescence change after the addition of valinomycin versus concentrations of Ca²⁺ for data in (a). (c) Dose-dependent Fpn-specific Ca²⁺ uptake in HEK cells expressing Fpn measured by Fluo-4 loaded inside cells. The raw traces of the empty control group at a certain [Ca²⁺] were subtracted from the corresponding traces of the Fpn WT group. (d) Initial rates of fluorescence change versus concentrations of Ca²⁺ in HEK cells. The black solid lines in (b) and (d) are curve fits to a Michaelis–Menten equation.

Figure 2 with 3 supplements

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Structure of HsFpn bound to Ca²⁺.

(a) Cryo-EM map of HsFpn in complex with 11F9 in the presence of Ca²⁺. Densities for NTD, CTD, and 11F9 are colored in pale green, light orange, and slate purple, respectively. A smoothened map contoured at a low threshold (translucent grey) is overlaid to show the lipid nanodisc density around Fpn. (b) Structure of HsFpn with a bound Ca²⁺ in an outward-facing conformation. NTD and CTD are colored as described in (a). Ca²⁺ is shown as a green sphere. (c) Zoomed-in view of the Ca²⁺ binding site in the NTD. The five residues coordinating Ca²⁺ are labeled and shown as side chain sticks. The density for Ca²⁺ is contoured at 7.5σ as blue mesh. (d) Structural comparison of apo (grey, PDB ID 6W4S), Co²⁺-bound (pink, PDB ID 8DL8), and Ca²⁺-bound (green) HsFpn near S1. The side chains of D39 and H43 are shown as sticks. Co²⁺ is shown as a pink sphere. (e), (f), and (g) Three views of conformational changes in NTD induced by Ca²⁺ binding. The Co²⁺-bound (pink) and Ca²⁺-bound (green) structures are aligned and shown as cartoon with cylindrical helices. (e) Top view (from the extracellular side) of the alignment. The helical directions of TM1-6 are visualized by vectors inside cylinders, and the bending of these helices is indicated by black arrows. Bending of TMs viewed from the front (f) and the back (g). The displacement of the extracellular loop between TM3 and TM4 is marked with a black arrow and distance.

Figure 2—figure supplement 1

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Cryo-EM analysis of HsFpn-Ca²⁺ in nanodisc.

(a) Representative electron micrograph (upper panel) and 2D class averages (lower panel). (b) Workflow of single-particle data processing. (c) The gold-standard Fourier shell correlation (FSC) curves (left panel) and map-to-model FSC curves (right panel). (d) Direction distribution of particles used in the final 3D reconstruction. (e) Local resolution map.

Figure 2—figure supplement 2

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Cryo-EM densities of TM helices and amphipathic helices (AH).

Densities for HsFpn-Ca²⁺ are shown as green mesh. Residues within the ranges indicated below are rendered in stick representation and colored in pale green for NTD or light orange for CTD.

Figure 2—figure supplement 3

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Structural comparisons of Ca²⁺-bound HsFpn.

(a) Differences in Ca²⁺ coordination by HsFpn (green) and by BbFpn (deep blue, PDB ID 6BTX). (b) Comparison of Ca²⁺-bound (green) versus hepcidin-bound (deep purple, PDB ID 6WBV) structure of HsFpn shows further outward movements of NTD. Displacement of the loop between TM3 and TM4 is marked with an arrow and distance. Co²⁺ ions in the Fpn-hepcidin structure are shown as deep purple spheres.

Figure 3 with 3 supplements

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Mutations on the Ca²⁺ binding site.

(a) Ca²⁺ influx by WT (green), D39A (blue), and the triple mutant (Q99A_N100A_E219A, purple) of the Ca²⁺ binding site in proteoliposomes. (b) Comparison of initial rates of Ca²⁺ influx in (a). (c) Ca²⁺ uptake in HEK cells expressing WT (green) or the triple mutant (purple) Fpn. (d) Fpn-specific Ca²⁺ transport activities of WT and mutants. Initial rates are subtracted from the empty control. For statistical significances in (b) and (d), Dunnett’s test was used as a post hoc test following one-way ANOVA with the WT as the control. 500 µM Ca²⁺ was used in (a–d). Binding of Ca²⁺ to WT (e), D39A (f), and H43A (g) HsFpn measured by ITC. Upper plot: raw thermogram showing the heat during binding and baseline (red line). Lower plot: integrated heat of each injection (black square) and the fit of data (red line).

Figure 3—figure supplement 1

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Expression of WT and mutant Fpns in HEK cells assessed by western blot.

Figure 3—figure supplement 1—source data 1 Uncropped western blot image of Fpn expressed in HEK cells. Black dashed rectangle indicates the region of interest.: https://cdn.elifesciences.org/articles/82947/elife-82947-fig3-figsupp1-data1-v2.zip
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Figure 3—figure supplement 2

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**Binding of Ca²⁺, Co²⁺**, **or Zn²⁺ to WT and mutant Fpns measured by ITC**.

Binding of Ca²⁺ to HsFpn-11F9 complex (a), TsFpn (b), and S2 mutant (c). (d) Binding of Co²⁺ to the alanine mutant of Q99 in the Ca²⁺ binding site. Titration of Ca²⁺ into the alanine mutants of Q99 (e), N100 (f), N212 (g), and E219 (h) in the Ca²⁺ binding site. Binding of Zn²⁺ to WT (i) and the alanine mutant of N212 (j) in the Ca²⁺ binding site.

Figure 3—figure supplement 3

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Effect of D39A on Co²⁺ transport by Fpn.

(a) Co²⁺ influx in proteoliposomes with symmetrical NaCl buffer. 500 µM Co²⁺ was added at time zero. (b) Comparison of initial rates of Co²⁺ transport. Statistical significances were analyzed with unpaired t-test with Welch correction.

Figure 4 with 3 supplements

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Interplay between Fe²⁺/Co²⁺ and Ca²⁺ in HsFpn.

(a) Ca²⁺ binding in the presence of 2 mM Co²⁺. (b) Co²⁺ binding in the presence of 2 mM Ca²⁺. (c) Fe²⁺ transport into proteoliposomes in the presence or absence of Ca²⁺. The external [Fe²⁺] is 50 µM. ‘1:1’ indicates symmetrical [Ca²⁺] at 1.25 mM and “10:1” indicates 1.25 mM Ca²⁺ inside and 0.125 mM Ca²⁺ outside. 1 mM sodium ascorbate was included in all samples. All fluorescence traces are subtracted from corresponding blank controls using vesicles with no HsFpn. (d) Comparison of initial rates of Fe²⁺ transport in (c). One-way ANOVA was used for statistical analysis. (e) Ca²⁺ (1 mM) uptake by HEK cells expressing HsFpn in the presence (dark yellow) or absence (green) of 80 µM Fe²⁺ that has been pre-loaded into the cells. Uptake is monitored by jGCaMP7s which is co-expressed in cytosol. Cells transfected with an empty vector (dark and light gray) serve as negative controls. Ca²⁺ transport at different concentrations of Fe²⁺ (f) or Co²⁺ (g). Normalized initial rates of Fpn-specific Ca²⁺ uptake were used to represent relative Ca²⁺ transport activities. Data were fitted (black solid line) to an inhibitory dose-response equation to calculate IC₅₀ values.

Figure 4—figure supplement 1

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Fe²⁺/Co²⁺ transport into proteoliposomes by Fpn in the presence or absence of Ca²⁺.

(a) Co²⁺ transport with or without 1.25 mM of intra-vesicular Ca²⁺. (c) Fe²⁺ transport with or without 0.5 mM of intra-vesicular Ca²⁺. (e) Co²⁺ transport with or without 0.5 mM of intra-vesicular Ca²⁺. (b), (d), and (f) Comparison of initial rates in (a), (c), and (e). Liposome samples were diluted (10×) into outside buffers containing 50 µM Co²⁺ or Fe²⁺. ‘1:1’ or ‘10:1’ denotes the ratio of [Ca²⁺] inside:outside achieved after 10×dilution. 1 mM sodium ascorbate was included in (c). All fluorescence traces were subtracted from their corresponding no protein controls. One-way ANOVA was used for statistical analysis.

Figure 4—figure supplement 2

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Ca²⁺ transport in the presence of Fe²⁺ or Co²⁺ in HEK cells.

(a) Schematic of the Ca²⁺ transport assay in Fe²⁺-loaded HEK cells co-expressing Fpn and the Ca²⁺ sensor jGCaMP7s. (b) Transition metal ions do not interact with jGCaMP7s as indicated by unaltered fluorescence readings during loading of Fe²⁺ (top) or Co²⁺ (bottom) into HEK cells expressing Fpn (orange or pink traces) or transfected with empty vectors (gray traces). Black arrows indicate additions of metal ions. (c) Ca²⁺ transport at different extracellular pHs in the presence of 20 µM Co²⁺. Two-way ANOVA: among different pHs, p=0.717; among empty, WT, and S2 mutant, p<0.0001; interaction, p=0.130. (d) and (e) Fpn-specific Ca²⁺ transport in the presence of Fe²⁺ or Co²⁺ loaded inside HEK cells. The concentrations of Fe²⁺ or Co²⁺ used during the loading step are indicated to the right of each trace. The Fe²⁺ stock was prepared with a 10-fold molar excess of sodium ascorbate. The raw traces of the empty control group were subtracted from the corresponding traces of the Fpn WT group. Normalized initial rates calculated from the datasets are shown in Figure 4f–g.

Figure 4—figure supplement 3

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Proposed transport mechanisms of metal ions in Fpn.

(a) Electrogenic Ca²⁺ uniport in the absence of Fe²⁺. (b) Electroneutral Fe²⁺/2H⁺ antiport in the absence of Ca²⁺. (c) Reduced Ca²⁺ flux in the presence of Fe²⁺. Higher concentration of Fe²⁺ (orange ramp) shows larger inhibition on Ca²⁺ transport. The inward-facing model is generated based on the bacterial homolog structure (PDB ID 6BTX). The four-helical bundle (pale green) in NTD, and TM7 and TM11 (light orange) in CTD are shown as cylinders and overlaid on silhouettes (grey) of the whole transporter. S1 and S2 of Fe²⁺ are indicated as black forks with two tines to represent two ligand residues in each site. The Ca²⁺ binding site is indicated as a black circle. When Fe²⁺ is bound to S1, the Ca²⁺ binding site is drawn as an incomplete circle to indicate its partial disruption. Similarly, the S1 becomes a half fork when Ca²⁺ is bound. H⁺ (blue), Fe²⁺ (orange), and Ca²⁺ (green) are shown as spheres. The black dashed line and shaded green spheres of Ca²⁺ in (c) indicate reduced Ca²⁺ release and occupancy.

Author response image 1

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Titration of Ca²⁺ to Fluo-4 used in liposome transport assays.

(a) Emission spectra of 100 µM Fluo-4 in the inside buffer at 37 °C titrated with increasing [Ca²⁺] (from blue to red). (b) Correlation of F/F0 to [Ca²⁺]. The blue line is the linear fit to data in the region of [Ca²⁺] within 2 µM.

Tables

Table 1

Summary of cryo-EM data collection, processing, and refinement.

Sample	HsFpn-Ca²⁺–11 F9
Cryo-EM data collection
Voltage (kV)	300
Magnification (×)	81,000
Pixel Size (Å)	1.10
Electron exposure (e^-/Å²/frame)	1.25
Defocus range (µm)	[-2.5,–0.8]
Number of image stacks	4,498
Number of frames per stack	40
Cryo-EM data processing
Initial number of particles	2,184,301
Final number of particles	437,959
Symmetry imposed	C1
Map resolution (Å)	3.0
Map resolution range (Å)	2.4–3.6
FSC threshold	0.143
Model refinement
Number of amino acids	875
Total non-hydrogen atoms	6,192
Average B factor (Å²)	171.4
Bond length RMSD (Å)	0.008
Bond angle RMSD (°)	0.939
Ramachandran Plot
Favored (%)	93.53
Allowed (%)	6.24
Outliers (%)	0.23
Rotamer outliers (%)	1.25
MolProbity Score	1.98

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Chemical compound, druC	n-Dodecyl-β-D-Maltopyranoside	Anatrace	Cat#D310
Chemical compound, drug	Lauryl maltose neopentyl glycol	Anatrace	Cat#NG310
Chemical compound, drug	1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC)	Avanti Polar Lipids	Cat#850457 C
Chemical compound, drug	1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE)	Avanti Polar Lipids	Cat#850457 C
Chemical compound, drug	1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG)	Avanti Polar Lipids	Cat#840457 C
Chemical compound, drug	Calcein	Invitrogen	Cat#C481
Chemical compound, drug	Fluo-4, Pentapotassium Salt, cell impermeant	Invitrogen	Cat#F14200
Chemical compound, drug	Valinomycin	Sigma	Cat#V0627
Peptide, recombinant protein	Hepcidin-25 (human)	Sigma	Cat#SML1118
Commercial assay, kit	TALON Metal Affinity Resin	TaKaRa	Cat#635504
Commercial assay, kit	SRT-10C SEC-300	Sepax Technologies	Cat#239300–10030
Commercial assay, kit	Q Sepharose Fast Flow	GE Healthcare	Cat#17051010
Commercial assay, kit	Biobeads SM2	Bio-Rad	Cat#1528920
Commercial assay, kit	PD-10 Desalting Column	GE Healthcare	Cat#17085101
Commercial assay, kit	400 nm NanoSizer Extruder	T&T Scientific Corporation	Cat#TT-004–0010
Commercial assay, kit	KOD Hot Start DNA polymerase	Novagen	Cat#71086–3
Commercial assay, kit	Fluo-4, AM, cell permeant	Invitrogen	Cat#F14201
Commercial assay, kit	pHrodo Red AM	Invitrogen	Cat#P35372
Recombinant DNA reagent	pGP-CMV-jGCaMP7s	Addgene	Plasmid #104463	RRID:Addgene_104463
Software, algorithm	MotionCorr2	Zheng et al., 2017	https://msg.ucsf.edu/em/software/motioncor2.html
Software, algorithm	Gctf	Zhang, 2016	https://www.mrc-lmb.cam.ac.uk/kzhang/Gctf/	RRID:SCR_016500
Software, algorithm	Relion 3.0	Kimanius et al., 2016	https://www3.mrc-lmb.cam.ac.uk/relion	RRID:SCR_016274
Software, algorithm	CryoSPARC	Punjani et al., 2017	https://cryosparc.com/	RRID:SCR_016501
Software, algorithm	ChimeraX	Pettersen et al., 2021	https://www.cgl.ucsf.edu/chimerax/	RRID:SCR_015872
Software, algorithm	COOT	Emsley et al., 2010	https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/	RRID:SCR_014222
Software, algorithm	PHENIX	Adams et al., 2010	http://www.phenix-online.org/	RRID:SCR_014224
Software, algorithm	EMringer	Barad et al., 2015	http://fraserlab.com/2015/02/18/EMringer/
Other	Grids: R1.2/1.3 Cu 300 mesh	Quantifoil	Cat#Q325CR1.3	Cryo-EM grid