1. Structural Biology and Molecular Biophysics

Cryo-EM structures and functional characterization of murine Slc26a9 reveal mechanism of uncoupled chloride transport

  1. Justin D Walter
  2. Marta Sawicka
  3. Raimund Dutzler  Is a corresponding author
  1. University of Zurich, Switzerland
https://doi.org/10.7554/eLife.46986
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Cite this article
  1. Justin D Walter
  2. Marta Sawicka
  3. Raimund Dutzler
(2019)
Cryo-EM structures and functional characterization of murine Slc26a9 reveal mechanism of uncoupled chloride transport
eLife 8:e46986.
https://doi.org/10.7554/eLife.46986
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8 figures, 5 videos, 3 tables and 1 additional file

Figures

Figure 1 with 3 supplements
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Functional properties of Slc26a9.

(A) Cl– transport of Slc26a9T reconstituted into proteoliposomes, monitored by the fluorescence change of the pH gradient-sensitive fluorophore ACMA. (*) Indicates addition of the H+ ionophore CCCP, …

https://doi.org/10.7554/eLife.46986.003
Figure 1—figure supplement 1
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Sequence alignment and topology.

(A) Sequence alignment of the murine Slc26 paralogs Slc26a2 (NP_031911.1), Slc26a4 (NP_035997.1), Slc26a5 (NP_109652.3), and Slc26a9 (NP_796217.2). Identical residues are highlighted in green, …

https://doi.org/10.7554/eLife.46986.004
Figure 1—figure supplement 2
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Expression, purification and functional characterization of Slc26a9T.

(A) Fluorescence-microscopy images of HEK293T cells transfected with different Slc26a9 constructs (From left to right: full-length Slc26a9, the ΔIVSΔCT truncation construct Slc26a9T, the ΔIVS …

https://doi.org/10.7554/eLife.46986.005
Figure 1—figure supplement 3
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Functional properties of Slc26a9T.

(A) I–V relationships for excised inside-out patches from HEK293T cells expressing Slc26a9T-vYFP, under varying NaCl gradients. The perfusate (intracellular) NaCl concentration was varied from 0 to …

https://doi.org/10.7554/eLife.46986.006
Figure 2 with 4 supplements
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Slc26a9T structure.

(A) Cryo-EM density of Slc26a9T in the detergent GDN at 3.96 Å contoured at 5σ. Density corresponding to distinct subunits in the dimeric protein is colored in blue and red respectively. Residual …

https://doi.org/10.7554/eLife.46986.007
Figure 2—figure supplement 1
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Cryo-EM reconstruction of Slc26a9T in detergent at 3.96 Å.

(A) Representative cryo-EM micrograph acquired with Titan Krios microscope. (B) 2D class averages of Slc26a9T. (C) Angular distribution plot of all particles included in the final C2-symmetrized …

https://doi.org/10.7554/eLife.46986.008
Figure 2—figure supplement 2
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Cryo-EM density of the Slc26a9T structure in detergent.

(A) Sections of the cryo-EM density (gray, 7σ) superimposed on the refined structure of Slc26a9T. Secondary structure elements are labeled. (B) Stereo view of cryo-EM density (7σ) of the …

https://doi.org/10.7554/eLife.46986.009
Figure 2—figure supplement 3
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Cryo-EM reconstruction of Slc26a9T in nanodiscs at 7.8 Å.

(A) Representative cryo-EM micrograph acquired with Tecnai G2 Polara microscope. (B) 2D class averages of Slc26a9T. (C) Angular distribution plot of all particles included in the final …

https://doi.org/10.7554/eLife.46986.010
Figure 2—figure supplement 4
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Cryo-EM density of the Slc26a9T structure in nanodiscs.

(A) Cryo-EM density (6σ) of Slc26a9T in lipid nanodiscs at 7.77 Å. Density corresponding to distinct subunits is colored in cyan and salmon respectively. (B) Left, Cα-representation of the refined …

https://doi.org/10.7554/eLife.46986.011
Figure 3 with 2 supplements
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STAS domain and dimer interface.

(A) Ribbon representation of the STAS domain dimer and interacting parts of the N-terminus and the TMD. The view is as in Figure 2B. (B) Elements of mutual STAS domain interactions and contacts with …

https://doi.org/10.7554/eLife.46986.016
Figure 3—figure supplement 1
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Effect of N-terminal truncation of Slc26a9T on function and stability.

(A) Depiction of the interaction of residues 5–25 of both subunits with the STAS domain dimer. The N-termini are displayed as ribbon interacting with the STAS domain depicted by its molecular …

https://doi.org/10.7554/eLife.46986.017
Figure 3—figure supplement 2
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Structural features of the Slc26a9 STAS domain.

(A) Ribbon representation of the STAS domain of a single subunit of Slc26a9T with secondary structure elements labeled. (B) Superposition of the STAS domain of Slc26a9T with the X-ray structures of …

https://doi.org/10.7554/eLife.46986.018
Figure 4 with 1 supplement
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Transmembrane domain.

(A) Ribbon representation of the TMD of a single subunit of Slc26a9T viewed from the extracellular side. (B) View of the gate and (C) the core module from within the membrane. Green sphere indicates …

https://doi.org/10.7554/eLife.46986.019
Figure 4—figure supplement 1
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Structural features of the TMD.

(A) Ribbon representation of the TMD of a single subunit of Slc26a9T viewed from within the membrane. The N-terminal (α1-α7) and C-terminal (α8-α14) halves are colored in green and magenta …

https://doi.org/10.7554/eLife.46986.020
Figure 5
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Comparison of the TMD.

(A) Structural superposition of the TMDs of Slc26a9T (beige and blue) and SLC26Dg (PDBID: 5DA0, cyan). (B) Superposition of the gate domains and (C) core domains of Slc26a9T and SLC26Dg. N-terminal …

https://doi.org/10.7554/eLife.46986.021
Figure 6 with 1 supplement
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Electrostatic properties of the intracellular cavity.

(A) View of the intracellular cavity leading to the Cl–-binding site. Section of the molecular surface is shown with contact region of acidic residues colored in red and basic residues in blue. (B) …

https://doi.org/10.7554/eLife.46986.023
Figure 6—figure supplement 1
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Functional properties of mutants of basic residues.

(A) Introduction of negative charges at positions of basic residues distal to the putative anion-binding site show negligible effects on the linear I–V relation. Shown are I–V relationships of …

https://doi.org/10.7554/eLife.46986.024
Figure 7 with 3 supplements
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Substrate binding site.

(A) Structure of the Cl– binding site. The molecular surface of the binding pocket is shown. Selected residues are displayed as sticks. Bound Cl– is shown as a green sphere. (B) I-V relationships of …

https://doi.org/10.7554/eLife.46986.025
Figure 7—figure supplement 1
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I-V-relationships of anion binding site mutants.

(A–G) Alanine mutants investigated by inside-out patch-clamp electrophysiology of HEK392T cells expressing Slc26a9T anion binding-site mutations. Binding-site mutations do not alter Cl– selectivity, …

https://doi.org/10.7554/eLife.46986.026
Figure 7—figure supplement 2
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Anion selectivity of binding site mutants.

Currents were recorded by inside-out patch-clamp electrophysiology at asymmetric conditions containing 150 mM extracellular Cl– and 150 mM of the indicated anion. (A–G) Top, Bi-ionic I–V …

https://doi.org/10.7554/eLife.46986.027
Figure 7—figure supplement 3
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Kinetic properties of anion binding site mutants.

Data was recorded from excised patches. (A–E) Relative permeabilities (Px/PCl, green) and macroscopic conductivities (Gx/GCl, orange) for (A) Q88A, (B) F92A, (C) T127A, (D) L391A and (E) N441A. …

https://doi.org/10.7554/eLife.46986.028
Figure 8 with 3 supplements
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Transport mechanism.

(A) Molecular surface of Slc26a9T viewed towards the long dimension of the molecule. (B) Sections of the TMD in the inward-facing conformation defined by the Slc26a9T detergent structure (left), an …

https://doi.org/10.7554/eLife.46986.031
Figure 8—figure supplement 1
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Dimer architecture of transport proteins sharing the 7 + 7 inverted repeat topology.

The TMDs of selected transporters are shown as representatives for their respective family as ribbon. (A), SLC26 family: Slc26a9, (B), SLC4 family: SLC4A1/Band 3 (PDBID: 4YZF), (C), SLC23 family: …

https://doi.org/10.7554/eLife.46986.032
Figure 8—figure supplement 2
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Ion binding by SLC26 proteins.

Putative anion binding region of murine Slc26 paralogs are shown as Cα-trace with selected side-chains in interaction distance with the bound anion displayed as sticks. Numbering corresponds to the …

https://doi.org/10.7554/eLife.46986.033
Figure 8—figure supplement 3
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Mechanistic relationships within the SLC26 family.

Schematic depiction of plausible kinetic cycles underlying different transport modes observed in the SLC26 family. States occupied in a channel-like uncoupled bidirectional uniport as observed in …

https://doi.org/10.7554/eLife.46986.034

Videos

Video 1
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Cryo-EM density map of the dimeric Slc26a9T.

Shown is the cryo-EM map of the protein in detergent with the refined model of the inward-facing state superimposed.

https://doi.org/10.7554/eLife.46986.013
Video 2
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Cryo-EM density map of the dimeric Slc26a9T in nanodiscs.

Shown is the cryo-EM map of the protein in nanodiscs with the modeled structure of the intermediate state superimposed.

https://doi.org/10.7554/eLife.46986.014
Video 3
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The structure of Slc26a9T.

Shown are unique features of a mammalian SLC26 transporter. The oligomerization interface is minimal between the transmembrane domains and is predominantly mediated by the swapped STAS domains. The …

https://doi.org/10.7554/eLife.46986.015
Video 4
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Conformational transition from the inward-facing to the intermediate state.

Ribbon representation of the transmembrane domain as a morph between the inward-facing and intermediate states. The structure is viewed from the peripheral. Residues 276 to 312 are removed for …

https://doi.org/10.7554/eLife.46986.022
Video 5
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Structure of the presumed ion binding site.

Ribbon representation of the anion binding site with side-chains of interacting residues displayed as sticks. Surface of the binding pocket around a modeled chloride (green sphere) is shown. View is …

https://doi.org/10.7554/eLife.46986.030

Tables

Table 1
Cryo-EM data collection, refinement and validation statistics.
https://doi.org/10.7554/eLife.46986.012
Dataset 1
Slc26a9T in detergent
(EMD-4997)
(PDB 6RTC)		Dataset 2
Slc26a9T in nanodiscs
(EMD-4998)
(PDB 6RTF)
Data collection and processing
MicroscopeFEI Titan KriosFEI Tecnai G2 Polara
CameraGatan K2 Summit + GIFGatan K2 Summit + GIF
Magnification6,51137,313
Voltage (kV)300300
Electron exposure (e–/Å2)7060
Defocus range (μm)−0.5 to −3.0−0.5 to −3.0
Pixel size (Å)*1.075 (0.5375)1.34
Symmetry imposedC2C2
Initial particle images (no.)416,164711,032
Final particle images (no.)112,93017,442
Map resolution (Å)
FSC threshold 0.143		3.967.77
Map resolution range (Å)3.0–4.26.0–10.0
Refinement
Model resolution (Å)
FSC threshold 0.5		4.08.0
Model resolution range (Å)118–3.96
Map sharpening b-factor (Å2)−205−512
Model composition
Non-hydrogen atoms
Protein residues		9570
1242		9570
1242
B factors (Å2)
Protein		7373
Refmac FSCavg/Rfactor0.851/0.351
R.m.s. deviations
Bond lengths (Å)
Bond angles (°)		0.005
0.888		0.006
1.195
Validation
MolProbity score
Clashscore
Poor rotamers (%)		1.10
1.19
0		1.07
0.78
0
Ramachandran plot
Favored (%)
Allowed (%)
Disallowed (%)		96.10
3.90
0		95.28
4.72
0

  1. *Values in parentheses indicate the pixel size in super-resolution.

Table 2
Transport properties of WT and mutated Slc26a9T.
https://doi.org/10.7554/eLife.46986.029
WT*Q88AF92AT127AF128AL391AS392AN441A
SCN–Erev (mV)†
Px/PCl
Gx/GCl		50.5
7.5
0.43		58.4
10.6
0.55		37.8
4.5
0.44		59.3
10.7
0.80		68.7
14.9
1.46		61.5
12.0
1.10		71.5
18.0
1.39		52.9
8.2
0.57
I–Erev (mV)
Px/PCl
Gx/GCl		26.6
2.9
0.23		16.3
1.9
0.14		14.1
1.8
0.25		9.5
1.5
0.17		31.0
3.5
0.53		23.5
2.6
0.54		53.2
8.4
1.31		29.1
3.2
0.35
Br–Erev (mV)
Px/PCl
Gx/GCl		21.3
2.4
0.60		3.7
1.2
0.32		5.1
1.2
0.72		7.6
1.4
0.40		24.6
2.7
1.13		16.1
1.9
1.11		40.7
5.2
1.78		20.0
2.2
0.70
NO3–Erev (mV)
Px/PCl
Gx/GCl		9.4
1.5
0.49		32.6
3.7
1.20		11.2
1.6
0.52		29.0
3.2
1.46		28.5
3.1
1.04		20.7
2.3
0.70		39.1
4.8
1.31		16.9
2.0
0.77
Cl–Erev (mV)
Px/PCl
Gx/GCl		−0.1
1
1		−1.4
1
1		−1.5
1
1		−0.5
1
1		0
1
1		−0.7
1
1		0.3
1
1		−0.3
1
1
F–Erev (mV)
Px/PCl
Gx/GCl		−81.1
.05
nd		−64.2
.08
nd		−81.0
.04
nd		−74.5
.07
nd		−67.6
.07
nd		−81.0
.05
nd		−58.8
0.11
nd		−86.7
0.03
nd
HCO3–Erev (mV)
Px/PCl
Gx/GCl		−81.7
0.05
nd		−54.4
0.12
nd		−69.5
0.07
nd		−84.7
0.04
nd		−47.3
0.16
nd		−93.1
0.03
nd		−57.4
0.10
nd		−76.0
0.05
nd
SO42–Erev (mV)
Px/PCl
Gx/GCl		−96.9
0.01
nd		−102.2
0.01
nd		−78.0
0.01
nd		−104.5
0
nd		−70.5
0.02
nd		−93.6
0.01
nd		−71.2
0.02
nd		−82.4
0.01
nd
E30mM Cl‡−37.7−39.3−38.5−39.8−42.1−37.9−39.0−38.3
Km,Cl (mM) §29.52.319.24.718.644.054.131.3
Km,SCN (mM) §0.50.20.81.65.229.43.80.7
VSCN/VCl §0.440.630.440.741.081.080.830.68

  1. *WT refers to non-mutated Slc26a9T.

    †Erev values, Px/PCl, and Gx/GCl were derived from bi-ionic data in Figure 1—figure supplement 3B–F, and Figure 7—figure supplement 2.

  2. ‡Measured reversal potential, in mV, under a 5-fold NaCl gradient (ECl = –40.2 mV). Extracted from data in Figure 1C and Figure 7—figure supplement 1A–H.

    §Km and VSCN/VCl values are derived from conductance–concentration data presented in Figure 1D, Figure 7, and Figure 7—figure supplement 3F–J.

  3. nd, not determined.

Key resources table
Reagent type
(species) or resource		DesignationSource or referenceIdentifierAdditional
information
Chemical compound, drugPro293S-CDM mediumLonzaCat#BE02-025Q
Chemical compound, drugHyClone HyCell TransFx-H mediumGE HealthcareCat#SH30939.02
Chemical compound, drugL-glutamineMillipore SigmaCat#G7513
Chemical compound, drugPenicillin-streptomycinMillipore SigmaCat#P0781
Chemical
compound, drug		Fetal bovine serumMillipore SigmaCat#F7524
Chemical compound, drugPluronic F-68ThermoFisher ScientificCat#24040032
Chemical compound, drugPolyethylenimine 25 K MW, linearPolysciencesCat#23966–1
Chemical compound, drugDulbecco’s Modified Eagle’s Medium (DMEM)Millipore SigmaCat#D5546
Chemical compound, drugValproic acidMillipore SigmaCat#P4543
Chemical compound, drugcOmplete, EDTA-free Protease Inhibitor CocktailRocheCat#5056489001
Chemical compound, drugDigitoninAppliChemCat#A1905
Chemical compound, drugGlyco-diosgeninAnatraceCat#GDN101
Chemical compound, drugD-desthiobiotinMillipore SigmaCat#D1411
Chemical compound, drugn-dodecyl-β-D-maltoside (DDM)AnatraceCat#D310
Chemical compound, drugCholesteryl
hemisuccinate, tris salt (CHS)		AnatraceCat#CH210
Chemical compound, drug1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE)Avanti Polar Lipids, IncCat#850757
Chemical compound, drug1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG)Avanti Polar Lipids, IncCat#840457
Chemical compound, drugDiethyl etherMillipore SigmaCat#296082
Chemical compound, drugTriton X-100Millipore SigmaCat#T9284
Chemical compound, drugBiotinMillipore SigmaCat#B4501
Chemical compound, drug9-amino-6-chloro-2-methoxyacridine (ACMA)Thermo Fisher ScientificCat#A1324
Chemical compound, drugcarbonyl cyanide 3-chloropheny
lhydrazone (CCCP)		Merck MilliporeCat#C2759
Chemical compound, drug4,4’-Diisothiocyanatostilbene-2,2’-disulfonic acid (DIDS)Millipore SigmaCat#D3514
Commercial assay or kitQuikChange site-directed mutagenesis kitAgilentCat#200523
Commercial assay or kitNucleoBond Xtra Maxi kitMacherey-NagelCat#740416
Commercial assay or kitStrepTactin Superflow affinity resin slurryIBA LifesciencesCat#2-1206-002
Commercial assay or kitSuperose 6 10/300 GLGE HealthcareCat#17-5172-01
Commercial assay or kitZorbax GF-450AgilentCat#884973–902
Commercial assay or kitSuperose 6 5/150GE HealthcareCat#29091597
Commercial assay or kitPierce Streptavidin Plus UltraLink ResinThermo Fisher ScientificCat#53117
Commercial assay or kitBio-Beads SM-2Bio-RadCat# 1523920
Commercial assay or kitAvestin LiposoFast Liposome Factory BasicMillipore SigmaCat#Z373400
Commercial assay or kit400 nm polycarbonate filters for LiposoFastMillipore SigmaCat#Z373435
Commercial assay or kit96-well black-walled microplateThermo Fisher ScientificCat#M33089
Commercial assay or kit200 mesh Au 1.2/1.3 cryo-EM gridsQuantifoilCat#N1-C14nAu20-01
Commercial assay or kitAmicon 100 kDa MWCO centrifugal filterEMD MilliporeCat#UFC910008
Commercial assay or kit0.22 µm Ultrafree-MC Centrifugal FilterEMD MilliporeCat#UFC30GV
Commercial assay or kitBorosilicate glass capillary with filamentSutter InstrumentCat#BF150-86-10HP
Cell line (human)HEK293S GnTI-ATCCCRL-3022
Cell line (human)HEK-293TATCCCRL-1573
Recombinant DNAMus musculus Slc26a9 ORF shuttle cloneSource BioScienceORFeome# OCACo5052B0115D;
GenBank BC160193
Recombinant DNApcDNA 3.1 (+)vector, InvitrogenThermo Fisher ScientificCat# V79020
Recombinant DNAModified pcDNA 3.1 vector with C-terminal 3C protease cleavage site, Venus and Myc tags and streptavidin binding peptideRaimund Dutzler laboratoryN/A
Recombinant DNAModified pcDNA 3.1 vector with C-terminal 3C protease cleavage site, Myc tag and streptavidin binding peptideRaimund Dutzler laboratoryN/A
Recombinant DNAExpression vector encoding membrane scaffold protein (MSP) E3D1, pMSP1E3D1Denisov et al., 2007Addgene, Cat#20066
Software,algorithmSerialEM 3.5.0Mastronarde, 2005http://bio3d.colorado.edu/SerialEM/
Software, algorithmRELION-3.0Scheres, 2012https://www2.mrc-lmb.cam.ac.uk/relion/
Software, algorithmCTFFIND4.1Rohou and Grigorieff, 2015http://grigoriefflab.jan elia.org/ctf
Software, algorithmBsoft 1.9.5Heymann and Belnap, 2007https://lsbr.niams.nih.gov/bsoft/
Software, algorithmCoot 0.8.8Emsley and Cowtan, 2004https://www2.mrc-lmb.cam.ac.uk/person al/pemsley/coot/
Software, algorithmPHENIX 1.14Adams et al., 2002http://phenix-online.org/
Software, algorithmREFMAC5Murshudov et al., 2011http://www.ccpem.ac.uk/
Software, algorithmMSMSSanner et al., 1996http://mgltools.scripps.edu/packages/MSMS/
Software, algorithmDINO 0.9.4http://www.dino3d.orghttp://www.dino3d.org
Software, algorithmPyMOL 2.3.0DeLano, 2002https://pymol.org/2/
Software, algorithmChimera 1.13.1Pettersen et al., 2004http://www.cgl.ucsf.edu/chimera/
Software, algorithmChimeraX 0.7Goddard et al., 2018https://www.cgl.ucsf.edu/chimerax/
Software, algorithmCHARMMBrooks et al., 1983https://www.charmm.org/charmm/
Software, algorithmSWISS-MODELBiasini et al., 2014https://swissmodel.expasy.org/
Software, algorithmAxon Clampex 10.6Molecular DevicesN/A
Software, algorithmAxon Clampfit 10.6Molecular DevicesN/A
Software, algorithmPrism 7GraphPadhttps://www.graphpad.com/

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https://doi.org/10.7554/eLife.46986.035

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