advances.sciencemag.org/cgi/content/full/6/33/eabb4747/DC1

Supplementary Materials for

Molecular insights into the human CLC-7/Ostm1 transporter

Sensen Zhang, Yang Liu, Bing Zhang, Jun Zhou, Tianyu Li, Zhiqiang Liu*, Yang Li*, Maojun Yang*

*Corresponding author. Email: drliuzhiqiang@163.com (Z.L.); liyang@simm.ac.cn (Y.L.);

maojunyang@tsinghua.edu.cn (M.Y.)

Published 12 August 2020, Sci. Adv. 6, eabb4747 (2020) DOI: 10.1126/sciadv.abb4747

This PDF file includes:

Figs. S1 to S8 Tables S1 and S2

Fig. S1. Protein purification and structure determination of the CLC-7/Ostm1 complex.

A. Size-exclusion chromatography of CLC-7/Ostm1 in digitonin buffer. The peak corresponding to

the CLC-7/Ostm1 complex was subjected to SDS-PAGE and coomassie blue staining.

B. Representative cryo-EM micrograph of CLC-7/Ostm1 complex in digitonin buffer.

C. 2D class averages of the CLC-7/Ostm1 sample in digitonin buffer.

D. The workflow of 2D/3D reconstruction with CLC-7/Ostm1 cryo-EM data. In brief, 125,1 k

particles were kept after 2D classification, and subjected to three rounds of 3D classification. A

final dataset containing 148 k particles were used for high-resolution refinement (see methods for

more details).

Fig. S2. Reconstruction and representative cryo-EM densities of CLC-7/Ostm1

A. Gold-standard Fourier Shell correlation (FSC) curve of CLC-7/Ostm1 after 3D refinement. The

resolution estimation was based on the criterion of FSC 0.143 cutoff.

B. Angular distribution of the CLC-7/Ostm1 final reconstruction.

C. Local resolution map of the CLC-7/Ostm1 after the final 3D density map.

D. Cross-validation of the atomic model with the summed map and the half maps of CLC-7/Ostm1.

E. Density maps of representative transmembrane regions of CLC-7. Stick style atomic models (gold)

were fitted into the cryo-EM density maps (gray mesh). The density maps were contoured at 6.5

σ

F. Density maps of representative regions of Ostm1, similar to the panel (E).

Fig. S3. Sequence alignment of CLC and Ostm1 proteins.

A. Sequence alignment of human CLC-1 to CLC-7 using ESPript3.

B. Sequence alignment Ostm1 among different organisms (human, mouse, rat, bovine, and zebrafish)

using ESPript3. The labeled cysteine residues were identified to form disulfide bonds in human

Ostm1.

Fig. S4. Glycosylation and interactions in the CLC-7/Ostm1 complex.

A. Schematic representation of the CLC-7/Ostm1 complex docked into the cryo-EM density map

from a side view. Glycosylation modifications (Asn163-NAG and Asn263-NAG) were indicated in

the intravesicular cleft between Ostm1 and CLC-7.

B. The top view of the surface electrostatic potential of CLC-7.

C. The dimer of the intravesicular domain in Ostm1 with one subunit shown as surface (orange) and

the other shown as ribbon (blue).

D. Interaction between the dimer Ostm1 was contributed by Glu168 and Arg151. In addition, in the

intravesicular domain, Tyr228 in EH5 of Ostm1 interacts with Asp456 from an intravesicular loop

in CLC-7.

Fig. S5. CLC-7/Ostm1 expression level between WT and mutants.

A. The total expression level of the wild type, E416A (CLC-7), and Y300A (Ostm1) in HEK293F

cells with actin as the loading control. The CLC-7 proteins carry a strep tag and the Ostm1 proteins

carry a flag tag.

B. Fluorescence images of the WT and mutant CLC-7PM/Ostm1. CLC-7PM carries the leucine

mutations that partially target CLC-7 to the plasma membrane and Ostm1 carries a C terminal

EGFP fusion tag. The fluorescence intensities of the CLC-7PM_E416A mutants are lower than that

of WT.

C. Western blot of the surface protein expression levels between WT and mutant CLC-7PM/Ostm1

proteins in HEK293F cells with actin as the loading control. Surface expressions were detected

through the surface biotinylation experiments. The surface expression of the CLC-7PM_E416A

mutants are lower than that of WT proteins, consistent with the fluorescence intensities observed

in panel (B).

Fig. S6. Comparisons between CLC-7 and other CLC proteins.

A. Stereo view of the three proposed ion-binding sites. Cl- binding sites (Sext, Scen, and Sint) are shown

as blue spheres.

B-F. Comparisons between CLC-7 and other CLC proteins in the proposed ion-binding sites.

G. Comparisons of the SerC and TyrC between CLC-7 and other CLC proteins.

H. The intracellular vestibule of CLC-7 was contributed by the Tyr602, Val560, Met562, Phe310, and the

proton glutamate Glu314.

I-L. Comparison of the intracellular vestibule between CLC-7 and other CLC proteins.

Fig. S7. CLC-7/Ostm1 current traces and I-V curves between the WT and mutants.

A. Representative currents trace of mutants in the N terminus (E95A, D98A, Y99A, E103A, and

N104A). Voltage was clamped from -80 to +140 mV in 20 mV increment.

B. Representative currents trace of mutants in the CBS domain (N214A, R756A, Y746A, and

R784A). Voltage was clamped from -80 to +140 mV in 20 mV increment.

C. Representative currents trace of mutants in the TMD region (E313A, Q321A, and E313A/Q321A).

Voltage was clamped from -80 to +140 mV in 20 mV increment.

D-F. Comparisons of the I-V curves between the WT and mutants.

Fig. S8. Structural interpretation of the Y715C mutant.

A. Interaction between Tyr715 from the CBS domain and Asp282 from the TMD domain. Different

domains are color-coded for clarity. Purple, N terminus; Brown, CBS; Green, TMD.

B. The EM density map of the neighboring residues is shown at a contour level of 6σ.

Table S1. | Cryo-EM data collection, refinement, and validation statistics

CLC-7/Ostm1 complex

Data collection and processing

Magnification 130,000

Voltage (kV) 300

Electron exposure (e-/Å2) 50

Defocus range (μm) -1.3 ~ -2.3

Pixel size (Å) 1.08

Software Relion-3

Symmetry imposed C2

Initial particle images (no.) 1251,000

Final particles images (no.) 148,000

Map resolution (Å) 3.7

FSC threshold 0.143

Local map resolution range (Å) 4.8-3.0

Refinement

Software PHENIX 1.14

Initial model used (PDB code)

Model resolution (Å) 3.8

FSC threshold 0.5

Map sharpening B factor -202.4

Model composition

Non-hydrogen atoms 13338

Protein residues 1760

Ligand 0

B factors (Å2)

Protein 55.36

Ligand 0

R.m.s deviations

Bond length (Å) 0.008

Bond angles (°) 1.39

Validation

MolProbity score 1.77

Clashscore 4.49

Poor rotamers (%) 0.57

Ramachandran plot

Favored (%) 90.25

Allowed (%) 9.75

Disallowed (%) 0.00

Table S2. | Time constant (τactivation) of WT CLC-7/Ostm1 and different mutants

τactivation (ms) n

CLC-7wt/Ostm1wt 598.8±45.4 16

CLC-7E95A/Ostm1wt 170.4±22.9 10

CLC-7D98A/Ostm1wt 151.2±12.4 16

CLC-7Y99A/Ostm1wt 93.2±15.4 15

CLC-7E103A/Ostm1wt 370.7±30.9 15

CLC-7N104A/Ostm1wt 346.6±29.3 13

CLC-7N214/Ostm1wt 85.7±6.2 11

CLC-7R756/Ostm1wt 251.2±27.2 11

CLC-7Y746A/Ostm1wt 133.5±15.4 10

CLC-7R784A/Ostm1wt 28.9±2.7 12

CLC-7E313A/Ostm1wt 105.6±6.8 9

CLC-7Q321A/Ostm1wt 125.7±12.8 10

CLC-7E313AQ321A/Ostm1wt 25.6±4.3 13

CLC-7wt/Ostm1Y300A 795.3±36.4 13

CLC-7E416A/Ostm1wt 659.5±29.6 11

CLC-7E416A/Ostm1Y300A 756.4±48.3 10