Inside the cells of mammals, acidic compartments called lysosomes are responsible for breaking down large molecules and worn-out cells parts so their components can be used again. Similar to lysosomes, specialized cells called osteoclasts require an acidic environment to degrade tissues in the bone. Both osteoclasts and lysosomes rely on a two-component protein complex to help them digest molecules. Mutations in the genes for both proteins are directly linked to human diseases including neurodegeneration and osteopetrosis – a disease characterized by dense and brittle bones.

For the main protein in this complex, called CLC-7, to remain stable and perform its roles, it requires an accessory subunit known as OSTM1. CLC-7 is a transporter that funnels electrically charged particles into and out of the lysosome, which helps to maintain the environment inside the lysosome compartment. However, due to the tight partnership between CLC-7 and OTSM1, how they influence each other is poorly understood.

To determine the roles of CLC-7 and OSTM1, Schrecker et al. looked at the structure of the complex using a technique called single particle electron microscopy, which allows proteins to be visualized almost down to the individual atom. The analysis revealed that OSTM1 covers almost the entire inside surface of CLC-7, protecting it from the acidic environment inside the lysosome and contributing to its stability. When the two subunits are bound together, OSTM1 also slightly changes the structure of the pore formed by CLC-7, suggesting that OSTM1 may regulate CLC-7 activity.

Schrecker et al. have laid the foundation for understanding more about the activity and regulation of CLC-7 and OSTM1 in lysosomes and osteoclasts. The structures described also help explain previous findings, including why OSTM1 is important for the stability of CLC-7.

CLC-7 is a member of the CLC family of chloride (Cl^-) channels and chloride (Cl^-)/proton (H⁺) transporters and is expressed in the lysosome and the resorption lacuna of osteoclasts (Graves et al., 2008; Ishida et al., 2013; Kornak et al., 2001; Weinert et al., 2010). In the membranes of these acidic compartments, CLC-7 uses the large pH gradient to catalyze the uptake of two Cl^- ions for each H⁺ released (Graves et al., 2008; Leisle et al., 2011; Ludwig et al., 2013). Dysfunction of CLC-7 is associated with dysregulation of ion and pH homoeostasis of the lysosome and the resorption lacuna (Graves et al., 2008; Ishida et al., 2013; Kasper et al., 2005; Kornak et al., 2001; Lange et al., 2006; Undiagnosed Diseases Network et al., 2019; Steinberg et al., 2010; Weinert et al., 2010). As both of these compartments rely on high proton concentrations to perform their physiological roles, disruption of CLC-7 function is associated with human diseases driven by impaired lysosomal and/or osteoclast function (Kasper et al., 2005; Kornak et al., 2001; Lange et al., 2006; Undiagnosed Diseases Network et al., 2019; Pressey et al., 2010). In particular, osteopetrosis, a disease characterized by dense and brittle bones, is the most common disease associated with CLC-7 mutation, with more than 50 distinct pathogenic mutations identified to date (Chalhoub et al., 2003; Cleiren et al., 2001; Kasper et al., 2005; Kornak et al., 2001; Lange et al., 2006; Sartelet et al., 2014; Schulz et al., 2010; Weinert et al., 2010).

Extensive structural and functional characterization of prokaryotic and eukaryotic CLC channels and transporters have established a framework for Cl^-/H⁺ exchange and identified several key residues that participate in the transport cycle (Accardi et al., 2004; Accardi and Miller, 2004; Accardi et al., 2005; Basilio et al., 2014; Chavan et al., 2020; Dutzler et al., 2002; Dutzler et al., 2003; Feng et al., 2010; Feng et al., 2012; Jayaram et al., 2008; Park et al., 2017; Park and MacKinnon, 2018; Picollo et al., 2012; Wang et al., 2019; Zdebik et al., 2008). Within the Cl^--conduction pathway, the gating glutamate (Glu_gate) that is conserved in CLC transporters is proposed to oscillate between at least four different conformations (Chavan et al., 2020; Dutzler et al., 2002; Dutzler et al., 2003; Feng et al., 2010). The movement and changes in the protonation state of Glu_gate are coupled to the binding and release of Cl^- ions in the highly conserved external and central binding sites (Picollo et al., 2012). Near the center of the transporter, the anion and H⁺-conduction pathways diverge with the anion pathway passing through the internal binding site before reaching the cytosol, while the H⁺-conduction pathway passes through a hydrophobic gap before reaching a conserved internal glutamate (Glu_in) (Accardi and Miller, 2004; Accardi et al., 2005; Chavan et al., 2020; Leisle et al., 2020; Lim and Miller, 2009; Zdebik et al., 2008). This conserved Glu_in is dispensable for coupled transport and water molecules has been proposed to mediate H⁺ transport through the hydrophobic gap (Feng et al., 2010; Han et al., 2014; Wang and Voth, 2009). Despite these extensive efforts, the precise mechanisms by which Cl^- and H⁺ transport are coupled remains poorly understood as are the mechanisms that underlie the gating of CLC transporters.

Unique among mammalian CLC transporters, CLC-7 requires a β-subunit, osteopetrosis-associated transmembrane protein 1 (OSTM1), for transport activity (Lange et al., 2006; Leisle et al., 2011). CLC-7 and OSTM1 co-localize in lysosomes and the ruffled border of osteoclasts (Lange et al., 2006; Leisle et al., 2011; Schulz et al., 2010). There, CLC-7 and OSTM1 stabilize the expression of one another and are both required for Cl^-/H⁺ exchange (Lange et al., 2006; Leisle et al., 2011). OSTM1 is predicted to be a glycosylated, single-pass transmembrane protein and mutations in OSTM1, like mutations in CLC-7, can lead to osteopetrosis and neurodegeneration in humans and mice (Chalhoub et al., 2003; Kasper et al., 2005; Kornak et al., 2001; Lange et al., 2006; Majumdar et al., 2011; Pressey et al., 2010). However, the mechanisms by which OSTM1 and CLC-7 cooperate to enable proper ion transport remains an open question. To begin to understand the mechanisms of CLC-7 function and its unique requirement for OSTM1, we have determined electron cryomicroscopy (cryo-EM) structures of CLC-7 and of a CLC-7/OSTM1 complex.

In this study, we present structures of the lysosomal Cl^-/H⁺ exchanger CLC-7 alone and in complex with its obligatory β-subunit OSTM1. The structure of the CLC-7/OSTM1 complex reveals that OSTM1 forms a heavily-glycosylated cap that covers the luminal surface of CLC-7 (Figures 5 and 6). OSTM1 associates with CLC-7 largely through interactions mediated by the transmembrane domains, consistent with analyses that demonstrated that deletion of the transmembrane domain of OSTM1 phenocopies the Ostm1 null in mice (Pata and Vacher, 2018). When complexed with CLC-7, OSTM1 does not adopt the structure of a RING finger domain as had previously been suggested (Fischer et al., 2003). Instead, the luminal domain of OSTM1 forms a tightly packed core composed of helical bundles linked together by numerous disulfide bonds (Figure 6). This stable core, together with the glycosylated periphery make the luminal domain of OSTM1 well-suited to survive the harsh degradative environment of the lysosomal lumen. In contrast, CLC-7 alone among the human CLC transporters lacks any N-glycosylation sites of its own and is consequently unstable in the lysosome when expressed in the absence of OSTM1 (Lange et al., 2006). The structure of CLC-7/OSTM1 is thus consistent with OSTM1 serving a protective role to shield CLC-7 from proteolysis and degradation.

Comparison of the CLC-7 structures in the presence and absence of OSTM1 reveals that OSTM1 binding induces subtle changes to the conformations of the ion permeation pathways. Among the changes are the conformations of residues essential for proper transport activity and the occupancies of the Cl^--binding sites. Notably, these conformational changes appear to occur only in a subset of the CLC-7/OSTM1 complexes. It is therefore possible that OSTM1 binding alters the equilibrium between different CLC-7 conformations. However, the current data do not enable accurate modeling of alternative rotamers, and thus it is not possible to compare the fraction of CLC-7 transporters adopting each possible state in the presence and absence of OSTM1. Moreover, we do not know precisely to which functional state these conformations correspond. In the CLC-7/OSTM1 and ggCLC-7 structures, the Cl^--conduction pathways resemble those of the ecCLC E148Q mutant where the three Cl^--binding sites are occupied and the Glu_gate adopts the ‘up’ conformation where it can potentially exchange protons with the lumen (Dutzler et al., 2003). Because the ‘up’ conformation is a coordinate along the proposed transport cycle (Feng et al., 2012), and because CLC-7 functionality has been previously detected in the absence of OSTM1 using solid-supported membranes and in plant vacuoles (Costa et al., 2012; Schulz et al., 2010), it is possible that CLC-7 structures both in the presence and absence of OSTM1 represent states that are competent for Cl^-/H⁺ transport activity. Notably, even in conditions where CLC-7 activity could be detected without OSTM1, current levels were significantly increased by its co-expression, consistent with OSTM1 potentiating transport activity (Schulz et al., 2010). Future investigations will thus be necessary to precisely determine how OSTM1 stimulates CLC-7 activity - whether by inducing conformational changes in the ion conduction pathways or merely stabilizing lysosomal expression of CLC-7.

Initial characterizations of CLC-7/OSTM1 demonstrated that its activity is dependent on membrane potential and luminal pH (Leisle et al., 2011; Ludwig et al., 2013). Our structures reveal the presence of ATP and phosphatidylinositol-binding sites, suggesting that additional signals may also regulate CLC-7 activity. Indeed, ATP has been demonstrated to influence the activity of multiple CLCs, but the precise effect of ATP on transporter activity has been controversial with evidence supporting both stimulatory and inhibitory roles. For example, addition of ATP increased transporter activity of CLC-4 by two-fold but inhibited activity of CLC-2 channels (Stölting et al., 2013; Vanoye and George, 2002). Moreover, the particular adenine nucleotide species that can influence CLC activity has been unclear. Binding studies conducted with the CBS domains of CLC-5 detected affinities of ~100 µM for ATP, ADP and AMP (Meyer et al., 2007). Recent studies revealed that CLC-3, CLC-4 and CLC-5 are able to distinguish between different nucleotide moieties, and showed that Mg²⁺ ions modify the effect of ADP binding (Grieschat et al., 2020). In the structures of ggCLC-7 and CLC-7/OSTM1, we observe direct coordination of all three phosphates not only through interactions with the CBS domains but also with the N-terminal domain (Figure 3 and Figure 3—figure supplement 1). Based on our data, we suggest that the actual binding affinity of CLC-7 to ATP is much higher than that detected for the CBS domains alone. As ATP levels are in excess of 1 mM under physiological conditions, it is likely that ATP is constitutively bound to CLC-7 and may therefore serve a structural role rather than a regulatory role. While ATP is a regulatory factor for numerous proteins, a structural role for nucleotides has been previously described for AMP-activated protein kinase (AMPK), inositol 1,4,5-trisphosphate (IP3) receptors and some prokaryotic regulator of potassium conductance (RCK)-gated channels (Bezprozvanny and Ehrlich, 1993; Cao et al., 2013; Hardie and Hawley, 2001; Kong et al., 2012; Kröning et al., 2007; Teixeira-Duarte et al., 2019). Among the CLC family, the residues in the N-terminal domain of CLC-7 that interact with ATP and Mg²⁺ are not broadly conserved, suggesting that the structural divergence of the ATP-binding site may contribute to varied effects that been reported among CLC family members.

Dysregulated ATP binding to CLC-7 may play a role in human disease. While CLC-7 remains fully functional in the absence of ATP (Leisle et al., 2011), mapping disease mutations onto CLC-7/OSTM1 reveals a hotspot of mutations on CBS2 near the ATP-binding site (Figure 8). Several distinct mutations of Arg767, which directly participates in the ATP γ-phosphate coordination, as well as mutations of neighboring Gly765 and Leu766 have been identified as leading to osteopetrosis (Cleiren et al., 2001; Leisle et al., 2011; Sartelet et al., 2014). Previous work characterizing the function of the Arg767 mutants revealed distinct phenotypes, with the R767P and R767W mutants displaying no activity while the R767Q mutants displayed increased activation kinetics (Leisle et al., 2011). Together, these results indicate that while ATP may be constitutively bound and serve a structural role, disruption of the binding site has functional consequences for CLC-7.

Figure 8

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The second ligand co-purified with ggCLC-7 and CLC-7/OSTM1 is PI3P, a phosphatidylinositol (PI) lipid species enriched in endolysosomal membranes that constitutes between 0.1% and 0.5% of the total PI content in cells (Falasca and Maffucci, 2006). While PI3P has not been previously characterized as a regulator of ion transport protein activity, the related phosphatidylinositol 3,5-bisphosphate (PI(3,5)P₂) is potent modulator of ion transport proteins whose abundance is tightly regulated (Fine et al., 2018; Hasegawa et al., 2017; She et al., 2018; She et al., 2019). Under basal conditions, PI(3,5)P₂ concentrations are very low (<0.1% of cellular PI content), but in yeast can rise more than 20-fold upon hyperosmotic shock (Duex et al., 2006). In endosomes and lysosomes, PI(3,5)P₂ binding activates the Ca²⁺ channel TRPML1 (Dong et al., 2010) and the Na⁺ channel TPC1 (Wang et al., 2012). In plant vacuoles, which share many features with lysosomes, PI(3,5)P₂ potently regulates atCLC-a, inhibiting its activity with an IC₅₀ of ~10 nM (Carpaneto et al., 2017). Because the PI3P-binding site appears to be conserved between atCLC-a and CLC-7, we modeled in a PI(3,5)P₂ lipid into the binding site in the CLC-7/OSTM1 structure to gain insights into its effect (Figure 4—figure supplement 1). In the model, a phosphate at 5-position of inositol ring could not be accommodated due to steric clashes with Lys281 on helix αG. We therefore speculate that binding of the regulatory PI(3,5)P₂ may induce conformational changes to CLC-7 that may alter its activity, analogous to the inhibitory effect of PI(3,5)P₂ on atCLC-a. Consistent with the PI lipids influencing transporter activity, a mutation of Tyr715, which is located near the PI3P-binding site, to cysteine was recently identified in the gene encoding human CLC-7 that causes a novel lipid storage disease without osteopetrosis (Undiagnosed Diseases Network et al., 2019). Functional analysis of this mutant revealed that it displays increased current levels when expressed in oocytes compared to wild-type CLC-7 and leads to a hyper-acidification phenotype in lysosomes (Figure 8; Undiagnosed Diseases Network et al., 2019).

For decades CLC-7 was perhaps the most enigmatic CLC family member (Brandt and Jentsch, 1995). Functional and structural characterization was limited by its lysosomal expression and its absolute requirement for a β-subunit, OSTM1. OSTM1 has been shown to have dual functions, both stabilizing CLC-7 in the lysosome and serving as an essential activator of the transporter (Lange et al., 2006; Leisle et al., 2011; Stauber and Jentsch, 2010). Our studies reveal how OSTM1 interacts with CLC-7 protecting the transporter from the acidic environment of the lysosomal lumen and lay the groundwork for future studies to elucidate how it, as well as ATP and the lipids identified in the structures, regulate CLC-7 transport activity and contribute to pH homeostasis in the lysosome and osteoclasts.

Cryo-EM maps and atomic coordinates have been deposited with the EMDB and PDB under accession codes EMD-22386 and PDB ID 7JM6 for ggCLC-7 and EMD-22389 and PDB ID 7JM7 for human CLC-7/OSTM1.

1. 1. Schrecker M
   2. Hite RK

   (2020) RCSB Protein Data Bank

   ID 7JM6. Structure of chicken CLC-7.

   https://www.rcsb.org/structure/7JM6
2. 1. Schrecker M
   2. Hite RK

   (2020) Electron Microscopy Data Bank

   ID EMD-22386. Structure of chicken CLC-7.

   https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-22386
3. 1. Schrecker M
   2. Hite R

   (2020) RCSB Protein Data Bank

   ID 7JM7. Structure of human CLC-7/OSTM1 complex.

   https://www.rcsb.org/structure/7JM7
4. 1. Schrecker M
   2. Hite R

   (2020) Electron Microscopy Data Bank

   ID EMD-22389. Structure of human CLC-7/OSTM1 complex.

   https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-22389

1. 1. Dutzler R
   2. Campbell EB
   3. MacKinnon R

   (2003) RCSB Protein Data Bank

   ID 1OTS. Structure of the Escherichia coli ClC Chloride channel and Fab Complex.

   https://www.rcsb.org/structure/1OTS
2. 1. Dutzler R
   2. Campbell EB
   3. MacKinnon R

   (2003) RCSB Protein Data Bank

   ID 1OTU. Structure of the Escherichia coli ClC Chloride channel E148Q mutant and Fab Complex.

   https://www.rcsb.org/structure/1OTU
3. 1. Feng L
   2. MacKinnon R

   (2010) RCSB Protein Data Bank

   ID 3ORG. Crystal Structure of a eukaryotic CLC transporter.

   https://www.rcsb.org/structure/3ORG
4. 1. Maduke M
   2. Mathews II
   3. Chavan TS

   (2020) RCSB Protein Data Bank

   ID 6V2J. Crystal structure of ClC-ec1 triple mutant (E113Q, E148Q, E203Q).

   https://www.rcsb.org/structure/6V2J

1. 1. Accardi A
   2. Kolmakova-Partensky L
   3. Williams C
   4. Miller C

   (2004) Ionic currents mediated by a prokaryotic homologue of CLC cl- channels

   Journal of General Physiology 123:109–119.

   https://doi.org/10.1085/jgp.200308935

   • PubMed
   • Google Scholar
2. 1. Accardi A
   2. Walden M
   3. Nguitragool W
   4. Jayaram H
   5. Williams C
   6. Miller C

   (2005) Separate ion pathways in a cl-/H+ exchanger

   Journal of General Physiology 126:563–570.

   https://doi.org/10.1085/jgp.200509417

   • PubMed
   • Google Scholar
3. 1. Accardi A
   2. Miller C

   (2004) Secondary active transport mediated by a prokaryotic homologue of ClC cl- channels

   Nature 427:803–807.

   https://doi.org/10.1038/nature02314

   • PubMed
   • Google Scholar
4. 1. Adams PD
   2. Afonine PV
   3. Bunkóczi G
   4. Chen VB
   5. Davis IW
   6. Echols N
   7. Headd JJ
   8. Hung LW
   9. Kapral GJ
   10. Grosse-Kunstleve RW
   11. McCoy AJ
   12. Moriarty NW
   13. Oeffner R
   14. Read RJ
   15. Richardson DC
   16. Richardson JS
   17. Terwilliger TC
   18. Zwart PH

   (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution

   Acta Crystallographica Section D Biological Crystallography 66:213–221.

   https://doi.org/10.1107/S0907444909052925

   • PubMed
   • Google Scholar
5. 1. Basilio D
   2. Noack K
   3. Picollo A
   4. Accardi A

   (2014) Conformational changes required for H(+)/Cl(-) exchange mediated by a CLC transporter

   Nature Structural & Molecular Biology 21:456–463.

   https://doi.org/10.1038/nsmb.2814

   • PubMed
   • Google Scholar
6. 1. Bezprozvanny I
   2. Ehrlich BE

   (1993) ATP modulates the function of inositol 1,4,5-trisphosphate-gated channels at two sites

   Neuron 10:1175–1184.

   https://doi.org/10.1016/0896-6273(93)90065-Y

   • PubMed
   • Google Scholar
7. 1. Brandt S
   2. Jentsch TJ

   (1995) ClC-6 and ClC-7 are two novel broadly expressed members of the CLC chloride channel family

   FEBS Letters 377:15–20.

   https://doi.org/10.1016/0014-5793(95)01298-2

   • PubMed
   • Google Scholar
8. 1. Cao Y
   2. Pan Y
   3. Huang H
   4. Jin X
   5. Levin EJ
   6. Kloss B
   7. Zhou M

   (2013) Gating of the TrkH ion channel by its associated RCK protein TrkA

   Nature 496:317–322.

   https://doi.org/10.1038/nature12056

   • PubMed
   • Google Scholar
9. 1. Carpaneto A
   2. Boccaccio A
   3. Lagostena L
   4. Di Zanni E
   5. Scholz-Starke J

   (2017) The signaling lipid phosphatidylinositol-3,5-bisphosphate targets plant CLC-a anion/H⁺ exchange activity

   EMBO Reports 18:1100–1107.

   https://doi.org/10.15252/embr.201643814

   • PubMed
   • Google Scholar
10. 1. Chalhoub N
    2. Benachenhou N
    3. Rajapurohitam V
    4. Pata M
    5. Ferron M
    6. Frattini A
    7. Villa A
    8. Vacher J

    (2003) Grey-lethal mutation induces severe malignant autosomal recessive osteopetrosis in mouse and human

    Nature Medicine 9:399–406.

    https://doi.org/10.1038/nm842

    • PubMed
    • Google Scholar
11. 1. Chavan TS
    2. Cheng RC
    3. Jiang T
    4. Mathews II
    5. Stein RA
    6. Koehl A
    7. Mchaourab HS
    8. Tajkhorshid E
    9. Maduke M

    (2020) A CLC^-ec1 mutant reveals global conformational change and suggests a unifying mechanism for the CLC Cl^-/H⁺ transport cycle

    eLife 9:e53479.

    https://doi.org/10.7554/eLife.53479

    • PubMed
    • Google Scholar
12. 1. Cleiren E
    2. Bénichou O
    3. Van Hul E
    4. Gram J
    5. Bollerslev J
    6. Singer FR
    7. Beaverson K
    8. Aledo A
    9. Whyte MP
    10. Yoneyama T
    11. deVernejoul MC
    12. Van Hul W

    (2001) Albers-Schönberg disease (autosomal dominant osteopetrosis, type II) results from mutations in the ClCN7 chloride channel gene

    Human Molecular Genetics 10:2861–2867.

    https://doi.org/10.1093/hmg/10.25.2861

    • PubMed
    • Google Scholar
13. 1. Costa A
    2. Gutla PV
    3. Boccaccio A
    4. Scholz-Starke J
    5. Festa M
    6. Basso B
    7. Zanardi I
    8. Pusch M
    9. Schiavo FL
    10. Gambale F
    11. Carpaneto A

    (2012) The Arabidopsis central vacuole as an expression system for intracellular transporters: functional characterization of the cl-/H⁺ exchanger CLC-7

    The Journal of Physiology 590:3421–3430.

    https://doi.org/10.1113/jphysiol.2012.230227

    • PubMed
    • Google Scholar
14. 1. De Angeli A
    2. Monachello D
    3. Ephritikhine G
    4. Frachisse JM
    5. Thomine S
    6. Gambale F
    7. Barbier-Brygoo H

    (2006) The nitrate/proton antiporter AtCLCa mediates nitrate accumulation in plant vacuoles

    Nature 442:939–942.

    https://doi.org/10.1038/nature05013

    • Google Scholar
15. 1. Dong XP
    2. Shen D
    3. Wang X
    4. Dawson T
    5. Li X
    6. Zhang Q
    7. Cheng X
    8. Zhang Y
    9. Weisman LS
    10. Delling M
    11. Xu H

    (2010) PI(3,5)P2 controls membrane trafficking by direct activation of mucolipin Ca2+ release channels in the endolysosome

    Nature Communications 1:38.

    https://doi.org/10.1038/ncomms1037

    • PubMed
    • Google Scholar
16. 1. Duex JE
    2. Nau JJ
    3. Kauffman EJ
    4. Weisman LS

    (2006) Phosphoinositide 5-phosphatase fig 4p is required for both acute rise and subsequent fall in stress-induced phosphatidylinositol 3,5-bisphosphate levels

    Eukaryotic Cell 5:723–731.

    https://doi.org/10.1128/EC.5.4.723-731.2006

    • PubMed
    • Google Scholar
17. 1. Dutzler R
    2. Campbell EB
    3. Cadene M
    4. Chait BT
    5. MacKinnon R

    (2002) X-ray structure of a ClC chloride channel at 3.0 A reveals the molecular basis of anion selectivity

    Nature 415:287–294.

    https://doi.org/10.1038/415287a

    • PubMed
    • Google Scholar
18. 1. Dutzler R
    2. Campbell EB
    3. MacKinnon R

    (2003) Gating the selectivity filter in ClC chloride channels

    Science 300:108–112.

    https://doi.org/10.1126/science.1082708

    • PubMed
    • Google Scholar
19. 1. Emsley P
    2. Lohkamp B
    3. Scott WG
    4. Cowtan K

    (2010) Features and development of coot

    Acta Crystallographica. Section D, Biological Crystallography 66:486–501.

    https://doi.org/10.1107/S0907444910007493

    • PubMed
    • Google Scholar
20. 1. Emsley P
    2. Crispin M

    (2018) Structural analysis of glycoproteins: building N-linked glycans with Coot

    Acta Crystallographica Section D Structural Biology 74:256–263.

    https://doi.org/10.1107/S2059798318005119

    • Google Scholar
21. 1. Falasca M
    2. Maffucci T

    (2006) Emerging roles of phosphatidylinositol 3-monophosphate as a dynamic lipid second messenger

    Archives of Physiology and Biochemistry 112:274–284.

    https://doi.org/10.1080/13813450601094664

    • PubMed
    • Google Scholar
22. 1. Feng L
    2. Campbell EB
    3. Hsiung Y
    4. MacKinnon R

    (2010) Structure of a eukaryotic CLC transporter defines an intermediate state in the transport cycle

    Science 330:635–641.

    https://doi.org/10.1126/science.1195230

    • PubMed
    • Google Scholar
23. 1. Feng L
    2. Campbell EB
    3. MacKinnon R

    (2012) Molecular mechanism of proton transport in CLC cl-/H+ exchange transporters

    PNAS 109:11699–11704.

    https://doi.org/10.1073/pnas.1205764109

    • PubMed
    • Google Scholar
24. 1. Fine M
    2. Schmiege P
    3. Li X

    (2018) Structural basis for PtdInsP₂-mediated human TRPML1 regulation

    Nature Communications 9:4192.

    https://doi.org/10.1038/s41467-018-06493-7

    • PubMed
    • Google Scholar
25. 1. Fischer T
    2. De Vries L
    3. Meerloo T
    4. Farquhar MG

    (2003) Promotion of G alpha i3 subunit down-regulation by GIPN, a putative E3 ubiquitin ligase that interacts with RGS-GAIP

    PNAS 100:8270–8275.

    https://doi.org/10.1073/pnas.1432965100

    • PubMed
    • Google Scholar
26. 1. Goddard TD
    2. Huang CC
    3. Meng EC
    4. Pettersen EF
    5. Couch GS
    6. Morris JH
    7. Ferrin TE

    (2018) UCSF ChimeraX: meeting modern challenges in visualization and analysis

    Protein Science 27:14–25.

    https://doi.org/10.1002/pro.3235

    • PubMed
    • Google Scholar
27. 1. Goehring A
    2. Lee CH
    3. Wang KH
    4. Michel JC
    5. Claxton DP
    6. Baconguis I
    7. Althoff T
    8. Fischer S
    9. Garcia KC
    10. Gouaux E

    (2014) Screening and large-scale expression of membrane proteins in mammalian cells for structural studies

    Nature Protocols 9:2574–2585.

    https://doi.org/10.1038/nprot.2014.173

    • PubMed
    • Google Scholar
28. 1. Graves AR
    2. Curran PK
    3. Smith CL
    4. Mindell JA

    (2008) The cl-/H+ antiporter ClC-7 is the primary chloride permeation pathway in lysosomes

    Nature 453:788–792.

    https://doi.org/10.1038/nature06907

    • PubMed
    • Google Scholar
29. 1. Grieschat M
    2. Guzman RE
    3. Langschwager K
    4. Fahlke C
    5. Alekov AK

    (2020) Metabolic energy sensing by mammalian CLC anion/proton exchangers

    EMBO Reports 21:e47872.

    https://doi.org/10.15252/embr.201947872

    • PubMed
    • Google Scholar
30. 1. Han W
    2. Cheng RC
    3. Maduke MC
    4. Tajkhorshid E

    (2014) Water access points and hydration pathways in CLC H+/Cl- transporters

    PNAS 111:1819–1824.

    https://doi.org/10.1073/pnas.1317890111

    • PubMed
    • Google Scholar
31. 1. Hardie DG
    2. Hawley SA

    (2001) AMP-activated protein kinase: the energy charge hypothesis revisited

    BioEssays 23:1112–1119.

    https://doi.org/10.1002/bies.10009

    • PubMed
    • Google Scholar
32. 1. Hasegawa J
    2. Strunk BS
    3. Weisman LS

    (2017) PI5P and PI(3,5)P₂: minor, but essential phosphoinositides

    Cell Structure and Function 42:49–60.

    https://doi.org/10.1247/csf.17003

    • PubMed
    • Google Scholar
33. 1. Heymann JB

    (2018) Guidelines for using bsoft for high resolution reconstruction and validation of biomolecular structures from electron micrographs

    Protein Science 27:159–171.

    https://doi.org/10.1002/pro.3293

    • PubMed
    • Google Scholar
34. 1. Ishida Y
    2. Nayak S
    3. Mindell JA
    4. Grabe M

    (2013) A model of lysosomal pH regulation

    Journal of General Physiology 141:705–720.

    https://doi.org/10.1085/jgp.201210930

    • Google Scholar
35. 1. Jayaram H
    2. Accardi A
    3. Wu F
    4. Williams C
    5. Miller C

    (2008) Ion permeation through a cl--selective channel designed from a CLC cl-/H+ exchanger

    PNAS 105:11194–11199.

    https://doi.org/10.1073/pnas.0804503105

    • PubMed
    • Google Scholar
36. 1. Kasper D
    2. Planells-Cases R
    3. Fuhrmann JC
    4. Scheel O
    5. Zeitz O
    6. Ruether K
    7. Schmitt A
    8. Poët M
    9. Steinfeld R
    10. Schweizer M
    11. Kornak U
    12. Jentsch TJ

    (2005) Loss of the chloride channel ClC-7 leads to lysosomal storage disease and neurodegeneration

    The EMBO Journal 24:1079–1091.

    https://doi.org/10.1038/sj.emboj.7600576

    • Google Scholar
37. 1. Kong C
    2. Zeng W
    3. Ye S
    4. Chen L
    5. Sauer DB
    6. Lam Y
    7. Derebe MG
    8. Jiang Y

    (2012) Distinct gating mechanisms revealed by the structures of a multi-ligand gated K(+) channel

    eLife 1:e00184.

    https://doi.org/10.7554/eLife.00184

    • PubMed
    • Google Scholar
38. 1. Kornak U
    2. Kasper D
    3. Bösl MR
    4. Kaiser E
    5. Schweizer M
    6. Schulz A
    7. Friedrich W
    8. Delling G
    9. Jentsch TJ

    (2001) Loss of the ClC-7 chloride channel leads to osteopetrosis in mice and man

    Cell 104:205–215.

    https://doi.org/10.1016/S0092-8674(01)00206-9

    • PubMed
    • Google Scholar
39. 1. Kröning N
    2. Willenborg M
    3. Tholema N
    4. Hänelt I
    5. Schmid R
    6. Bakker EP

    (2007) ATP binding to the KTN/RCK subunit KtrA from the K+ -uptake system KtrAB of Vibrio alginolyticus: its role in the formation of the KtrAB complex and its requirement in vivo

    The Journal of Biological Chemistry 282:14018–14027.

    https://doi.org/10.1074/jbc.M609084200

    • PubMed
    • Google Scholar
40. 1. Lange PF
    2. Wartosch L
    3. Jentsch TJ
    4. Fuhrmann JC

    (2006) ClC-7 requires Ostm1 as a beta-subunit to support bone resorption and lysosomal function

    Nature 440:220–223.

    https://doi.org/10.1038/nature04535

    • PubMed
    • Google Scholar
41. 1. Last NB
    2. Stockbridge RB
    3. Wilson AE
    4. Shane T
    5. Kolmakova-Partensky L
    6. Koide A
    7. Koide S
    8. Miller C

    (2018) A CLC-type F-/H+ antiporter in ion-swapped conformations

    Nature Structural & Molecular Biology 25:601–606.

    https://doi.org/10.1038/s41594-018-0082-0

    • Google Scholar
42. 1. Leisle L
    2. Ludwig CF
    3. Wagner FA
    4. Jentsch TJ
    5. Stauber T

    (2011) ClC-7 is a slowly voltage-gated 2cl(-)/1H(+)-exchanger and requires Ostm1 for transport activity

    The EMBO Journal 30:2140–2152.

    https://doi.org/10.1038/emboj.2011.137

    • PubMed
    • Google Scholar
43. 1. Leisle L
    2. Xu Y
    3. Fortea E
    4. Lee S
    5. Galpin JD
    6. Vien M
    7. Ahern CA
    8. Accardi A
    9. Bernèche S

    (2020) Divergent cl^- and H⁺ pathways underlie transport coupling and gating in CLC exchangers and channels

    eLife 9:e51224.

    https://doi.org/10.7554/eLife.51224

    • PubMed
    • Google Scholar
44. 1. Liebschner D
    2. Afonine PV
    3. Baker ML
    4. Bunkóczi G
    5. Chen VB
    6. Croll TI
    7. Hintze B
    8. Hung LW
    9. Jain S
    10. McCoy AJ
    11. Moriarty NW
    12. Oeffner RD
    13. Poon BK
    14. Prisant MG
    15. Read RJ
    16. Richardson JS
    17. Richardson DC
    18. Sammito MD
    19. Sobolev OV
    20. Stockwell DH
    21. Terwilliger TC
    22. Urzhumtsev AG
    23. Videau LL
    24. Williams CJ
    25. Adams PD

    (2019) Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in phenix

    Acta Crystallographica Section D Structural Biology 75:861–877.

    https://doi.org/10.1107/S2059798319011471

    • PubMed
    • Google Scholar
45. 1. Lim HH
    2. Miller C

    (2009) Intracellular proton-transfer mutants in a CLC cl-/H+ exchanger

    Journal of General Physiology 133:131–138.

    https://doi.org/10.1085/jgp.200810112

    • PubMed
    • Google Scholar
46. 1. Lobet S
    2. Dutzler R

    (2006) Ion-binding properties of the ClC chloride selectivity filter

    The EMBO Journal 25:24–33.

    https://doi.org/10.1038/sj.emboj.7600909

    • PubMed
    • Google Scholar
47. 1. Ludwig CF
    2. Ullrich F
    3. Leisle L
    4. Stauber T
    5. Jentsch TJ

    (2013) Common gating of both CLC transporter subunits underlies voltage-dependent activation of the 2cl-/1H+ exchanger ClC-7/Ostm1

    The Journal of Biological Chemistry 288:28611–28619.

    https://doi.org/10.1074/jbc.M113.509364

    • PubMed
    • Google Scholar
48. 1. Majumdar A
    2. Capetillo-Zarate E
    3. Cruz D
    4. Gouras GK
    5. Maxfield FR

    (2011) Degradation of Alzheimer's amyloid fibrils by microglia requires delivery of ClC-7 to lysosomes

    Molecular Biology of the Cell 22:1664–1676.

    https://doi.org/10.1091/mbc.e10-09-0745

    • PubMed
    • Google Scholar
49. 1. Mastronarde DN

    (2005) Automated electron microscope tomography using robust prediction of specimen movements

    Journal of Structural Biology 152:36–51.

    https://doi.org/10.1016/j.jsb.2005.07.007

    • Google Scholar
50. 1. Meyer S
    2. Savaresi S
    3. Forster IC
    4. Dutzler R

    (2007) Nucleotide recognition by the cytoplasmic domain of the human chloride transporter ClC-5

    Nature Structural & Molecular Biology 14:60–67.

    https://doi.org/10.1038/nsmb1188

    • PubMed
    • Google Scholar
51. 1. Park E
    2. Campbell EB
    3. MacKinnon R

    (2017) Structure of a CLC chloride ion channel by cryo-electron microscopy

    Nature 541:500–505.

    https://doi.org/10.1038/nature20812

    • PubMed
    • Google Scholar
52. 1. Park E
    2. MacKinnon R

    (2018) Structure of the CLC-1 chloride channel from Homo sapiens

    eLife 7:e36629.

    https://doi.org/10.7554/eLife.36629

    • Google Scholar
53. 1. Pata M
    2. Vacher J

    (2018) Ostm1 bifunctional roles in osteoclast maturation: insights from a mouse model mimicking a human OSTM1 mutation

    Journal of Bone and Mineral Research 33:888–898.

    https://doi.org/10.1002/jbmr.3378

    • PubMed
    • Google Scholar
54. 1. Pettersen EF
    2. Goddard TD
    3. Huang CC
    4. Couch GS
    5. Greenblatt DM
    6. Meng EC
    7. Ferrin TE

    (2004) UCSF chimera--a visualization system for exploratory research and analysis

    Journal of Computational Chemistry 25:1605–1612.

    https://doi.org/10.1002/jcc.20084

    • PubMed
    • Google Scholar
55. 1. Picollo A
    2. Malvezzi M
    3. Houtman JC
    4. Accardi A

    (2009) Basis of substrate binding and conservation of selectivity in the CLC family of channels and transporters

    Nature Structural & Molecular Biology 16:1294–1301.

    https://doi.org/10.1038/nsmb.1704

    • PubMed
    • Google Scholar
56. 1. Picollo A
    2. Xu Y
    3. Johner N
    4. Bernèche S
    5. Accardi A

    (2012) Synergistic substrate binding determines the stoichiometry of transport of a prokaryotic H(+)/Cl(-) exchanger

    Nature Structural & Molecular Biology 19:525–531.

    https://doi.org/10.1038/nsmb.2277

    • PubMed
    • Google Scholar
57. 1. Pravda L
    2. Sehnal D
    3. Toušek D
    4. Navrátilová V
    5. Bazgier V
    6. Berka K
    7. Svobodová Vareková R
    8. Koca J
    9. Otyepka M

    (2018) MOLEonline: a web-based tool for analyzing channels, tunnels and pores (2018 update)

    Nucleic Acids Research 46:W368–W373.

    https://doi.org/10.1093/nar/gky309

    • PubMed
    • Google Scholar
58. 1. Pressey SN
    2. O'Donnell KJ
    3. Stauber T
    4. Fuhrmann JC
    5. Tyynelä J
    6. Jentsch TJ
    7. Cooper JD

    (2010) Distinct neuropathologic phenotypes after disrupting the chloride transport proteins ClC-6 or ClC-7/Ostm1

    Journal of Neuropathology & Experimental Neurology 69:1228–1246.

    https://doi.org/10.1097/NEN.0b013e3181ffe742

    • PubMed
    • Google Scholar
59. 1. Punjani A
    2. Rubinstein JL
    3. Fleet DJ
    4. Brubaker MA

    (2017) cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination

    Nature Methods 14:290–296.

    https://doi.org/10.1038/nmeth.4169

    • PubMed
    • Google Scholar
60. Preprint

    1. Punjani A
    2. Zhang H
    3. Fleet DJ

    (2019) Non-uniform refinement: adaptive regularization improves single particle cryo-EM reconstruction

    bioRxiv.

    https://doi.org/10.1101/2019.12.15.877092

    • Google Scholar
61. Preprint

    1. Punjani A
    2. Fleet DJ

    (2020) 3d variability analysis: directly resolving continuous flexibility and discrete heterogeneity from single particle cryo-EM images

    bioRxiv.

    https://doi.org/10.1101/2020.04.08.032466

    • Google Scholar
62. 1. Rohou A
    2. Grigorieff N

    (2015) CTFFIND4: fast and accurate defocus estimation from electron micrographs

    Journal of Structural Biology 192:216–221.

    https://doi.org/10.1016/j.jsb.2015.08.008

    • PubMed
    • Google Scholar
63. 1. Sartelet A
    2. Stauber T
    3. Coppieters W
    4. Ludwig CF
    5. Fasquelle C
    6. Druet T
    7. Zhang Z
    8. Ahariz N
    9. Cambisano N
    10. Jentsch TJ
    11. Charlier C

    (2014) A missense mutation accelerating the gating of the lysosomal cl-/H+-exchanger ClC-7/Ostm1 causes osteopetrosis with gingival hamartomas in cattle

    Disease Models & Mechanisms 7:119–128.

    https://doi.org/10.1242/dmm.012500

    • PubMed
    • Google Scholar
64. 1. Scheres SHW

    (2016) Processing of structurally heterogeneous Cryo-EM data in RELION

    Method Enzymol 579:125–157.

    https://doi.org/10.1016/bs.mie.2016.04.012

    • Google Scholar
65. Software

    1. Schrödinger LLC

    (2020)

    The PyMOL Molecular Graphics System, version 1.2r3pre

    Schrödinger, LLC.
66. 1. Schulz P
    2. Werner J
    3. Stauber T
    4. Henriksen K
    5. Fendler K

    (2010) The G215R mutation in the cl-/H+-antiporter ClC-7 found in ADO II osteopetrosis does not abolish function but causes a severe trafficking defect

    PLOS ONE 5:e12585.

    https://doi.org/10.1371/journal.pone.0012585

    • PubMed
    • Google Scholar
67. 1. She J
    2. Guo J
    3. Chen Q
    4. Zeng W
    5. Jiang Y
    6. Bai X

    (2018) Structural insights into the voltage and phospholipid activation of the mammalian TPC1 channel

    Nature 556:130–134.

    https://doi.org/10.1038/nature26139

    • Google Scholar
68. 1. She J
    2. Zeng W
    3. Guo J
    4. Chen Q
    5. Bai XC
    6. Jiang Y

    (2019) Structural mechanisms of phospholipid activation of the human TPC2 channel

    eLife 8:e45222.

    https://doi.org/10.7554/eLife.45222

    • PubMed
    • Google Scholar
69. 1. Stauber T
    2. Jentsch TJ

    (2010) Sorting motifs of the endosomal/lysosomal CLC chloride transporters

    Journal of Biological Chemistry 285:34537–34548.

    https://doi.org/10.1074/jbc.M110.162545

    • PubMed
    • Google Scholar
70. 1. Steinberg BE
    2. Huynh KK
    3. Brodovitch A
    4. Jabs S
    5. Stauber T
    6. Jentsch TJ
    7. Grinstein S

    (2010) A cation counterflux supports lysosomal acidification

    The Journal of Cell Biology 189:1171–1186.

    https://doi.org/10.1083/jcb.200911083

    • PubMed
    • Google Scholar
71. 1. Stölting G
    2. Teodorescu G
    3. Begemann B
    4. Schubert J
    5. Nabbout R
    6. Toliat MR
    7. Sander T
    8. Nürnberg P
    9. Lerche H
    10. Fahlke C

    (2013) Regulation of ClC-2 gating by intracellular ATP

    Pflügers Archiv - European Journal of Physiology 465:1423–1437.

    https://doi.org/10.1007/s00424-013-1286-0

    • Google Scholar
72. 1. Suloway C
    2. Pulokas J
    3. Fellmann D
    4. Cheng A
    5. Guerra F
    6. Quispe J
    7. Stagg S
    8. Potter CS
    9. Carragher B

    (2005) Automated molecular microscopy: the new leginon system

    Journal of Structural Biology 151:41–60.

    https://doi.org/10.1016/j.jsb.2005.03.010

    • PubMed
    • Google Scholar
73. 1. Teixeira-Duarte CM
    2. Fonseca F
    3. Morais-Cabral JH

    (2019) Activation of a nucleotide-dependent RCK domain requires binding of a cation cofactor to a conserved site

    eLife 8:e50661.

    https://doi.org/10.7554/eLife.50661

    • PubMed
    • Google Scholar
74. Preprint

    1. Terwilliger TC
    2. Ludtke SJ
    3. Read RJ
    4. Adams PD
    5. Afonine PV

    (2019) Improvement of cryo-EM maps by density modification

    bioRxiv.

    https://doi.org/10.1101/845032

    • Google Scholar
75. 1. Undiagnosed Diseases Network
    2. Nicoli ER
    3. Weston MR
    4. Hackbarth M
    5. Becerril A
    6. Larson A
    7. Zein WM
    8. Baker PR
    9. Burke JD
    10. Dorward H
    11. Davids M
    12. Huang Y
    13. Adams DR
    14. Zerfas PM
    15. Chen D
    16. Markello TC
    17. Toro C
    18. Wood T
    19. Elliott G
    20. Vu M
    21. Zheng W
    22. Garrett LJ
    23. Tifft CJ
    24. Gahl WA
    25. Day-Salvatore DL
    26. Mindell JA
    27. Malicdan MCV

    (2019) Lysosomal storage and albinism due to effects of a de novo CLCN7 variant on lysosomal acidification

    The American Journal of Human Genetics 104:1127–1138.

    https://doi.org/10.1016/j.ajhg.2019.04.008

    • PubMed
    • Google Scholar
76. 1. Vanoye CG
    2. George AL

    (2002) Functional characterization of recombinant human ClC-4 chloride channels in cultured mammalian cells

    The Journal of Physiology 539:373–383.

    https://doi.org/10.1113/jphysiol.2001.013115

    • PubMed
    • Google Scholar
77. 1. Wang X
    2. Zhang X
    3. Dong XP
    4. Samie M
    5. Li X
    6. Cheng X
    7. Goschka A
    8. Shen D
    9. Zhou Y
    10. Harlow J
    11. Zhu MX
    12. Clapham DE
    13. Ren D
    14. Xu H

    (2012) TPC proteins are phosphoinositide- activated sodium-selective ion channels in endosomes and lysosomes

    Cell 151:372–383.

    https://doi.org/10.1016/j.cell.2012.08.036

    • PubMed
    • Google Scholar
78. 1. Wang K
    2. Preisler SS
    3. Zhang L
    4. Cui Y
    5. Missel JW
    6. Grønberg C
    7. Gotfryd K
    8. Lindahl E
    9. Andersson M
    10. Calloe K
    11. Egea PF
    12. Klaerke DA
    13. Pusch M
    14. Pedersen PA
    15. Zhou ZH
    16. Gourdon P

    (2019) Structure of the human ClC-1 chloride channel

    PLOS Biology 17:e3000218.

    https://doi.org/10.1371/journal.pbio.3000218

    • PubMed
    • Google Scholar
79. 1. Wang D
    2. Voth GA

    (2009) Proton transport pathway in the ClC cl-/H+ antiporter

    Biophysical Journal 97:121–131.

    https://doi.org/10.1016/j.bpj.2009.04.038

    • PubMed
    • Google Scholar
80. 1. Waterhouse AM
    2. Procter JB
    3. Martin DM
    4. Clamp M
    5. Barton GJ

    (2009) Jalview version 2--a multiple sequence alignment editor and analysis workbench

    Bioinformatics 25:1189–1191.

    https://doi.org/10.1093/bioinformatics/btp033

    • PubMed
    • Google Scholar
81. 1. Weinert S
    2. Jabs S
    3. Supanchart C
    4. Schweizer M
    5. Gimber N
    6. Richter M
    7. Rademann J
    8. Stauber T
    9. Kornak U
    10. Jentsch TJ

    (2010) Lysosomal pathology and osteopetrosis upon loss of H+-driven lysosomal cl- accumulation

    Science 328:1401–1403.

    https://doi.org/10.1126/science.1188072

    • PubMed
    • Google Scholar
82. 1. Zdebik AA
    2. Zifarelli G
    3. Bergsdorf EY
    4. Soliani P
    5. Scheel O
    6. Jentsch TJ
    7. Pusch M

    (2008) Determinants of anion-proton coupling in mammalian endosomal CLC proteins

    Journal of Biological Chemistry 283:4219–4227.

    https://doi.org/10.1074/jbc.M708368200

    • PubMed
    • Google Scholar
83. 1. Zheng SQ
    2. Palovcak E
    3. Armache JP
    4. Verba KA
    5. Cheng Y
    6. Agard DA

    (2017) MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy

    Nature Methods 14:331–332.

    https://doi.org/10.1038/nmeth.4193

    • PubMed
    • Google Scholar

Author details

1. Marina Schrecker

   Structural Biology Program, Memorial Sloan Kettering Cancer Center, New York, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8542-6657

2. Julia Korobenko

   Structural Biology Program, Memorial Sloan Kettering Cancer Center, New York, United States

   Contribution

   Data curation, Formal analysis, Investigation

   Competing interests

   No competing interests declared

3. Richard K Hite

   Structural Biology Program, Memorial Sloan Kettering Cancer Center, New York, United States

   Contribution

   Conceptualization, Formal analysis, Supervision, Funding acquisition, Visualization, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   hiter@mskcc.org

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0496-0669

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