Structural and functional investigation of the human CLC-7/OSTM1 complex

• Applicant: Marina Schrecker, Ph.D.
• Subject Area: Biochemistry ; Structural Biology
• Term: from 2020 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 443618349

• Final Report Year: 2024

Final Report Abstract

CLC-7, distinct from other mammalian CLC transporters, uniquely requires the β-subunit OSTM1 for transport activity[1, 2]. Co-localized within lysosomes and osteoclasts, CLC-7 and OSTM1 stabilize each other's expression and facilitate Cl -/H+ exchange. OSTM1, single-pass transmembrane protein, is crucial for ion transport. Mutations in CLC-7 and OSTM1 are linked to osteopetrosis and neurodegeneration. However, the collaborative mechanisms enabling ion transport remain elusive. To address this, we've investigated the cryo -EM structures of CLC-7 and a CLC-7/OSTM1 complex. The structures illustrate how OSTM1, heavily glycosylated, extends into the lysosomal lumen to shield CLC-7 from its degradative environment. The analysis underscores the conserved role of nucleotide binding sites and PI lipids in the CLC family and suggest that the conformation of pores for Cl - and H + ions depends on the presence of OSTM1. The structures reveal ATP and phosphatidylinositol-binding sites, suggesting that besides the functional dependency of CLC-7/OSTM1 on membrane potential and pH, other signals may regulate CLC-7 activity. While ATP may be constitutively bound and serve a structural role, disruption of the binding site has functional consequences for CLC -7. The second ligand copurified with CLC-7 and CLC-7/OSTM1 is phosphatidylinositol 3-phosphate (PI3P). While PI3P wasn't previously known to regulate ion transport protein activity, phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), modulates ion transport proteins. We identified conserved residues responsible for PI3P coordination, also found in CLC -6. PI(3,5)P2 inhibits activity of CLC-a from Arabidopsis thaliana. We modelled a PI(3,5)P2 lipid into the CLC-7/OSTM1 structure's binding site to assess its effects. A phosphate at the 5-position of the inositol ring clashed with protein. We speculate that PI(3,5)P2 binding may induce conformational changes in CLC-7, similar to its inhibitory effect on CLC-a. Subsequent to my findings published in eLife, another group discovered that the lysosomal phosphoinositide PI(3,5)P2 tonically inhibits the CLC-7/OSTM1 chloride/proton antiporter. These results validate our hypotheses regarding the regulatory role of PI lipids for the CLC - 7/OSTM1 complex and lysosome homeostasis. In addition to my primary project, I collaborated with Dr. Dirk Remus's group at MSK to explore the selective loading of DNA sliding clamps onto specific DNA segments. These clamps, like the canonical PCNA, recruit crucial DNA-modifying enzymes for replication and repair. Loaded onto DNA by hetero-pentameric clamp loaders from the AAA+ family of ATPases, their mechanism was unclear due to the absence of resolved structures of a eukaryotic clamp-loader complex in the presence of DNA. Our work unveiled how the 9-1-1 checkpoint clamp uniquely loads onto damaged DNA to activate ATR kinase and initiate the DNA damage checkpoint. We identified the Rad17 checkpoint clamp loader's distinct recognition of DNA with recessed 5' ends, common after DNA damage or replication fork collapse, triggering a global conformational change facilitating 9-1-1 clamp opening for DNA loading. Our study introduced a new paradigm for recognizing unique DNA structures. Furthermore, I investigated the loading of the canonical clamp, PCNA, onto DNA with recessed 3' ends and nicked DNA. Our study clarified the multistep process by which DNA is loaded onto PCNA, resolving several longstanding controversies in the field.

Publications

• Cryo-EM structure of the lysosomal chloride-proton exchanger CLC-7 in complex with OSTM1. eLife, 9.
  Schrecker, Marina; Korobenko, Julia & Hite, Richard K.
  
• Presentation: ‚Structural and functional investigation of the human CLC-7/OSTM1 complex‘ during Molecular Biophysics Training Program annual Seminar in June 2020 at WCM in New York, NY.
  Marina Schrecker
  
• Mechanisms of loading and release of the 9-1-1 checkpoint clamp. Nature Structural & Molecular Biology, 29(4), 369-375.
  Castaneda, Juan C.; Schrecker, Marina; Remus, Dirk & Hite, Richard K.
  
• Multistep loading of a DNA sliding clamp onto DNA by replication factor C. eLife, 11.
  Schrecker, Marina; Castaneda, Juan C.; Devbhandari, Sujan; Kumar, Charanya; Remus, Dirk & Hite, Richard K.