Cryo-EM structure of the lysosomal chloride-proton exchanger CLC-7 in complex with OSTM1

Elife. 2020 Aug 4;9:e59555. doi: 10.7554/eLife.59555.

Abstract

The chloride-proton exchanger CLC-7 plays critical roles in lysosomal homeostasis and bone regeneration and its mutation can lead to osteopetrosis, lysosomal storage disease and neurological disorders. In lysosomes and the ruffled border of osteoclasts, CLC-7 requires a β-subunit, OSTM1, for stability and activity. Here, we present electron cryomicroscopy structures of CLC-7 in occluded states by itself and in complex with OSTM1, determined at resolutions up to 2.8 Å. In the complex, the luminal surface of CLC-7 is entirely covered by a dimer of the heavily glycosylated and disulfide-bonded OSTM1, which serves to protect CLC-7 from the degradative environment of the lysosomal lumen. OSTM1 binding does not induce large-scale rearrangements of CLC-7, but does have minor effects on the conformation of the ion-conduction pathway, potentially contributing to its regulatory role. These studies provide insights into the role of OSTM1 and serve as a foundation for understanding the mechanisms of CLC-7 regulation.

Keywords: CLC; chloride transport; human; molecular biophysics; proton transport; structural biology.

Plain Language Summary

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Chickens
  • Chloride Channels* / chemistry
  • Chloride Channels* / ultrastructure
  • Cryoelectron Microscopy
  • HEK293 Cells
  • Humans
  • Lysosomes / metabolism*
  • Membrane Proteins* / chemistry
  • Membrane Proteins* / ultrastructure
  • Molecular Dynamics Simulation
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / ultrastructure
  • Ubiquitin-Protein Ligases* / chemistry
  • Ubiquitin-Protein Ligases* / ultrastructure

Substances

  • CLCN7 protein, human
  • Chloride Channels
  • Membrane Proteins
  • OSTM1 protein, human
  • Recombinant Proteins
  • Ubiquitin-Protein Ligases