Collagen IVα345 dysfunction in glomerular basement membrane diseases. III. A functional framework for α345 hexamer assembly.

We identified a genetic variant, an 8-residue appendage, of the α345 hexamer of collagen IV present in patients with glomerular basement membrane (GBM) disease, Goodpasture's disease (GP) and Alport syndrome (AS) (see Companion Paper I), and determined the long-awaited crystal structure of hexamer (see Companion Paper II). We sought to elucidate how variants cause GBM disease by exploring the mechanism of hexamer assembly. Chloride ions induced in vitro hexamer assembly in a composition-specific manner in the presence of equimolar concentrations of α3, α4 and α5 NC1 monomers. Chloride ions, together with sulfilimine crosslinks, stabilized the assembled hexamer. Furthermore, the chloride ion-dependent assembly revealed conformational plasticity of the LCL bioactive sites, a critical property underlying bioactivity and pathogenesis. We explored the native mechanism by expressing recombinant α345 mini-protomers in cell culture and characterizing the expressed proteins. Our findings revealed: NC1-directed trimerization, forming protomers inside the cell, and hexamerization forming scaffolds outside the cell; and a Cl gradient signaled hexamerization. This assembly detail, along with a crystal structure, provides a framework for understanding hexamer dysfunction. Restoration of the native conformation of bioactive sites or α345 hexamer replacement are prospective approaches to therapeutic intervention.