Topology and Structure of Membrane-Inserted APOL1

Background: ApoL1 is an amphipathic protein that enters membranes at low pH and after titration to neutral pH, functions as a non-selective cation channel. Prior studies provide a model of membrane topology of active channel (Schaub et al., JBC (2021) 297: 101009): two loops span the membrane between positions 177 and 228 and positions 257 and 356, generating two segments exposed to the trans compartment. The segment from 335 to 356 forms a homodimer with the same segment of another molecule, forming the ion pore. A leucine zipper structure in the C-terminus is implicated in stabilizing the homo-dimer. We sought gain a fuller understanding of the sequences and environmental determinants that allow ApoL1 to form a functional pore. Methods: Using purified recombinant ApoL1 constructs and pre-formed phospholipid membranes: 1. Accessibility of 26 single cysteine substitution mutants for modification from the cis and trans compartment was determined under a variety of conditions 2. Proximity of individual cysteines in membrane-inserted protein complexes was determined with crosslinking reagents 3. Effect of elimination of charged amino acids in the first transmembrane loop on membrane topology, activity, and pH sensitivity of membrane association was assessed 4. Effect of disruption of putative leucine zipper motif on dimerization, topology and activity was determined Results: Cysteine accessibility confirms the topology model and demonstrates an additional trans-accessible segment in protein associated under optimal Cl permeability conditions. Crosslinking membrane-inserted cysteine-labelled protein identifies multiple sites of proximity between the dimers in addition to the pore-forming transmembrane segment. Elimination of the three glutamates in the first transmembrane loop alters the pH sensitivity of membrane association, but also eliminates activity and does not allow stable formation of the first transmembrane loop. Disruption of the leucine zipper motif eliminates activity, crosslinking at the pore segment, and stable formation of the first transmembrane loop. Conclusion: The data provide new constraints on any model of membrane inserted ApoL1 structure. Most importantly, the leu zipper data indicates a direct interaction between the C-terminal segment and the first transmembrane loop, suggesting a structural basis for the effects of the C-terminally located disease-associated sequence variants that activate the channel. Funding: NIDDK Support