Structural Determinants of Membrane Insertion and Activity of ApoL1

Background: ApoL1 is an amphitropic protein that inserts into membrane at low pH and after titration to neutral pH serves as a cation channel. Prior work supports a model of membrane-inserted ApoL1 in which two hairpin loops, positions 177 to 208 and 257 to 356, span the membrane and generate segments exposed to the trans compartment. The first loop is homologous to colicin A and may play a role in the membrane insertion. 3 Glu residues postioned near the tip of the hairpin have been proposed to function in pH-regulated membrane insertion: acidification of the cis compartment would eliminate the negative charge and facilitating transfer through the hydrophobic membrane. Exposure to neutral pH in the trans compartment would the deprotonate those positions, restoring negative charge and potentially preventing the loop for leaving the membrane. We tested this hypothesis. Methods: ApoL1 constructs were created in which Glu 201, 209, or 213 were changed to Cys or to Gln. The constructs were expressed, purified and assayed. The Cys constructs were assessed for modification of Cys by membrane impermeant AF488-maleimide. Cation channel activity of constructs was measured with vesicle based KCl efflux assay in the presence of chloride ionophore. Effect of introduced fixed negative change was assayed after modification of E201C construct with (2-sulfonatoethyl)methanethiosulfonate. Proximity of individual cysteines in homodimers were determined by crosslinking with MTS-1-MTS. Results: 1. Cys substitution mutants E201C and E209C are both modified from the trans comparment; E213C is not. 2. Single Gln substitution mutants all retain cation channel activity. Double Gln substitution at 201 and 209 eliminates activity while the other double substitutions are active. Triple substitution eliminates activity. 3. Introduction of untitratable negative charge at position 201 eliminates activity. 4. E201C is crosslinked in membrane inserted protein. Conclusion: The data provide new insight into membrane inserted ApoL1 structure. 1) Glu at positions 201 and 209 but not 213 are exposed to the trans compartment and ionization at those sites are necessary, consistent with ionic trapping. 2) Fixed negative charge at 201 eliminates activity; titration at that site is necessary for membrane insertion. 3) In membrane inserted dimer, the the trans segment around E201 in the two molecules are in close proximity. Funding: NIDDK Support