Abstract
Apolipoprotein L-I (APOL1) is a channel-forming effector of innate immunity. The common human APOL1 variant G0 provides protection against infection with certain Trypanosoma and Leishmania parasite species, but it cannot protect against the trypanosomes responsible for human African trypanosomiasis. Human APOL1 variants G1 and G2 protect against human-infective trypanosomes but also confer a higher risk of developing chronic kidney disease. Trypanosome-killing activity is dependent on the ability of APOL1 to insert into membranes at acidic pH and form pH-gated cation channels. We previously mapped the channel’s pore-lining region to the C-terminal domain (residues 332–398) and identified a membrane-insertion domain (MID, residues 177–228) that facilitates acidic pH-dependent membrane insertion. In this article, we further investigate structural determinants of cation channel formation by APOL1. Using a combination of site-directed mutagenesis and targeted chemical modification, our data indicate that the C-terminal heptad-repeat sequence (residues 368–395) is a bona fide leucine zipper domain (ZIP) that is required for cation channel formation as well as lysis of trypanosomes and mammalian cells. Using protein-wide cysteine-scanning mutagenesis, coupled with the substituted cysteine accessibility method, we determined that, in the open channel state, both the N-terminal domain and the C-terminal ZIP domain are exposed on the intralumenal/extracellular side of the membrane and provide evidence that each APOL1 monomer contributes four transmembrane domains to the open cation channel conformation. Based on these data, we propose an oligomeric topology model in which the open APOL1 cation channel is assembled from the coiled-coil association of C-terminal ZIP domains.
Highlights
Present address for Penny Lee: John Jay College, City University of New York, New York, USA
The C-terminus of APOL1 is integral for protein function, because truncations result in the loss of trypanolytic and cation channel activity [6, 14, 35]
Using site-directed mutagenesis and chemical modification, we show that a C-terminal heptad-repeat leucine motif constitutes a bona fide zipper domain (ZIP) domain that mediates coiled-coil interdigitation facilitating dimerization during APOL1 channel formation
Summary
Substitution of the four heptad-repeat leucines in the C-terminal ZIP domain results in loss of APOL1 open ion channel-forming activity. We reasoned that APOL1 residues that must insert into membranes to form functional open APOL1 cation channels might be identified upon modification with the large, hydrophilic MPB moiety, which would prevent their insertion into membranes and result in inhibition of trypanolytic activity.
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