Structure of Calcineurin bound to PI4KA reveals dual interface in both PI4KA and FAM126A

Phosphatidylinositol 4 kinase alpha (PI4KA, or PI4KIIIα), is crucial for maintaining the PI4P and phosphatidylserine pools of the plasma membrane. A key regulator of PI4KA is its association into a trimeric complex with TTC7 and FAM126 regulatory proteins. The activity of this complex can be regulated by the lipidated CNAβ1 isoform of the protein phosphatase Calcineurin. We previously identified that CNAβ1 directly binds to FAM126A. Here, we report a cryo-EM structure of a truncated PI4KA complex bound to Calcineurin, which reveals a direct Calcineurin interaction with PI4KA, forming a dimer of pentamers (PI4KA-TTC7B-FAM126A-CNA-CNB). HDX-MS, cryo-EM and computational modelling show that Calcineurin forms a direct complex with evolutionarily conserved IKISVT and LVPP sequences in PI4KA’s horn and dimerization domains. We also characterised conserved LTLT and PSISIT Calcineurin binding sequences in the C-terminus of FAM126A. These sites are located in close spatial proximity to multiple phosphorylation sites in the PI4KA complex, suggesting key roles of Calcineurin-regulated phosphosites in PI4KA regulation. This work reveals novel insight into how Calcineurin can regulate PI4KA activity at the plasma membrane.