The Use of Enhanced Sampling Molecular Dynamics (ES-MD) to Determine PIP2 Binding in SK2-Calmodulin Complex

Abstract

Small conductance Ca2+-activated K+ (SK) channels are unique since they are gated solely by changes in intracellular Ca2+ and are known to be upregulated in heart failure and cardiac arrhythmias. SK channels are regulated via the ubiquitous Ca2+ sensing protein, calmodulin (CaM). New evidence suggests that the anionic lipid phosphatidylinositol 4,5-bisphosphate (PIP2) is also essential for the activation for SK channels. Recent cryo-EM structures with a nearly complete tetrameric SK4-CaM complex (PDB: 6CNM, 6CNN, 6CNO) allowed us to generate nearly full-length SK2-CaM homology models (fSK2-CaM). In addition to PIP2 binding sites previously predicted in a crystal structure of SK2 peptide complexed with CaM (PDB: 4QNH), we have identified areas of basic amino acid clustering in the fSK2-CaM structure around the lower leaflet of the surrounding lipid bilayer. Dockings of PIP2 onto fSK2-CaM were performed to assess possible PIP2 binding sites. We further used all-atom enhanced sampling molecular dynamics (ES-MD) simulations on Frontera CPU nodes to refine the proposed binding sites for 3 separate states of fSK2-CaM (closed, open/inactivated, and open/activated). Orientation of PIP2 in the binding sites, corresponding binding affinities and mechanisms of channel activation were determined from ES-MD runs and further tested biochemically. The use of ES-MD has provided novel insights into PIP2 binding and regulation of SK2-CaM complexes.