A Novel Plate‐Binding Array to Characterize CaMKII Protein‐Protein Interactions
Authors: Perfitt Tyler L , Marks Christian R , Colbran Roger J 
Published: November 2023

Ca2+/calmodulin‐dependent protein kinase II (CaMKII) is a multifunctional Ser/Thr kinase with a large interactome. Improved mechanistic understanding of how various CaMKII‐associated proteins (CaMKAPs) interact with CaMKII can provide invaluable insight into their biological roles. Previous work by our lab and others has examined the functional relevance of single protein‐protein interactions with CaMKII, but the large CaMKII interactome has made the physiological consequences of specific interactions difficult to study. To address this problem, we aim to identify point mutations in CaMKII that disrupt binding interactions with specific CaMKAPs while retaining essentially normal binding to others. In order to identify CaMKII mutants with specific binding deficits, we have designed a medium‐throughput screen to selectively disrupt specific interactions. Positive hits from this screen will be further characterized in biochemical and physiological studies.A library of GST‐tagged CaMKAPs is immobilized in glutathione‐coated 96 well plates. Lysates of HEK293FT cells expressing CaMKIIα (wt or mutant) fused to a fluorescent protein are then incubated in these wells, and unbound lysate is washed off. Bound CaMKII is quantified using a fluorescence plate reader, allowing us to compare relative binding strength between wild‐type and mutant CaMKII. Initial studies showed that eGFP‐CaMKIIα binding to NMDA receptor GluN2B subunit required CaMKII activation by Ca2+/calmodulin and adenosine nucleotide, and was blocked by a CaMKII inhibitor peptide (CaMKIIN‐tide). Switching to a monomeric mApple fluorophore reduced background noise in our GST negative control and enhanced the bound fluorescence signal. As an additional control, we found no significant binding of soluble mApple to any GST‐fusion proteins. Moreover, levels of bound fluorescence depend on the amount of immobilized GST‐fusion protein as well as the concentration of mApple‐CaMKIIα in HEK293FT cell lysates, demonstrating that this array is concentration‐dependent.As proof of concept, we designed a library of 19 CaMKIIα mutants with single point mutations in the catalytic domain based on available functional and structural information. Each mutant was expressed as an eGFP‐or mApple fusion protein; CaMKII concentrations in lysates were quantified and normalized by immunoblotting and by the levels of eGFP/mApple fluorescence. GST fusion proteins containing CaMKII‐binding domains from GluN2B, densin‐180 (2 domains), the CaV1.3 N‐terminal domain, the β2a subunit of voltage‐gated calcium channels, spinophilin, and SAP97 were tested. As expected based on prior studies, an I205K mutation disrupted binding with GluN2B, but also disrupted binding to densin‐180 IN domain and CaV1.3 NTD. We identified other mutations in this domain that also broadly disrupted interactions with CaMKAPs. However, we found that a V102E mutation selectively disrupted CaMKII binding to the CaV1.3 NTD, with no significant effect on binding to other CaMKAPs. In addition, we found that an ASD‐associated CaMKIIα mutation globally reduced binding to CaMKAPs. This method can also be used to detect phosphorylation‐dependent changes in binding using phospho‐mimetic and phospho‐null mutants. Our experimental setup can be applied to other proteins and provides a novel approach to study single protein‐protein interactions within a large interactome.Support or Funding InformationSupport was generously provided by the NIH (R01‐MH063232) and the Molecular Endocrinology Training Program (T32‐DK007563).