Proteomic Snapshots of Structural Cross-Linking Rearrangements in Ca ^{2 <b>+</b>} /Calmodulin-Dependent Kinase-1-Delta Associated with Its Regulation by ATP, Ca ^{2 <b>+</b>} /Calmodulin, and Reduction Potential

Ca²⁺/calmodulin-dependent kinase 1 delta (CaMK1δ) plays a central role in regulatory pathways associated with ATP, reduction potential, and Ca²⁺/calmodulin (CaM). Mass spectrometry (MS)-based structural proteomics incorporating FragPipe and pLink cross-link analysis was used to reveal conformation selection induced by dialysis with ATP, reducing agents, and CaM. The structural changes were mediated via cysteine and phosphate cross-linking and loop-linking of the activation loop within the C-terminal. Phosphate loop-linking was validated by β-elimination and Michael addition (BEMAD) reactions, aligning these findings with phosphoproteomics analyses of phosphorylation events. Oxidizing conditions inhibited the functionality of CaMK1δ wild-type. A novel mechanism of autoinhibition via cysteine cross-linking between the activation loop (αT) and C-terminal (αI) helices was identified. The microenvironment associated with CaMK1δ, including ATP availability, CaM concentration, and reduction potential, modulates the structural rearrangements underlying autophosphorylation. Phosphoproteomics, cysteine and phosphate cross-linking MS, and structural molecular modeling were used to describe kinase activation, allowing the activation of regulatory kinases to be reevaluated. We propose that regulatory kinases respond to an array of kinase family-specific distinct second messengers which can be studied using this multiomics framework, giving significant new insights into PTMs as well as the associated protein structure rearrangements.