Structural Characterization of Calcium-Dependent Calmodulin-Calmidazolium Binding using Capillary Vibrating...

Capillary vibrating sharp-edge spray ionization (cVSSI)-based native mass spectrometry (nMS) and in-droplet hydrogen deuterium exchange (HDX-MS) were used to evaluate calcium-dependent interactions between calmodulin and calmidazolium (CDZ). cVSSI was found to produce a narrow charge-state distribution (CSD) with low average charge states, indicating that this method preserved native-like states. cVSSI was also able to resolve stepwise Ca ²⁺ binding for species containing one to four Ca ²⁺ ions. In the absence of Ca ²⁺ , no detectable CDZ binding was observed. However, CDZ binding was observed when calmodulin was fully loaded with Ca ²⁺ . CDZ binding to the protein caused marked redistribution of the CSD toward lower charge states, consistent with ligand-induced stabilization of the protein into a more compact conformational ensemble. The apparent dissociation constant ( K _d ) of the interaction was determined to be 261 ± 29 nM and 126 ± 17 nM from Langmuir and quadratic binding models, respectively. Complementary in-droplet HDX-MS revealed that deuterium uptake was correlated with charge state across the CSD, corroborating the nMS evidence for stepwise conformational compaction of CaM. Within the individual charge states, two distinct populations representing CDZ-bound and unbound CaM were resolved by HDX-MS, which were not detectable by nMS alone. Comparison of these populations within each of the 6+, 5+ and 4+ charge states revealed reductions in deuterium uptake by approximately 37%, 22%, and 11% upon CDZ binding, respectively, consistent with ligand occlusion and local structural tightening at the binding interface. The gradient in protection across charge states further reflects differences in binding interface accessibility among CaM conformers spanning a range of CDZ-induced conformational states. Together, these results demonstrate that cVSSI-based nMS coupled with in-droplet HDX-MS provides an integrated platform for simultaneously resolving metal loading, ligand binding, binding affinity, and ligand-induced conformational changes. This approach complements traditional structural methods by enabling direct interrogation of dynamic, metal-dependent protein-ligand interactions in their native states.