CaMKK2 kinase domain interacts with the autoinhibitory region through the N-terminal lobe including the RP insert

alcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2), a member of the Ca$^{⁠2+}$/calmodulin-dependent kinase (CaMK) family, functions as an upstream activator of CaMKI, CaMKIV and AMP-activated protein kinase. Thus, CaMKK2 is involved in the regulation of several key physiological and pathophysiological processes. Previous studies have suggested that Ca$^{⁠2+}$/CaM binding may cause unique conformational changes in the CaMKKs compared with other CaMKs. However, the underlying mechanistic details remain unclear.MethodsIn this study, hydrogen-deuterium exchange coupled to mass spectrometry, time-resolved fluorescence spectroscopy, small-angle x-ray scattering and chemical cross-linking were used to characterize Ca$^{⁠2+}$/CaM binding-induced structural changes in CaMKK2.ResultsOur data suggest that: (i) the CaMKK2 kinase domain interacts with the autoinhibitory region (AID) through the N-terminal lobe of the kinase domain including the RP insert, a segment important for targeting downstream substrate kinases; (ii) Ca$^{⁠2+}$/CaM binding affects the structure of several regions surrounding the ATP-binding pocket, including the activation segment; (iii) although the CaMKK2:Ca$^{⁠2+}$/CaM complex shows high conformational flexibility, most of its molecules are rather compact; and (iv) AID-bound Ca$^{⁠2+}$/CaM transiently interacts with the CaMKK2 kinase domain.ConclusionsInteractions between the CaMKK2 kinase domain and the AID differ from those of other CaMKs. In the absence of Ca$^{⁠2+}$/CaM binding the autoinhibitory region inhibits CaMKK2 by both blocking access to the RP insert and by affecting the structure of the ATP-binding pocket.General significanceOur results corroborate the hypothesis that Ca$^{⁠2+}$/CaM binding causes unique conformational changes in the CaMKKs relative to other CaMKs