Characterization of CaMKIIα holoenzyme stability

Abstract

Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) is a Ser/Thr kinase necessary for long-term memory formation and other Ca 2+ -dependent signaling cascades such as fertilization. Here, we investigated the stability of CaMKIIα using a combination of differential scanning calorimetry (DSC), X-ray crystallography, and mass photometry (MP). The kinase domain has a low thermal stability (apparent T m = 36 C), which is slightly stabilized by ATP/MgCl 2 binding (apparent T m = 40 C) and significantly stabilized by regulatory segment binding (apparent T m = 60 C). We crystallized the kinase domain of CaMKII bound to p-coumaric acid in the active site. This structure reveals solvent-exposed hydrophobic residues in the substrate-binding pocket, which are normally buried in the autoinhibited structure when the regulatory segment is present. This likely accounts for the large stabilization that we observe in DSC measurements comparing the kinase alone with the kinase plus regulatory segment. The hub domain alone is extremely stable (apparent T m~9 0 C), and the holoenzyme structure has multiple unfolding transitions ranging from~60 C to 100 C. Using MP, we compared a CaMKIIα holoenzyme with different variable linker regions and determined that the dissociation of both these holoenzymes occurs at a higher concentration (is less stable) compared with the hub domain alone. We conclude that within the context of the holoenzyme structure, the kinase domain is stabilized, whereas the hub domain is destabilized. These data support a model where domains within the holoenzyme interact. K E Y W O R D SCaMKII, differential scanning calorimetry, mass photometry, oligomer dissociation, thermal stability

Conformational Spread Drives the Evolution of the Calcium-calmodulin Protein Kinase II

“…1B). This hinge is more rigid (0.29 + 0.002 nm) than the Lat-Dim hinge (0.355 + 0.016 nm) consistent with previous work 9,12 or the AD-KD "clip" contact (0.292 + 0.005 nm). I used principal component analysis (PCA) to classify long-range collective motions within the tetramer.…”

CaMKII binds both substrates and activators at the active site

“…This indicates that these hydrophobic residues shift to accommodate substrate binding, which is likely a large contribution to an observed gain in kinase domain stability upon GluN2B binding (Fig. S2e), similar to what we have previously observed with regulatory segment binding, which is mediated by L290 in the regulatory segment [36]. All effectors studied here have a leucine at the -5 position.…”

Breakage of the oligomeric CaMKII hub by the regulatory segment of the kinase

“…Consistent with this, a construct consisting of just the hub and the regulatory segment was shown to undergo spontaneous subunit exchange ( Bhattacharyya et al, 2016 ). Recent differential scanning calorimetry and mass photometry experiments have shown that while the isolated hub is very stable, the holoenzyme, especially with the linker present, is unstable and undergoes dissociation ( Torres-Ocampo et al, 2020 ).…”