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Publication Abstract from PubMed

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 CaMKIIalpha using a combination of differential scanning calorimetry (DSC), X-ray crystallography and Mass Photometry (MP). The kinase domain has a low thermal stability (apparent Tm = 36 degrees C), which is slightly stabilized by ATP/MgCl2 binding (apparent Tm = 40 degrees C), and significantly stabilized by regulatory segment binding (apparent Tm = 60 degrees 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 we observe in DSC measurements comparing the kinase alone to the kinase plus regulatory segment. The hub domain alone is extremely stable (apparent Tm ~90 degrees C), and the holoenzyme structure has multiple unfolding transitions ranging from ~60 degrees C - 100 degrees C. Using MP, we compared a CaMKIIalpha holoenzyme with different variable linker regions and determined that the dissociation of both these holoenzymes occurs at a higher concentration (is less stable) compared to 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. This article is protected by copyright. All rights reserved.

Characterization of CaMKIIalpha holoenzyme stability.,Torres-Ocampo AP, Ozden C, Hommer A, Gardella A, Lapinskas E, Samkutty A, Esposito E, Garman S, Stratton M Protein Sci. 2020 Apr 13. doi: 10.1002/pro.3869. PMID:32282091^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.