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Characterization of CaMKIIα holoenzyme stability.
Torres-Ocampo, Ana P; Özden, Can; Hommer, Alexandra; Gardella, Anne; Lapinskas, Emily; Samkutty, Alfred; Esposito, Edward; Garman, Scott C; Stratton, Margaret M.
Afiliação
  • Torres-Ocampo AP; Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, Massachusetts, USA.
  • Özden C; Molecular and Cellular Biology Graduate Program, University of Massachusetts, Amherst, Massachusetts, USA.
  • Hommer A; Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, Massachusetts, USA.
  • Gardella A; Molecular and Cellular Biology Graduate Program, University of Massachusetts, Amherst, Massachusetts, USA.
  • Lapinskas E; Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, Massachusetts, USA.
  • Samkutty A; Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, Massachusetts, USA.
  • Esposito E; Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, Massachusetts, USA.
  • Garman SC; Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, Massachusetts, USA.
  • Stratton MM; Malvern Panlytical, Northampton, Massachusetts, USA.
Protein Sci ; 29(6): 1524-1534, 2020 06.
Article em En | MEDLINE | ID: mdl-32282091
Ca2+ /calmodulin-dependent protein kinase II (CaMKII) is a Ser/Thr kinase necessary for long-term memory formation and other Ca2+ -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 Tm = 36°C), which is slightly stabilized by ATP/MgCl2 binding (apparent Tm = 40°C) and significantly stabilized by regulatory segment binding (apparent Tm = 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 Tm ~ 90°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.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Holoenzimas / Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina Tipo de estudo: Prognostic_studies Limite: Humans Idioma: En Revista: Protein Sci Assunto da revista: BIOQUIMICA Ano de publicação: 2020 Tipo de documento: Article País de afiliação: Estados Unidos País de publicação: Estados Unidos

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Holoenzimas / Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina Tipo de estudo: Prognostic_studies Limite: Humans Idioma: En Revista: Protein Sci Assunto da revista: BIOQUIMICA Ano de publicação: 2020 Tipo de documento: Article País de afiliação: Estados Unidos País de publicação: Estados Unidos