Ligand‐induced CaMKIIα hub Trp403 flip, hub domain stacking, and modulation of kinase activity

Narayanan D, Larsen A, Gauger S, Adafia R, Hammershøi R, Hamborg L, Bruus‐Jensen J, Griem‐Krey N, Gee C, Frølund B, Stratton M, Kuriyan J, Kastrup J, Langkilde A, Wellendorph P, Solbak S, Protein Science 33(10) (2024) DOI

SASDUS4 – Calcium/calmodulin-dependent protein kinase type II alpha (CaMKIIα) WT hub domain with PIPA (i)

Calcium/calmodulin-dependent protein kinase type II subunit alpha
2-(6-(4-chlorophenyl)imidazo[1,2-b]pyridazine-2-yl)acetic acid
MWexperimental 243 kDa
MWexpected 186 kDa
VPorod 396 nm3
log I(s) 2.06×10-1 2.06×10-2 2.06×10-3 2.06×10-4
Calcium/calmodulin-dependent protein kinase type II subunit alpha 2-(6-(4-chlorophenyl)imidazo[1,2-b]pyridazine-2-yl)acetic acid small angle scattering data  s, nm-1
ln I(s)
Calcium/calmodulin-dependent protein kinase type II subunit alpha 2-(6-(4-chlorophenyl)imidazo[1,2-b]pyridazine-2-yl)acetic acid Guinier plot ln 2.06×10-1 Rg: 4.8 nm 0 (4.8 nm)-2 s2
(sRg)2I(s)/I(0)
Calcium/calmodulin-dependent protein kinase type II subunit alpha 2-(6-(4-chlorophenyl)imidazo[1,2-b]pyridazine-2-yl)acetic acid Kratky plot 1.104 0 √3 sRg
Dmax: 16 nm

Data validation

Fits and models

log I(s)
 s, nm-1
Calcium/calmodulin-dependent protein kinase type II subunit alpha 2-(6-(4-chlorophenyl)imidazo[1,2-b]pyridazine-2-yl)acetic acid PYMOL model
Calcium/calmodulin-dependent protein kinase type II subunit alpha 2-(6-(4-chlorophenyl)imidazo[1,2-b]pyridazine-2-yl)acetic acid PYMOL model
SAXS data from solutions of calcium/calmodulin-dependent protein kinase type II alpha (CaMKIIα) WT hub domain with PIPA in 20 mM MES, 150 mM NaCl, 1 mM DTT, 200 µM PIPA, pH 6 were collected using a Xenocs BioXolver L with MetalJet (University of Copenhagen, Department of Drug Design and Pharmacology) equipped with a Pilatus3 R 300K detector at a sample-detector distance of 0.6 m and at a wavelength of λ = 0.13402 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). One solute concentration of 1.20 mg/ml was measured at 25°C. 40 successive 60 second frames were collected. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted.

The data is a merge from data collected at two different configurations. In addition to the listed, data was collected on the same sample with sample-detector distance 1.4558 m (60 frames of 120 s exposure). The resulting data is a merge of the data from the two configurations. Several samples of CaMKIIa WT hub with PIPA were collected. This is sample (i), lowest concentration. The sample is not monodisperse but fitted by a mixture of CaMKIIa WT hub dodecamers and multimers of self-associated (stacked) dodecamers. Only single and double dodecameric models shown.

Calcium/calmodulin-dependent protein kinase type II subunit alpha (CaMKIIα WT hub)
Mol. type   Protein
Organism   Homo sapiens
Olig. state   Dodecamer
Mon. MW   15.5 kDa
 
UniProt   Q9UQM7 (345-475)
Sequence   FASTA
 
2-(6-(4-chlorophenyl)imidazo[1,2-b]pyridazine-2-yl)acetic acid (PIPA)
Mol. type   Other
Olig. state   Monomer
Mon. MW   0.3 kDa