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Carna Inc camkiiα holoenzyme

PIPA binds to <t>CaMKIIα</t> WT hub and inhibits substrate phosphorylation. (a) Chemical structures of γ‐hydroxybutyric acid (GHB), 3‐hydroxycyclopenten‐1‐enecarboxylic acid (HOCPCA), 5‐hydroxydiclofenac (5‐HDC), ( E )‐2‐(5‐hydroxy‐2‐phenyl‐5,7,8,9‐tetrahydro‐6 H ‐benzo[7]annulen‐6‐ylidene)acetic acid (Ph‐HTBA), and 2‐(6‐(4‐chlorophenyl)imidazo[1,2‐ b ]pyridazine‐2‐yl)acetic acid (PIPA). (b) Concentration‐dependent inhibition of [ 3 H]HOCPCA binding by PIPA in whole‐cell homogenates from HEK293T cells transfected with CaMKIIα (pooled data, n = 4; GHB for comparison; Leurs et al., ). (c) Concentration‐dependent binding of PIPA to immobilized CaMKIIα WT hub measured by SPR, and (d) associated Langmuir‐binding isotherm (representative data of n = 3). (e) Right‐shifted TSA melting curves of CaMKIIα WT hub upon binding of PIPA (representative data of n = 3). (f) Saturation isotherm (representative). (g) Concentration‐dependent inhibition of CaMKIIα syntide‐2 by PIPA using sub‐maximal (30 nM) CaM concentration in the luminescence‐based ADP‐Glo kinase assay (pooled data, n = 4, mean ± SEM). (h) Right‐shifted CaM curve of CaMKIIα syntide‐2 phosphorylation in the presence of PIPA (pooled data, n = 3). For clarity, only mean values without variation are given; all mean values can be found in the respective results section.

Camkiiα Holoenzyme, supplied by Carna Inc, used in various techniques. Bioz Stars score: 96/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/02-109/CaMK2a/pmc11400628-184-12-15
Average 96 stars, based on 5 article reviews

camkiiα holoenzyme - by Bioz Stars, 2026-09

96/100 stars

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1) Product Images from "Ligand‐induced CaMKIIα hub Trp403 flip, hub domain stacking, and modulation of kinase activity"

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

Journal: Protein Science : A Publication of the Protein Society

doi: 10.1002/pro.5152

PIPA binds to CaMKIIα WT hub and inhibits substrate phosphorylation. (a) Chemical structures of γ‐hydroxybutyric acid (GHB), 3‐hydroxycyclopenten‐1‐enecarboxylic acid (HOCPCA), 5‐hydroxydiclofenac (5‐HDC), ( E )‐2‐(5‐hydroxy‐2‐phenyl‐5,7,8,9‐tetrahydro‐6 H ‐benzo[7]annulen‐6‐ylidene)acetic acid (Ph‐HTBA), and 2‐(6‐(4‐chlorophenyl)imidazo[1,2‐ b ]pyridazine‐2‐yl)acetic acid (PIPA). (b) Concentration‐dependent inhibition of [ 3 H]HOCPCA binding by PIPA in whole‐cell homogenates from HEK293T cells transfected with CaMKIIα (pooled data, n = 4; GHB for comparison; Leurs et al., ). (c) Concentration‐dependent binding of PIPA to immobilized CaMKIIα WT hub measured by SPR, and (d) associated Langmuir‐binding isotherm (representative data of n = 3). (e) Right‐shifted TSA melting curves of CaMKIIα WT hub upon binding of PIPA (representative data of n = 3). (f) Saturation isotherm (representative). (g) Concentration‐dependent inhibition of CaMKIIα syntide‐2 by PIPA using sub‐maximal (30 nM) CaM concentration in the luminescence‐based ADP‐Glo kinase assay (pooled data, n = 4, mean ± SEM). (h) Right‐shifted CaM curve of CaMKIIα syntide‐2 phosphorylation in the presence of PIPA (pooled data, n = 3). For clarity, only mean values without variation are given; all mean values can be found in the respective results section.

Figure Legend Snippet: PIPA binds to CaMKIIα WT hub and inhibits substrate phosphorylation. (a) Chemical structures of γ‐hydroxybutyric acid (GHB), 3‐hydroxycyclopenten‐1‐enecarboxylic acid (HOCPCA), 5‐hydroxydiclofenac (5‐HDC), ( E )‐2‐(5‐hydroxy‐2‐phenyl‐5,7,8,9‐tetrahydro‐6 H ‐benzo[7]annulen‐6‐ylidene)acetic acid (Ph‐HTBA), and 2‐(6‐(4‐chlorophenyl)imidazo[1,2‐ b ]pyridazine‐2‐yl)acetic acid (PIPA). (b) Concentration‐dependent inhibition of [ 3 H]HOCPCA binding by PIPA in whole‐cell homogenates from HEK293T cells transfected with CaMKIIα (pooled data, n = 4; GHB for comparison; Leurs et al., ). (c) Concentration‐dependent binding of PIPA to immobilized CaMKIIα WT hub measured by SPR, and (d) associated Langmuir‐binding isotherm (representative data of n = 3). (e) Right‐shifted TSA melting curves of CaMKIIα WT hub upon binding of PIPA (representative data of n = 3). (f) Saturation isotherm (representative). (g) Concentration‐dependent inhibition of CaMKIIα syntide‐2 by PIPA using sub‐maximal (30 nM) CaM concentration in the luminescence‐based ADP‐Glo kinase assay (pooled data, n = 4, mean ± SEM). (h) Right‐shifted CaM curve of CaMKIIα syntide‐2 phosphorylation in the presence of PIPA (pooled data, n = 3). For clarity, only mean values without variation are given; all mean values can be found in the respective results section.

Techniques Used: Concentration Assay, Inhibition, Binding Assay, Transfection, Comparison, Kinase Assay

PIPA bound to CaMKIIα 6x hub promotes Trp403 flip while acetate does not. (a) X‐ray crystal structure of tetradecameric CaMKIIα 6x hub (PDB entry 9EOY ) in schematic representation, with two subunits bound to PIPA (orange), 10 subunits to acetate (ACT) only (green), and two to ACT + PIPA (yellow). Trp403 is shown in red. (b) The structure of CaMKIIα 6x hub with seven subunits A–G in the asymmetric unit of the crystal, forming a tetradecamer with symmetry‐related molecules, is shown in white cartoon representation with PIPA (orange carbon atoms), ACT (green), PEG (cyan), Arg453 (white), and Trp403 (white) shown in sticks representation. (c) Zoom‐in on binding mode and interactions of acetate in subunit A. Salt bridges and hydrogen bonds between acetate and corresponding amino‐acid residues are shown as black dashed lines and with compounds and residues as sticks representation. In addition, a hydrogen bond between the C‐terminal carboxylate group of Val475 and Trp403 is shown. The 2 F o − F c electron densities for the ligands are contoured at 1.0 sigma and carved at 1.6 Å. (d) Alternative binding mode and interactions of PIPA in subunit C. Hydrogen‐bonding interactions between PIPA, ACT, and corresponding amino‐acid residues are shown as black dashed lines and with compounds and residues as sticks representation. The 2 F o − F c electron densities for PIPA (carved at 1.6 Å), ACT (carved at 1.75 Å), and PEG (carved at 2 Å) have been contoured at 0.5 sigma. In addition, Trp403 and Val475 are shown as sticks representation. (e) Binding mode and interactions of PIPA in subunit D. Salt bridges and hydrogen bonds between PIPA and corresponding amino‐acid residues (as sticks representation) are shown as black dashed lines. In addition, Trp403 and Val475 are shown as sticks representation. The 2 F o − F c electron density for PIPA has been contoured at 0.5 sigma and carved at 1.8 Å. (f–h) Binding site interactions between CaMKIIα 6x hub and acetate (f) and PIPA (g, h) analyzed using the LigPlot+ software (Laskowski & Swindells, ). Hydrogen bonds and hydrophobic interactions between amino‐acid residues and ligands are depicted as green dotted lines and red semicircles, respectively.

Figure Legend Snippet: PIPA bound to CaMKIIα 6x hub promotes Trp403 flip while acetate does not. (a) X‐ray crystal structure of tetradecameric CaMKIIα 6x hub (PDB entry 9EOY ) in schematic representation, with two subunits bound to PIPA (orange), 10 subunits to acetate (ACT) only (green), and two to ACT + PIPA (yellow). Trp403 is shown in red. (b) The structure of CaMKIIα 6x hub with seven subunits A–G in the asymmetric unit of the crystal, forming a tetradecamer with symmetry‐related molecules, is shown in white cartoon representation with PIPA (orange carbon atoms), ACT (green), PEG (cyan), Arg453 (white), and Trp403 (white) shown in sticks representation. (c) Zoom‐in on binding mode and interactions of acetate in subunit A. Salt bridges and hydrogen bonds between acetate and corresponding amino‐acid residues are shown as black dashed lines and with compounds and residues as sticks representation. In addition, a hydrogen bond between the C‐terminal carboxylate group of Val475 and Trp403 is shown. The 2 F o − F c electron densities for the ligands are contoured at 1.0 sigma and carved at 1.6 Å. (d) Alternative binding mode and interactions of PIPA in subunit C. Hydrogen‐bonding interactions between PIPA, ACT, and corresponding amino‐acid residues are shown as black dashed lines and with compounds and residues as sticks representation. The 2 F o − F c electron densities for PIPA (carved at 1.6 Å), ACT (carved at 1.75 Å), and PEG (carved at 2 Å) have been contoured at 0.5 sigma. In addition, Trp403 and Val475 are shown as sticks representation. (e) Binding mode and interactions of PIPA in subunit D. Salt bridges and hydrogen bonds between PIPA and corresponding amino‐acid residues (as sticks representation) are shown as black dashed lines. In addition, Trp403 and Val475 are shown as sticks representation. The 2 F o − F c electron density for PIPA has been contoured at 0.5 sigma and carved at 1.8 Å. (f–h) Binding site interactions between CaMKIIα 6x hub and acetate (f) and PIPA (g, h) analyzed using the LigPlot+ software (Laskowski & Swindells, ). Hydrogen bonds and hydrophobic interactions between amino‐acid residues and ligands are depicted as green dotted lines and red semicircles, respectively.

Techniques Used: Binding Assay, Software

Solution structures of CaMKIIα WT hub reveals PIPA‐induced structured self‐association. (a) SAXS scattering curves for the CaMKIIα WT, 6x, and W403L hub domains, with corresponding Guinier fits (insert). Full concentration series of CaMKIIα WT hub and CaMKIIα 6x hub are included in Figure . (b) Comparison of CaMKIIα WT hub and CaMKIIα 6x hub curves to atomic models of the dodecamer (PDB entry 5IG3 ; Bhattacharyya et al., ) and tetradecamer (PDB entry 6OF8 ; McSpadden et al., ), respectively. The scattering curves have been translated for clarity. (c) SAXS scattering curves of CaMKIIα WT hub in the presence and absence of PIPA, including oligomer fit using model of self‐associated dodecamers. (d) SAXS scattering curves of CaMKIIα W403L hub in the presence and absence of PIPA. (e) Examples of the stacked self‐associated dodecamers used to model CaMKIIα WT hub with PIPA data. A single dodecamer is shown in two orientations. In addition, a dimer of dodecamers and a stacked model including 10 dodecamers are illustrated. Models and figure were created using PyMOL. (f) Representative MP histograms showing CaMKIIα WT hub diluted to 500 nM with and without the addition of PIPA. (g) Representative MP histograms showing W403L CaMKIIα hub diluted to 500 nM, with and without PIPA. Each count indicates a single molecule. Additional MP data are shown in Figure .

Figure Legend Snippet: Solution structures of CaMKIIα WT hub reveals PIPA‐induced structured self‐association. (a) SAXS scattering curves for the CaMKIIα WT, 6x, and W403L hub domains, with corresponding Guinier fits (insert). Full concentration series of CaMKIIα WT hub and CaMKIIα 6x hub are included in Figure . (b) Comparison of CaMKIIα WT hub and CaMKIIα 6x hub curves to atomic models of the dodecamer (PDB entry 5IG3 ; Bhattacharyya et al., ) and tetradecamer (PDB entry 6OF8 ; McSpadden et al., ), respectively. The scattering curves have been translated for clarity. (c) SAXS scattering curves of CaMKIIα WT hub in the presence and absence of PIPA, including oligomer fit using model of self‐associated dodecamers. (d) SAXS scattering curves of CaMKIIα W403L hub in the presence and absence of PIPA. (e) Examples of the stacked self‐associated dodecamers used to model CaMKIIα WT hub with PIPA data. A single dodecamer is shown in two orientations. In addition, a dimer of dodecamers and a stacked model including 10 dodecamers are illustrated. Models and figure were created using PyMOL. (f) Representative MP histograms showing CaMKIIα WT hub diluted to 500 nM with and without the addition of PIPA. (g) Representative MP histograms showing W403L CaMKIIα hub diluted to 500 nM, with and without PIPA. Each count indicates a single molecule. Additional MP data are shown in Figure .

Techniques Used: Concentration Assay, Comparison

CaMKIIα hub mutation confirms a central role for Trp403 in PIPA binding. (a) Quenching of intrinsic tryptophan fluorescence caused by Trp403 flip in the CaMKIIα 6x hub with increasing concentrations of PIPA: 0.43 μM (black), 1 μM (red), 2.3 μM (orange), 5.3 μM (pink), 12.1 μM (purple), and (b) resulting normalized inhibition curve (pooled data, n = 3). (c) Right‐shifted TSA melting curves of CaMKIIα W403L hub upon binding of PIPA (representative data). (d) Thermal melting point ( T m ) of CaMKIIα WT hub (blue) and CaMKIIα W403L hub (gray) plotted against increasing concentrations of PIPA (representative data of n = 3). (e) Concentration‐dependent binding of PIPA to immobilized CaMKIIα W403L hub measured by SPR (representative data), and (f) associated Langmuir‐binding isotherm (representative data of n = 2).

Figure Legend Snippet: CaMKIIα hub mutation confirms a central role for Trp403 in PIPA binding. (a) Quenching of intrinsic tryptophan fluorescence caused by Trp403 flip in the CaMKIIα 6x hub with increasing concentrations of PIPA: 0.43 μM (black), 1 μM (red), 2.3 μM (orange), 5.3 μM (pink), 12.1 μM (purple), and (b) resulting normalized inhibition curve (pooled data, n = 3). (c) Right‐shifted TSA melting curves of CaMKIIα W403L hub upon binding of PIPA (representative data). (d) Thermal melting point ( T m ) of CaMKIIα WT hub (blue) and CaMKIIα W403L hub (gray) plotted against increasing concentrations of PIPA (representative data of n = 3). (e) Concentration‐dependent binding of PIPA to immobilized CaMKIIα W403L hub measured by SPR (representative data), and (f) associated Langmuir‐binding isotherm (representative data of n = 2).

Techniques Used: Mutagenesis, Binding Assay, Fluorescence, Inhibition, Concentration Assay

Model for sequential two‐step process of PIPA binding to CaMKIIα 6x hub (PDB entry 9EOY ). Surface representation of subunit A, where Trp403 (marked in red) is flipped inward as shown to the left. PIPA (magenta sticks) has been modeled into site 1 to illustrate entrance of PIPA into the CaMKIIα 6x hub. Subunit D is shown to the right where PIPA (cyan sticks) binds at the conventional binding site 2, where Trp403 is flipped outward as shown to the right.

Figure Legend Snippet: Model for sequential two‐step process of PIPA binding to CaMKIIα 6x hub (PDB entry 9EOY ). Surface representation of subunit A, where Trp403 (marked in red) is flipped inward as shown to the left. PIPA (magenta sticks) has been modeled into site 1 to illustrate entrance of PIPA into the CaMKIIα 6x hub. Subunit D is shown to the right where PIPA (cyan sticks) binds at the conventional binding site 2, where Trp403 is flipped outward as shown to the right.

Techniques Used: Binding Assay

Inhibition:

Article Title: The oral Ca/calmodulin‐dependent kinase II inhibitor RA608 improves contractile function and prevents arrhythmias in heart failure
Article Snippet: .. Ca/calmodulin‐dependent kinase II inhibition was measured by capillary electrophoresis mobility shift assay technology from Caliper Life Sciences (PerkinElmer) using purified human CaMKII protein isoforms: CaMKIIα (Carna Biosciences, 02‐109), CaMKIIβ (Millipore, 14‐718), CaMKIIɣ (Millipore, 14‐719), and CaMKIIδ (Millipore, 14‐723) as previously described. .. Myocytes on laminin‐coated recording chambers were loaded with 10 μmol/L Fluo‐4 acetoxymethyl ester in the presence of 0.02% (w/v) pluronic acid (Molecular Probes; 15 min incubation), mounted on stage of a laser‐scanning confocal microscope (LSM 7, Zeiss), and superfused with normal Tyrode solution.

Article Title: The oral Ca/calmodulin-dependent kinase II inhibitor RA608 improves contractile function and prevents arrhythmias in heart failure.
Article Snippet: .. Ca/calmodulin-dependent kinase II enzymatic assays Ca/calmodulin-dependent kinase II inhibition was measured by capillary electrophoresis mobility shift assay technology from Caliper Life Sciences (PerkinElmer) using purified human CaMKII protein isoforms: CaMKIIα (Carna Biosciences, 02- 109), CaMKIIβ (Millipore, 14-718), CaMKIIɣ (Millipore, 14- 719), and CaMKIIδ (Millipore, 14-723) as previously described.18 ESC Heart Failure (2020) DOI: 10.1002/ehf2.12895 .. Myocytes on laminin-coated recording chambers were loaded with 10 μmol/L Fluo-4 acetoxymethyl ester in the presence of 0.02% (w/v) pluronic acid (Molecular Probes; 15 min incubation), mounted on stage of a laser-scanning confocal microscope (LSM 7, Zeiss), and superfused with normal Tyrode solution.

Electrophoresis:

Mobility Shift:

Purification:

Article Title: Ligand‐induced CaMKIIα hub Trp403 flip, hub domain stacking, and modulation of kinase activity
Article Snippet: SPR measurements were performed at 25°C using a Pioneer FE instrument (Sartorius). .. Recombinant purified CaMKIIα WT hub, CaMKIIα 6x hub, CaMKIIα W403L hub, and CaMKIIα holoenzyme (#02‐109, Carna Biosciences) were immobilized by amine coupling on to a biosensor using 20 mM NaAc, pH 5 buffer. ..

Recombinant:

Article Title: GHB analogs confer neuroprotection through specific interaction with the CaMKIIα hub domain
Article Snippet: SPR measurements were performed at 25 °C using a Pioneer FE instrument (Molecular Devices, FortéBio). .. 6x Hub human protein, WT hub or full-length human recombinant CaMKIIa (#02-109, Carna Biosciences) were immobilized on to a biosensor surfaces by amine coupling using a 20 mM NaAc pH 5 immobilization buffer. ..

Article Title: Ligand-induced CaMKIIα hub Trp403 flip, hub domain stacking and kinase inhibition
Article Snippet: SPR measurements were performed at 25 °C using a Pioneer FE instrument (Sartorius). .. Recombinant CaMKIIα WT hub, CaMKIIα 6x hub, CaMKIIα W403L hub, and CaMKIIα holoenzyme (#02-109, Carna Biosciences) were immobilized by amine coupling on to a biosensor using 20 mM NaAc, pH 5 immobilization buffer. ..

Article Title: GHB confers neuroprotection by stabilizing the CaMKIIα hub domain
Article Snippet: SPR measurements were performed at 25 °C using a Pioneer FE instrument (Molecular Devices, FortéBio). .. 6x Hub human protein, WT hub or full-length human recombinant CaMKIIα (#02-109, Carna Biosciences) were immobilized on to a biosensor surfaces by amine coupling using a 20 mM NaAc pH 5 immobilization buffer. ..

Phospho-proteomics:

Article Title: GHB analogs confer neuroprotection through specific interaction with the CaMKIIα hub domain
Article Snippet: .. Syntide-II substrate phosphorylation was assayed using the ADP-Glo Assay kit (#V9101, Promega) using CaMKIIa full-length protein with a N-terminal GST-tag (#02-109, Carna Biosciences) or CaMKIIa full-length protein with a C-terminal 6xHis-tag (#PR4586C, Thermo Fisher). .. All experiments were performed in 384-well white polypropylene plates (#784075, Greiner) with a working volume of 25 μL.

Article Title: GHB confers neuroprotection by stabilizing the CaMKIIα hub domain
Article Snippet: .. Syntide-II substrate phosphorylation was assayed using the ADP-Glo Assay kit (#V9101, Promega) using CaMKIIα full-length protein with a N-terminal GST-tag (#02-109, Carna Biosciences) or CaMKIIα full-length protein with a C-terminal 6xHis-tag (#PR4586C, Thermo Fisher). .. All experiments were performed in 384-well white polypropylene plates (#784075, Greiner) with a working volume of 25 μL.

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