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Allosteric factors in the calcium/calmodulin-responsive kinase II hub domain determine selectivity of GHB ligands for CaMKIIα.

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Title: Allosteric factors in the calcium/calmodulin-responsive kinase II hub domain determine selectivity of GHB ligands for CaMKIIα.
Authors: Gauger, Stine J.1, Palmelund, Line B.1, Yongsong Tian1,2, Marek, Ales2, Namini, Mathias R.1, Griem-Krey, Nane1, Kickinger, Stefanie1, Mortensen, Jonas S.1, Frølund, Bente1, Wellendorph, Petrine1 pw@sund.ku.dk
Source: Journal of Biological Chemistry. Jun2025, Vol. 301 Issue 6, p1-10. 10p.
Subjects: Site-specific mutagenesis, Ligands (Biochemistry), Protein kinases, Isoenzymes, Proteins
Abstract: The Ca2+/CaM-dependent protein kinase II alpha (CaMKIIa) is a highly important synaptic protein, which comprises a unique holoenzyme structure organized via the central hub domain. Recently, a distinct binding pocket in the CaMKIIa hub domain was identified for the endogenous neuromodulator g-hydroxybutyric acid (GHB) and related synthetic analogs. Intriguingly, of the four native CaMKII isozymes, only CaMKIIa accommodates GHB ligands. Key interacting residues in CaMKIIa were revealed, but their involvement in selectivity toward the alpha variant of CaMKII has remained unresolved. Aimed at elucidating the molecular determinants for this selectivity, we here conducted binding studies to CaMKII-HEK whole-cell homogenates using two different in-house-developed GHB-related radioligands, 3-hydroxycyclopent-1-enecarboxylic acid ([3H]HOCPCA) and [3H]O-5-hydroxydiclofenac, in combination with site-directed mutagenesis. Binding to CaMKIIa with the smaller type radioligand [3H]HOCPCA validated key involvement of the four known residues (His395, Arg433, Arg453, and Arg469), but also revealed a role for the upper hub flexible loop containing the CaMKIIa-specific residue Trp403 (Leu in all other CaMKII isozymes) previously suggested to be involved in holoenzyme stability. Insertion of the corresponding residues (L467W/C533R) into CaMKIIb failed to induce [3H]HOCPCA binding. However, with the larger type radioligand, [3H]O-5-hydroxydiclofenac, specific binding in CaMKIIb (L467W/C533R) was achieved. Thus, the study confirms involvement of central binding residues and identifies the CaMKIIa flexible pocket loop as a distantly located allosteric factor in determining selectivity of GHB analogs for CaMKIIa. It sheds light on a remarkable interplay of the entire hub cavity for accommodation of ligands and corroborates GHB analogs as CaMKIIa-selective. [ABSTRACT FROM AUTHOR]
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Database: Engineering Source
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Abstract:The Ca2+/CaM-dependent protein kinase II alpha (CaMKIIa) is a highly important synaptic protein, which comprises a unique holoenzyme structure organized via the central hub domain. Recently, a distinct binding pocket in the CaMKIIa hub domain was identified for the endogenous neuromodulator g-hydroxybutyric acid (GHB) and related synthetic analogs. Intriguingly, of the four native CaMKII isozymes, only CaMKIIa accommodates GHB ligands. Key interacting residues in CaMKIIa were revealed, but their involvement in selectivity toward the alpha variant of CaMKII has remained unresolved. Aimed at elucidating the molecular determinants for this selectivity, we here conducted binding studies to CaMKII-HEK whole-cell homogenates using two different in-house-developed GHB-related radioligands, 3-hydroxycyclopent-1-enecarboxylic acid ([3H]HOCPCA) and [3H]O-5-hydroxydiclofenac, in combination with site-directed mutagenesis. Binding to CaMKIIa with the smaller type radioligand [3H]HOCPCA validated key involvement of the four known residues (His395, Arg433, Arg453, and Arg469), but also revealed a role for the upper hub flexible loop containing the CaMKIIa-specific residue Trp403 (Leu in all other CaMKII isozymes) previously suggested to be involved in holoenzyme stability. Insertion of the corresponding residues (L467W/C533R) into CaMKIIb failed to induce [3H]HOCPCA binding. However, with the larger type radioligand, [3H]O-5-hydroxydiclofenac, specific binding in CaMKIIb (L467W/C533R) was achieved. Thus, the study confirms involvement of central binding residues and identifies the CaMKIIa flexible pocket loop as a distantly located allosteric factor in determining selectivity of GHB analogs for CaMKIIa. It sheds light on a remarkable interplay of the entire hub cavity for accommodation of ligands and corroborates GHB analogs as CaMKIIa-selective. [ABSTRACT FROM AUTHOR]
ISSN:00219258
DOI:10.1016/j.jbc.2025.108543