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

The Ca²⁺/CaM-dependent protein kinase II alpha (CaMKIIα) 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 CaMKIIα hub domain was identified for the endogenous neuromodulator γ-hydroxybutyric acid (GHB) and related synthetic analogs. Intriguingly, of the four native CaMKII isozymes, only CaMKIIα accommodates GHB ligands. Key interacting residues in CaMKIIα 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 CaMKIIα 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 CaMKIIα-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 CaMKIIβ failed to induce [³H]HOCPCA binding. However, with the larger type radioligand, [³H]O-5-hydroxydiclofenac, specific binding in CaMKIIβ (L467W/C533R) was achieved. Thus, the study confirms involvement of central binding residues and identifies the CaMKIIα flexible pocket loop as a distantly located allosteric factor in determining selectivity of GHB analogs for CaMKIIα. It sheds light on a remarkable interplay of the entire hub cavity for accommodation of ligands and corroborates GHB analogs as CaMKIIα-selective.