Biology subjects

Gauger, S. J.

Publications and source records attributed to Gauger, S. J..

3 recordsLinked to original sources

bioRxiv2025

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

The Ca2+/CaM-dependent protein kinase II alpha (CaMKII) 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 CaMKII hub domain was identified for the endogenous neuromodulator {gamma}-hydroxybutyric acid (GHB) and related synthetic analogues. Key interacting residues in CaMKII were revealed, but the pronounced selectivity towards 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, [3H]HOCPCA and [3H]O-5-HDC, in combination with site-directed mutagenesis. Binding to CaMKII 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 CaMKII-specific residue Trp403 (Leu in all other CaMKII isozymes). Insertion of the corresponding residues (L467W/C533R) into CaMKII{beta} failed to introduce [3H]HOCPCA binding. However, with the larger-type radioligand, [3H]O-5-HDC, specific binding in CaMKII{beta} (L467W/C533R) was achieved. Thus, of the four native CaMKII isozymes, only CaMKII accommodates GHB ligands. The study identifies the CaMKII flexible pocket loop as a distantly located "allosteric" factor in determining selectivity of GHB analogues for CaMKII. It sheds light on a remarkable interplay of the entire hub cavity for accommodation of ligands, and corroborates GHB analogues as CaMKII-selective.

biochemistry↗

bioRxiv2024

Ligand-induced CaMKIIα hub Trp403 flip, hub domain stacking and kinase inhibition

{gamma}-Hydroxybutyric acid (GHB) analogs are small molecules that bind competitively to a specific cavity in the oligomeric CaMKII hub domain. Binding affects conformation and stability of the hub domain, which may explain the neuroprotective action of some of these compounds. Here, we describe molecular details of interaction of the larger-type GHB analog 2-(6-(4-chlorophenyl)imidazo[1,2-b]pyridazine-2-yl)acetic acid (PIPA). Like smaller-type analogs, PIPA binding to the CaMKII hub domain promoted thermal stability. PIPA additionally inhibited CaMKII kinase activity by reducing CaM sensitivity. A high-resolution X-ray crystal structure of a stabilized CaMKII (6x mutant) hub construct revealed details of the binding mode of PIPA, which involved outward placement of tryptophan 403 (Trp403), a central residue in a flexible loop close to the upper hub cavity. Small-angle X-ray scattering (SAXS) solution structures and mass photometry of the CaMKII wildtype hub domain in the presence of PIPA revealed a high degree of ordered self-association (stacks of CaMKII hub domains). This stacking neither occurred with the smaller compound 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA), nor when Trp403 was replaced with leucine (W403L). Additionally, CaMKII W403L hub was stabilized to a larger extent by PIPA compared to CaMKII hub wildtype, indicating that loop flexibility is important for holoenzyme stability. Thus, we propose that ligand-induced outward placement of Trp403 by PIPA, which promotes an unforeseen mechanism of hub domain stacking, may be involved in the observed reduction in CaMKII kinase activity. Altogether, this sheds new light on allosteric regulation of CaMKII activity via the hub domain.

biophysics↗

bioRxiv2020

GHB confers neuroprotection by stabilizing the CaMKIIα hub domain

Ca2+/calmodulin-dependent protein kinase II alpha (CaMKII) is an abundant neuronal signaling protein involved in synaptic plasticity and memory formation1,2. The central hub domain regulates the activity of CaMKII by organizing the holoenzyme complex into functional oligomers3-6. Recent findings have suggested that the hub is also an allosteric determinant of kinase activity7, and is thus an emerging target for therapies to correct CaMKII dysregulation8,9. However, pharmacological modulation of the hub domain has never been demonstrated. Here we show that stabilization of the CaMKII hub domain confers neuroprotection. By combining photoaffinity labeling and chemical proteomics using small molecule analogs of the natural metabolite {gamma}-hydroxybutyrate (GHB)10 we reveal that CaMKII is the selective target for GHB. We further find that these GHB analogs bind to the hub interior by solving a 2.2 [A] crystal structure of CaMKII with bound ligand. Using differential scanning fluorimetry, we show that binding of ligands to the hub interior increases the thermal stability of hub oligomers in a concentration-dependent manner. Moreover, we demonstrate the functional significance of this hub stabilization by showing substantial neuroprotective effects in cellular excitotoxicity assays and in a mouse model of cerebral ischemia. Together, our results reveal that CaMKII hub stabilization is the mechanism by which GHB provides endogenous neuroprotection and that small-molecule CaMKII-selective ligands have therapeutic potential.

biochemistry↗