GHB confers neuroprotection by stabilizing the CaMKIIα hub domain

Abstract

Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα) is an abundant neuronal signaling protein involved in synaptic plasticity and memory formation1,2. The central hub domain regulates the activity of CaMKIIα 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 CaMKIIα dysregulation8,9. However, pharmacological modulation of the hub domain has never been demonstrated. Here we show that stabilization of the CaMKIIα hub domain confers neuroprotection. By combining photoaffinity labeling and chemical proteomics using small molecule analogs of the natural metabolite γ-hydroxybutyrate (GHB)10 we reveal that CaMKIIα is the selective target for GHB. We further find that these GHB analogs bind to the hub interior by solving a 2.2 Å crystal structure of CaMKIIα 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 CaMKIIα hub stabilization is the mechanism by which GHB provides endogenous neuroprotection and that small-molecule CaMKIIα-selective ligands have therapeutic potential.

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

“…S3). Of note, in previous studies GHB analogs were all found to be inactive in the ADP-Glo kinase assay when performed at maximal CaM concentrations (1781 nM) 2 .…”

“…Thermal stabilization of the CaMKIIα hub domain by PIPA. An increased thermal stability of the purified CaMKIIα WT hub has been shown as a characteristic upon binding of GHB and several other known GHB analogs 2,22 . By using the described thermal shift assay based on differential scanning fluorometry, the Tm for the CaMKIIα WT hub was found to be 77.4 ± 0.2°C (Fig.…”

“…The human CaMKIIa WT hub (UniprotKB Q9UQM7, residues 345-475), CaMKIIa W403L hub and CaMKIIa 6x hub (containing the six mutations Thr354Asn, Glu355Gln, Thr412Asn, Ile414Met, Ile464His, and Phe467Met) were expressed and purified as previously described 2,18,32 . In brief, recombinant CaMKIIa hub domains with an Nterminal 6x His-precision protease expression tag were inserted into pSKB2 (6x hub) and pET28a (WT hub and W403L hub) vectors with kanamycin resistance, respectively.…”

“…We recently revealed the first structural details of the small-molecule binding pocket of CaMKIIα hub from a 2.2 Å resolution co-crystal structure of the tetradecamer-stabilized human CaMKIIα hub mutant (CaMKIIα 6x hub 18 ) with the GHB analog 5-hydroxydiclofenac (5-HDC; Fig. 1A) 2 . 5-HDC was found bound to 12 out of 14 subunits of the tetradecameric CaMKIIα hub oligomer, and to interact specifically with the positively charged residues Arg433, Arg453, and Arg469 as well as His395 that can be either neutral or positively charged.…”

“…This was surprising since GHB is a natural metabolite of γ-aminobutyric acid (GABA) involved in inhibitory neurotransmission. Intriguingly, GHB and synthetic small-molecule analogs were found to bind to a novel pocket in the hub domain of the multimeric CaMKIIα, leading to oligomeric stabilization, proposed as the mechanistic basis for the reported neuroprotective action of such analogs [2][3][4] . CaMKIIα holds a key role in synaptic plasticity, learning and memory 5 , and mutations in CaMKII human genes have been uncovered to cause intellectual disability [6][7][8][9][10] .…”