Molecular modeling and binding function of the RGS12 PDZ domain variant associated with familial bipolar disorder

Purpose –

Bipolar disorder (BD) is a highly heritable neuropsychiatric disease characterized by recurrent episodes of depression and mania. BD is considered one of the most vexing health disorders to medicate appropriately, yet affects ~2% of U.S. adults and is the most costly mental health condition for American health insurers nationwide [1]. In reporting the whole-exome sequencing of 81 individuals from 27 multiply-affected BD families, Forstner et al. [2] discovered a single nucleotide polymorphism co-segregating with BD in the gene encoding “Regulator of G protein Signaling” (RGS) type 12, predicted to cause a missense change (arginine-59 to glutamine) in the sequence of the encoded protein’s PDZ domain. RGS proteins are negative regulators of neurotransmitter signaling via G protein-coupled receptors (GPCRs) [3], specifically serving to accelerate signaling shutoff by increasing the GTP hydrolysis rate of GPCR-associated G-alpha subunits [4]. In prior mouse genetics studies, the Siderovski lab has demonstrated that RGS12 acts to regulate kappa opioid receptor signaling and extracellular dopamine levels in the basal ganglia of the brain, and that RGS12 is able to bind to various protein targets in neurons, such as the kinase MEK2 via its PDZ domain. To address whether the R59Q amino-acid variation in the RGS12 PDZ domain associated with BD affects the function of the RGS12 protein, molecular modeling and biochemical binding experiments were performed.

Methods –

Schrodinger’s software suite of molecular modeling and dynamics tools was employed to create structural models of the R59Q variant of RGS12’s PDZ domain, starting from the published NMR-derived structure of the domain with wildtype sequence (i.e., Protein Data Bank record id 2KV8). Desmond molecular dynamics software was used to relax both structural models and then assess their overall stability over 50 nanoseconds of elapsed simulation. Models of the wildtype and R59Q variant PDZ domain bound to short polypeptides from the C-termini of candidate RGS12-interacting proteins were similarly created and molecular dynamics simulations performed. Both PDZ domains were expressed as glutathione-S-transferase (GST) fusion proteins by E. coli cultures, purified by fast protein liquid chromatography (FPLC), and then tested for their binding to biotinylated polypeptides from the C-termini of candidate RGS12-interacting proteins using surface plasmon resonance (SPR).

Results and Conclusion –

The position of the missense change within RGS12 was not predicted to directly engage the polypeptide binding-site of the PDZ domain; however, when tested using SPR, the fusion protein bearing the R59Q-substituted PDZ domain exhibited less binding affinity for target polypeptide partners than the wildtype sequence. These initial in silico and in vitro findings suggest that the R59Q variant associated with BD may lead to reduced RGS12 protein function in vivo.

References:

[1] PMID: 35816713; DOI: 10.7326/AITC202207190

[2] PMID: 32066727; PMCID: PMC7026119; DOI: 10.1038/s41398-020-0732-y

[3] PMID: 21737532; PMCID: PMC3141876; DOI: 10.1124/pr.110.003038

[4] PMID: 20351284; PMCID: PMC2872438; DOI: 10.1073/pnas.0912934107