Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2023.10.24.563811

Crystallographic, kinetic, and calorimetric investigation of PKA interactions with L-type calcium channels and Rad GTPase

Yoo, R.·Haji-Ghassemi, O.·Van Petegem, F.·McFarlane, C.·Xu, J.
View original source ↗Export metadata ↓

Abstract

{beta}-adrenergic signalling leads to activation of cAMP-dependent protein kinase (PKA), which can regulate the activity of L-type voltage-gated calcium channels (CaVs) in multiple tissues. In CaV1.2, various sites have been proposed to be involved, including Ser1981 in the C-terminal tail. Its phosphorylation is linked to diabetes progression, synaptic plasticity, and the augmentation of Ca2+ currents in smooth muscle. Its role in augmenting cardiac Ca2+ currents has been heavily scrutinized, with alternative models including the sites Ser1718 and Ser1535. Recently, the GTPase Rad has been identified as a critical PKA target that mediates the augmentation of cardiac CaV1.2 currents upon its phosphorylation. However, it is unclear which of the four potential sites (Ser25, Ser38, Ser272, and Ser300) are favored by PKA. Using quantitative binding experiments and enzyme kinetics, we show that there are two Tiers of target sites, with CaV1.2 residue Ser1981 and Rad residues Ser25 and Ser272 forming Tier 1 substrates for PKA. The other sites form a second Tier, with PKA only showing minimal detectable activity. The Tier 1 substrates share a common feature with two arginine residues that anchor the peptide into the active site of PKA. We report crystal structures of the PKA catalytic subunit (PKAc) with and without a CaV1.2 substrate that represent different successive conformations prior to product turnover. Different target sites utilize different anchoring residues, highlighting the plasticity of PKAc to recognize substrates. SummaryStress signals can alter the electrical properties of excitable cells. cAMP-dependent protein kinase A (PKA) is a key enzyme that is activated upon {beta}-adrenergic stimulation and can alter the function of L-type voltage-gated calcium channels (CaVs) in various tissues. There is a lot of controversy surrounding the exact recognition and specificity of PKA towards CaV1.2, a key calcium channel located in neuronal, cardiac, and smooth muscle tissue, among others. Using a quantitative and unbiased approach, we determined the substrate specificities of PKA towards various sites in CaV1.2 and Rad, an inhibitory protein. Our work highlights two Tiers of substrates, suggesting a potential graded response. Using X-ray crystallography, we determined a high-resolution structure of PKA bound to its strongest target site in CaV1.2, showing how PKA undergoes multiple structural transitions towards binding and how it makes use of a unique anchoring residue.

Source connections

Explore related subjects

biochemistryPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Yoo, R., Haji-Ghassemi, O., Van Petegem, F., McFarlane, C., Xu, J.. 2023-10-24. Crystallographic, kinetic, and calorimetric investigation of PKA interactions with L-type calcium channels and Rad GTPase. https://doi.org/10.1101/2023.10.24.563811

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Modular assembly of mitochondrial complex III from a dynamic matrix domain pool

Mitochondrial complex III is a central component of the cellular energy metabolism. It is an obligate dimer of catalytic and accessory subunits that are encoded by two genomes. How its large matrix domain, formed by the core proteins Qcr1 and Qcr2, is built and joined to the membrane part and during assembly is not known. Here we show that Qcr1 and Qcr2 preassemble independently of complex III into matrix domain modules. Cryo-EM structures from a cardiolipin-deficient yeast strain and of recombinant protein resolved single, double and triple matrix domains, which are linked by the C-terminal tail of Qcr1. In the mature complex III, the same tail binds cytochrome b and cardiolipin. The modules form a dynamic pool, in which assembly species interconvert and exchange subunits. Mutations at the module interfaces, including mimics of human disease variants, reshape this pool and deletion of the Qcr1 tail impairs respiratory growth. Substitution of a tyrosine residue linked to parkinsonism exposes a conserved cysteine residue to oxidation. A cryo-EM structure of an assembly intermediate shows a cap of two matrix domains bound to a monomeric membrane module of cytochrome b, Qcr7, Qcr8 and cytochrome c1. We propose that complex III receives its matrix domain from a dynamic pool of prebuilt modules, which organizes the dimerization of the complex.

biochemistry↗
bioRxiv2026

Vasopressin V2 receptor phosphopeptide reshapes the conformational landscape of arrestin-2 beyond C-tail detachment

Binding of phosphorylated GPCR tails influences arrestin conformation, but the contribution of this interaction to the interdomain rotation associated with activation remains incompletely characterised. In particular, it remains unclear whether displacement of the arrestin C-tail is sufficient to favor larger interdomain rotations or whether the bound phosphotail exerts an additional effect on the conformational landscape. Here, we used enhanced-sampling simulations to characterise the conformational free-energy landscapes of basal (unbound) and V2Rpp-bound arrestin-2. We find that V2Rpp binding broadened the range of energetically accessible interdomain rotation angles relative to basal arrestin-2. However, the pronounced rotation observed in the experimental structures, while accessible, was not strongly favored. Strikingly, C-tail displacement alone does not reproduce this effect, as arrestin-2 retains a strong preference for small interdomain rotation angles in the absence of V2Rpp. Moreover, the C-tail-detached state is energetically unfavorable in the absence of V2Rpp. Together, these findings quantify the effects of V2Rpp binding on the interdomain rotation of arrestin-2 and disentangle the effects of phosphopeptide binding from those of C-tail detachment.

biochemistry↗
bioRxiv2026

Interventional breath analysis reveals oral microbial conversion of 1,2-Propanediol into Propanal and 1-Propanol

Background: Orally exhaled breath contains volatile organic compounds (VOCs) that may reflect local microbial metabolism as well as systemic processes. Consequently, the breath sampling route can influence VOC profiles and complicate biomarker interpretation. Previous studies have shown that 1-propanol is substantially enriched in orally compared with nasally exhaled breath. Objective: To determine whether a controlled oral challenge with 1,2-propanediol (1,2-PD) induces sampling route-dependent changes in breath 1-propanol concentrations. Methods: Ten healthy volunteers received 0.1 mL 1,2-PD diluted in approximately 50 mL water. The solution was used as an oral rinse for approximately 30 s and then expectorated. Matched oral and nasal end-tidal breath samples were collected before administration, immediately afterwards, and at 15-min intervals. Propanal, 1-propanol, acetol, acetone, and isoprene were analysed using gas chromatograph-ion mobility spectrometer (GC-IMS). Results: Oral 1,2-PD exposure produced a rapid and transient increase in exhaled propanal and 1-propanol signals, with the largest responses immediately after administration and at the second post-intervention measurement. In contrast, nasal signals remained comparatively stable. Acetone and isoprene showed no consistent intervention-related changes, regardless of the sampling route. No reproducible increase in acetol was detected, although interpretation was limited by analytical interference. Conclusion: A controlled oral 1,2-PD challenge elicited a route-specific propanal and 1-propanol response, consistent with local microbial metabolism of 1,2-PD within the oral cavity. Both fucose and 1,2-PD have been previously reported in human saliva, including elevated concentrations in selected disease-associated contexts. Bacteria can degrade salivary mucin glycans, releasing sugars including fucose. Together, these observations motivate investigation of a proposed pathway linking mucin-derived fucose to 1,2-PD and subsequent propanal and 1-propanol formation in the human oral cavity.

biochemistry↗