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Divalent Metal Ions Mg²? and Ca²? Have Distinct Effects on Protein Kinase A Activity and Regulation.

ABSTRACT: cAMP-dependent protein kinase (PKA) is regulated primarily in response to physiological signals while nucleotides and metals may provide fine-tuning. PKA can use different metal ions for phosphoryl transfer, yet some, like Ca(2+), do not support steady-state catalysis. Fluorescence Polarization (FP) and Surface Plasmon Resonance (SPR) were used to study inhibitor and substrate interactions with PKA. The data illustrate how metals can act differentially as a result of their inherent coordination properties. We found that Ca(2+), in contrast to Mg(2+), does not induce high-affinity binding of PKA to pseudosubstrate inhibitors. However, Ca(2+) works in a single turnover mode to allow for phosphoryl-transfer. Using a novel SPR approach, we were able to directly monitor the interaction of PKA with a substrate in the presence of Mg(2+)ATP. This allows us to depict the entire kinase reaction including complex formation as well as release of the phosphorylated substrate. In contrast to Mg(2+), Ca(2+) apparently slows down the enzymatic reaction. A focus on individual reaction steps revealed that Ca(2+) is not as efficient as Mg(2+) in stabilizing the enzyme:substrate complex. The opposite holds true for product dissociation where Mg(2+) easily releases the phospho-substrate while Ca(2+) traps both reaction products at the active site. This explains the low steady-state activity in the presence of Ca(2+). Furthermore, Ca(2+) is able to modulate kinase activity as well as inhibitor binding even in the presence of Mg(2+). We therefore hypothesize that the physiological metal ions Mg(2+) and Ca(2+) both play a role in kinase activity and regulation. Since PKA is localized close to calcium channels and may render PKA activity susceptible to Ca(2+), our data provide a possible mechanism for novel crosstalk between cAMP and calcium signaling.

SUBMITTER: Knape MJ

PROVIDER: S-EPMC4714867 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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Divalent Metal Ions Mg²⁺ and Ca²⁺ Have Distinct Effects on Protein Kinase A Activity and Regulation.

Knape Matthias J MJ Ahuja Lalima G LG Bertinetti Daniela D Burghardt Nicole C G NC Zimmermann Bastian B Taylor Susan S SS Herberg Friedrich W FW

ACS chemical biology 20150805 10

cAMP-dependent protein kinase (PKA) is regulated primarily in response to physiological signals while nucleotides and metals may provide fine-tuning. PKA can use different metal ions for phosphoryl transfer, yet some, like Ca(2+), do not support steady-state catalysis. Fluorescence Polarization (FP) and Surface Plasmon Resonance (SPR) were used to study inhibitor and substrate interactions with PKA. The data illustrate how metals can act differentially as a result of their inherent coordination ...[more]

PMID: 26200257

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Project description:Divalent cations, especially Ca2+ and Mg2+, play a vital role in the function of biomolecules and making them important to be constituents in samples for in vitro biophysical and biochemical characterizations. Although lipid nanodiscs are becoming valuable tools for structural biology studies on membrane proteins and for drug delivery, most types of nanodiscs used in these studies are unstable in the presence of divalent metal ions. To avoid the interaction of divalent metal ions with the belt of the nanodiscs, synthetic polymers have been designed and demonstrated to form stable lipid nanodiscs under such unstable conditions. Such polymer-based nanodiscs have been shown to provide an ideal platform for structural studies using both solid-state and solution NMR spectroscopies because of the near-native cell-membrane environment they provide and the unique magnetic-alignment behavior of large-size nanodiscs. In this study, we report an investigation probing the effects of Ca2+ and Mg2+ ions on the formation of polymer-based lipid nanodiscs and the magnetic-alignment properties using a synthetic polymer, styrene maleimide quaternary ammonium (SMA-QA), and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipids. Phosphorus-31 NMR experiments were used to evaluate the stability of the magnetic-alignment behavior of the nanodiscs for varying concentrations of Ca2+ or Mg2+ at different temperatures. It is remarkable that the interaction of divalent cations with lipid headgroups promotes the stacking up of nanodiscs that results in the enhanced magnetic alignment of nanodiscs. Interestingly, the reported results show that both the temperature and the concentration of divalent metal ions can be optimized to achieve the optimal alignment of nanodiscs in the presence of an applied magnetic field. We expect the reported results to be useful in the design of nanodisc-based nanoparticles for various applications in addition to atomic-resolution structural and dynamics studies using NMR and other biophysical techniques.

Dataset Information

Divalent Metal Ions Mg²? and Ca²? Have Distinct Effects on Protein Kinase A Activity and Regulation.

Publications

Divalent Metal Ions Mg²⁺ and Ca²⁺ Have Distinct Effects on Protein Kinase A Activity and Regulation.

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