A tandem motif-based and structural approach can identify hidden functional phosphodiesterases

Area of Science:

• Plant molecular biology
• Biochemistry
• Cell signaling

Background:

• Cyclic nucleotide monophosphates (cNMPs) are vital signaling molecules.
• Degradation of cNMPs is critical for cellular regulation.
• Cyclic nucleotide phosphodiesterases (PDEs) are elusive in higher plants.

Purpose of the Study:

• To identify and characterize novel plant cyclic nucleotide phosphodiesterases (PDEs).
• To investigate the role of PDEs in cyclic adenosine monophosphate (cAMP) signaling in plants.

Main Methods:

• Sequence analysis and motif development to identify candidate PDEs.
• Structural and molecular docking analysis of candidate proteins.
• Liquid chromatography tandem mass spectrometry (LC-MS/MS) to confirm PDE activity.
• Surface plasmon resonance (SPR) to confirm protein complex formation.

Main Results:

• Identified a novel PDE in Arabidopsis thaliana K+-Uptake Permease (AtKUP5).
• Structural analysis revealed a suitable binding pocket for cyclic adenosine monophosphate (cAMP).
• Confirmed PDE activity using LC-MS/MS and demonstrated Ca2+/CaM stimulation.
• AtKUP5 possesses dual adenylate cyclase (AC) and PDE activities.

Conclusions:

• AtKUP5 exhibits dual AC-PDE moonlighting activity, enabling intramolecular regulation of cAMP.
• This dual function allows for fine-tuning of cAMP signaling in potassium (K+) homeostasis.
• The discovery of AtKUP5's PDE activity provides new insights into plant signaling pathways.