A monomer-dimer switch modulates the activity of plant adenosine kinase

Adenosine undergoes ATP-dependent phosphorylation catalyzed by adenosine kinase (ADK). In plants, ADK also phosphorylates cytokinin ribosides, transport forms of the hormone. Here, we investigated the substrate preferences, oligomeric states and structures of ADKs from moss (Physcomitrella patens) and maize (Zea mays) alongside metabolomic and phenotypic analyses. We showed that dexamethasone-inducible ZmADK overexpressor lines in Arabidopsis can benefit from a higher number of lateral roots and larger root areas under nitrogen starvation. We discovered that maize and moss enzymes can form dimers upon increasing protein concentration, setting them apart from the monomeric human and protozoal ADKs. Structural and kinetic analyses revealed a catalytically inactive unique dimer. Within the dimer, both active sites are mutually blocked. The activity of moss ADKs, exhibiting a higher propensity to dimerize, was tenfold lower compared to maize ADKs. Two monomeric structures in a ternary complex highlight the characteristic transition from an open to a closed state upon substrate binding. This suggests that the oligomeric state switch can modulate the activity of moss ADKs and likely other plant ADKs. Moreover, dimer association represents a novel negative feedback mechanism, helping to maintain steady levels of adenosine and AMP.

Methods
This collection of datasets is related to molecular properties, ligand interactions and enzyme kinetics. Data measurements are given in Materials and methods. Other data are part of the Supplement of the manuscript.

Gel permeation chromatography

Gel permeation chromatography of studied plant Aadenosine kinases was performed on an NGC Medium-Pressure Liquid Chromatography System (https://www.bio-rad.com)on a Superdex 200 10/30 HR column in 20 mM Tris-HCl buffer, pH 7.5, 100 mM NaCl, with calibration performed using a gel filtration standard (Bio-Rad).

Affinity and thermal stability measurements

The MST method was used to determine the binding affinity of various ribosides to ZmADK2 and PpADK1. Proteins were fluorescently labeled with RED-tris-NTA dye (www.nanotemper-technologies.com) using a 1:1 dye/protein molar ratio. The labeled protein was adjusted to 100-300 nM in 50 mM HEPES buffer pH 7.5, 1 mM MgCl2 and 0.2% Tween. Measurements were performed in premium capillaries on a Monolith NT.115 instrument at 30°C with 5 sec/30 sec/5 sec laser off/on/off times, respectively, with continuous sample fluorescence recording.

Thermal stability was measured by nano-differential scanning fluorimetry on a Prometheus NT.48 instrument (www.nanotemper-technologies.com) in various buffers covering a pH (7.0-9.5) and temperature range (25 to 95°C), and with a heating rate of 1 °C min-1 and using NT melting control software. Protein unfolding was measured by detecting the temperature-induced change in tryptophan fluorescence intensity at emission wavelengths of 330 and 350 ± 5 nm. The melting temperature (Tm) was deduced from the maximum of the first derivative of the fluorescence ratios F350/F330.

Sedimentation coefficient

The analytical ultracentrifugation (AUC) in sedimentation velocity mode was performed using ProteomeLab XL-I analytical ultracentrifuge (Beckman Coulter, Indianapolis, IN, USA) equipped with an An-60 Ti rotor. Samples of ZmADK2 and PpADK1 were diluted in 20 mM Tris-HCl pH 7.5 with 150 mM NaCl, 10 mM MgCl2, 10 µM DTT and 1% glycerol and equilibrated at 4 °C overnight. Absorbance data were collected at 25 °C and at a rotor speed of 48000 rpm. Scans were collected at 280 nm in 5-min intervals and 0.003 cm spatial resolution in continuous scan mode. The partial specific volume of the protein and the solvent density and viscosity were calculated from the amino acid sequence and buffer composition, respectively, using the Sednterp software (http://bitcwiki.sr.unh.edu). The data were analyzed with the continuous c(s) distribution model implemented in the program SEDFIT 15.01b, using a confidence level of 0.95 for the regularization procedure. The plots of c(s) distributions were created in GUSSI 1.3.1.

DLS measurements

The hydrodynamic diameter was determined by DLS in 20 mM Tris-HCl pH 7.5 and 1 mM MgCl2 alone and in the presence of 2 mM ATP or 2 mM AP5A at 22 °C. Measurement was performed using the Zetasizer Nano ZS (Malvern Instruments, UK). Hydrodynamic diameter values were calculated by Zetasizer Software v7.13 (173° angle measurement, approximation fit to the sphere).