Adenosine Kinase couples sensing of cellular potassium depletion to purine metabolism

Adenosine Kinase (ADK) regulates the cellular levels of adenosine (ADO) by fine-tuning its metabolic clearance. The transfer of γ-phosphate from ATP to ADO by ADK involves regulation by the substrates and products, as well as by Mg2+ and inorganic phosphate. Here we present new crystal structures of mouse ADK (mADK) binary (mADK:ADO; 1.2 Å) and ternary (mADK:ADO:ADP; 1.8 Å) complexes. In accordance with the structural demonstration of ADO occupancy of the ATP binding site, kinetic studies confirmed a competitive model of auto-inhibition of ADK by ADO. In the ternary complex, a K+ ion is hexacoordinated between loops adjacent to the ATP binding site, where Asp310 connects the K+ coordination sphere to the ATP binding site through an anion hole structure. Nuclear Magnetic Resonance 2D 15N-1H HSQC experiments revealed that the binding of K+ perturbs Asp310 and residues of adjacent helices 14 and 15, engaging a transition to a catalytically productive structure. Consistent with the structural data, the mutants D310A and D310P are catalytically deficient and loose responsiveness to K+. Saturation Transfer Difference spectra of ATPγS provided evidence for an unfavorable interaction of the mADK D310P mutant for ATP. Reductions in K+ concentration diminish, whereas increases enhance the in vitro activity of mADK (maximum of 2.5-fold; apparent Kd = 10.4 mM). Mechanistically, K+ increases the catalytic turnover (Kcat) but does not affect the affinity of mADK for ADO or ATP. Depletion of intracellular K+ inhibited, while its restoration was accompanied by a full recovery of cellular ADK activity. Together, this novel dataset reveals the molecular basis of the allosteric activation of ADK by K+ and highlights the role of ADK in connecting depletion of intracellular K+ to the regulation of purine metabolism.

1 Overall quality at a glance i O The following experimental techniques were used to determine the structure:

X-RAY DIFFRACTION
The reported resolution of this entry is 1.80 Å.
Percentile scores (ranging between 0-100) for global validation metrics of the entry are shown in the following graphic. The table shows the number of entries on which the scores are based.

Metric
Whole archive (#Entries) Similar resolution (#Entries, resolution range(Å)) The table below summarises the geometric issues observed across the polymeric chains and their t to the electron density. The red, orange, yellow and green segments on the lower bar indicate the fraction of residues that contain outliers for >=3, 2, 1 and 0 types of geometric quality criteria. A grey segment represents the fraction of residues that are not modelled. The numeric value for each fraction is indicated below the corresponding segment, with a dot representing fractions <=5% The upper red bar (where present) indicates the fraction of residues that have poor t to the electron density. The numeric value is given above the bar.

Mol Chain Length
Quality of chain 1 A 363 O There are 9 unique types of molecules in this entry. The entry contains 2903 atoms, of which 0 are hydrogens and 0 are deuteriums.
In the tables below, the ZeroOcc column contains the number of atoms modelled with zero occupancy, the AltConf column contains the number of residues with at least one atom in alternate conformation and the Trace column contains the number of residues modelled with at most 2 atoms.
Molecule 1 is a protein called Adenosine kinase. 3 Residue-property plots i O These plots are drawn for all protein, RNA and DNA chains in the entry. The rst graphic for a chain summarises the proportions of the various outlier classes displayed in the second graphic. The second graphic shows the sequence view annotated by issues in geometry and electron density. Residues are color-coded according to the number of geometric quality criteria for which they contain at least one outlier: green = 0, yellow = 1, orange = 2 and red = 3 or more. A red dot above a residue indicates a poor t to the electron density (RSRZ > 2). Stretches of 2 or more consecutive residues without any outlier are shown as a green connector. Residues present in the sample, but not in the model, are shown in grey.

Mol Chain Residues
• Molecule 1: Adenosine kinase Chain A: Continued on next page...

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Full wwPDB X-ray Structure Validation Report 5KB5 Continued from previous page... The all-atom clashscore is dened as the number of clashes found per 1000 atoms (including hydrogen atoms). The all-atom clashscore for this structure is 2.

Mol Chain Non-H H(model) H(added) Clashes Symm-Clashes
All (11) close contacts within the same asymmetric unit are listed below, sorted by their clash magnitude. The Analysed column shows the number of residues for which the backbone conformation was analysed, and the total number of residues. There are no Ramachandran outliers to report.

Protein sidechains i O
In the following table, the Percentiles column shows the percent sidechain outliers of the chain as a percentile score with respect to all X-ray entries followed by that with respect to entries of similar resolution.
The Analysed column shows the number of residues for which the sidechain conformation was analysed, and the total number of residues. In the following table, the Counts columns list the number of bonds (or angles) for which Mogul statistics could be retrieved, the number of bonds (or angles) that are observed in the model and the number of bonds (or angles) that are dened in the chemical component dictionary. The Link column lists molecule types, if any, to which the group is linked. The Z score for a bond length (or angle) is the number of standard deviations the observed value is removed from the expected value. A bond length (or angle) with |Z| > 2 is considered an outlier worth inspection. RMSZ is the root-mean-square of all Z scores of the bond lengths (or angles). There are no chirality outliers.

Mol Type Chain Res Link
There are no torsion outliers.
There are no ring outliers.
No monomer is involved in short contacts.