Adenosine Kinase couples sensing of cellular potassium depletion to purine metabolism

Renata Rocha De Oliveira,Celisa Caldana Costa Tonoli,Artur Torres Cordeiro,Maurício Luis Sforça,Raphael Morales-Neto,Mário Tyago Murakami,Silvana Aparecida Rocco,Carla Cristina Polo,Gustavo Fernando Mercaldi,Kleber Gomes Franchini

doi:10.1038/s41598-018-30418-5

Abstract

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.

Highlights

Adenosine Kinase (ADK) regulates the cellular levels of adenosine (ADO) by fine-tuning its metabolic clearance
Crystal structures of Δ19 mouse ADK (mADK) complexed with adenosine and full- length-mADK complexed with adenosine and adenosine-5′-diphosphate were determined at 1.2 and 1.8 Å resolution, respectively (Table 1)
Crystals from mADK:ADO grew in the monoclinic space group P1,21,1, while crystals from mADK:ADO:adenosine diphosphate (ADP) grew in the orthorhombic space group P21,21,21

Summary

Introduction

Adenosine Kinase (ADK) regulates the cellular levels of adenosine (ADO) by fine-tuning its metabolic clearance. Adenosine Kinase (ADK; EC 2.7.1.20) catalyzes the phosphorylation of adenosine (ADO) to 5′-AMP using ATP as a source of phosphate[1]. This reaction is a committed step in the purine nucleotide salvage pathway, involved in maintaining the proper levels of cellular nucleotides[2]. ADO itself is a primary regulator of ADK, as increased levels of intracellular ADO inhibit the enzyme’s catalytic activity through a yet unclear mechanism[9]. In this respect, increases in ADO concentration from the nanomolar to the micromolar range are needed to www.nature.com/scientificreports/

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