Structural and functional plasticity of human adenylate kinase 4: Insights from enzymatic, biophysical, and...

Human adenylate kinase 4 (AK4) is a mitochondrial enzyme essential for cellular energy homeostasis and adaptation to metabolic and oxidative challenges. Although emerging as a therapeutic target in cancer and stress-related conditions, the molecular determinants governing its activity, stability, and conformational plasticity remain poorly understood. Here, we combined biochemical, biophysical, cell-based, and computational approaches to provide the first mechanistic insights into the regulation of human AK4. Recombinant wild-type AK4 and site-directed cysteine variants were characterized by nano-differential scanning fluorimetry, circular dichroism spectroscopy, enzymatic assays, and molecular dynamics simulations. Adenine nucleotides, particularly ATP, markedly enhanced AK4 thermal stability, induced conformational rearrangements, and increased its enzymatic competence, whereas pyrimidine nucleotides had minimal effects. Computational simulations corroborated that the closed, catalytically competent conformation is stabilized primarily by substrate binding. Importantly, these findings were validated in a biologically relevant model of hypoxia-exposed lung cancer cells, confirming the pronounced stabilizing effect of ATP on endogenous AK4. Reducing agents, such as dithiothreitol, enhanced the catalytic activity of AK4, whereas both reduced and oxidized glutathione at high concentrations inhibited AK4, indicating a distinct layer of redox regulation. Site-directed mutagenesis identified Cys22 as the principal cysteine residue mediating redox responsiveness of AK4. Importantly, all variants retained catalytic activity, thermal stability, and nucleotide-dependent structural properties comparable to the wild-type protein, demonstrating that redox regulation is not associated with global structural changes. Collectively, these findings provide mechanistic insights into the multifaceted regulation of human AK4 and underscore its potential as a redox- and ligand-responsive therapeutic target in human diseases.