A Unique Mode of Coenzyme A Binding to the Nucleotide Binding Pocket of Human Metastasis Suppressor NME1.

Coenzyme A (CoA) is a key cellular metabolite which participates in diverse metabolic pathways, regulation of gene expression and the antioxidant defense mechanism. Human NME1 (hNME1), which is a moonlighting protein, was identified as a major CoA-binding protein. Biochemical studies showed that hNME1 is regulated by CoA through both covalent and non-covalent binding, which leads to a decrease in the hNME1 nucleoside diphosphate kinase (NDPK) activity. In this study, we expanded the knowledge on previous findings by focusing on the non-covalent mode of CoA binding to the hNME1. With X-ray crystallography, we solved the CoA bound structure of hNME1 (hNME1-CoA) and determined the stabilization interactions CoA forms within the nucleotide-binding site of hNME1. A hydrophobic patch stabilizing the CoA adenine ring, while salt bridges and hydrogen bonds stabilizing the phosphate groups of CoA were observed. With molecular dynamics studies, we extended our structural analysis by characterizing the hNME1-CoA structure and elucidating possible orientations of the pantetheine tail, which is absent in the X-ray structure due to its flexibility. Crystallographic studies suggested the involvement of arginine 58 and threonine 94 in mediating specific interactions with CoA. Site-directed mutagenesis and CoA-based affinity purifications showed that arginine 58 mutation to glutamate (R58E) and threonine 94 mutation to aspartate (T94D) prevent hNME1 from binding to CoA. Overall, our results reveal a unique mode by which hNME1 binds CoA, which differs significantly from that of ADP binding: the α- and ß-phosphates of CoA are oriented away from the nucleotide-binding site, while 3'-phosphate faces catalytic histidine 118 (H118). The interactions formed by the CoA adenine ring and phosphate groups contribute to the specific mode of CoA binding to hNME1.

Regulation of metastasis suppressor NME1 by a key metabolic cofactor coenzyme A.

The metastasis suppressor protein NME1 is an evolutionarily conserved and multifunctional enzyme that plays an important role in suppressing the invasion and metastasis of tumour cells. The nucleoside diphosphate kinase (NDPK) activity of NME1 is well recognized in balancing the intracellular pools of nucleotide diphosphates and triphosphates to regulate cytoskeletal rearrangement and cell motility, endocytosis, intracellular trafficking, and metastasis. In addition, NME1 was found to function as a protein-histidine kinase, 3'-5' exonuclease and geranyl/farnesyl pyrophosphate kinase. These diverse cellular functions are regulated at the level of expression, post-translational modifications, and regulatory interactions. The NDPK activity of NME1 has been shown to be inhibited in vitro and in vivo under oxidative stress, and the inhibitory effect mediated via redox-sensitive cysteine residues. In this study, affinity purification followed by mass spectrometric analysis revealed NME1 to be a major coenzyme A (CoA) binding protein in cultured cells and rat tissues. NME1 is also found covalently modified by CoA (CoAlation) at Cys109 in the CoAlome analysis of HEK293/Pank1ß cells treated with the disulfide-stress inducer, diamide. Further analysis showed that recombinant NME1 is efficiently CoAlated in vitro and in cellular response to oxidising agents and metabolic stress. In vitro CoAlation of recombinant wild type NME1, but not the C109A mutant, results in the inhibition of its NDPK activity. Moreover, CoA also functions as a competitive inhibitor of the NME1 NDPK activity by binding non-covalently to the nucleotide binding site. Taken together, our data reveal metastasis suppressor protein NME1 as a novel binding partner of the key metabolic regulator CoA, which inhibits its nucleoside diphosphate kinase activity via non-covalent and covalent interactions.