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

TOSSOUNIAN, MARIA-ARMINEH; HRISTOV, STEFAN DENCHEV; SEMELAK, JONATHAN ALEXIS; YU, BESS YI KUN; BACZYNSKA, MARIA; ZHAO, YUHAN; ESTRIN, DARIO ARIEL; TRUJILLO, MADIA; FILONENKO, VALERIY; GOUGE, JEROME; GOUT, IVAN

INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES

MOLECULAR DIVERSITY PRESERVATION INTERNATIONAL-MDPI

Año: 2023 vol. 24

1422-0067

bstract: Coenzyme A (CoA) is a key cellular metabolite which participates in diverse metabolicpathways, 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. Bio-chemical studies showed that hNME1 is regulated by CoA through both covalent and non-covalentbinding, 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-covalentmode of CoA binding to the hNME1. With X-ray crystallography, we solved the CoA bound struc-ture of hNME1 (hNME1-CoA) and determined the stabilization interactions CoA forms within thenucleotide-binding site of hNME1. A hydrophobic patch stabilizing the CoA adenine ring, while saltbridges and hydrogen bonds stabilizing the phosphate groups of CoA were observed. With moleculardynamics studies, we extended our structural analysis by characterizing the hNME1-CoA structureand elucidating possible orientations of the pantetheine tail, which is absent in the X-ray structuredue to its flexibility. Crystallographic studies suggested the involvement of arginine 58 and threonine94 in mediating specific interactions with CoA. Site-directed mutagenesis and CoA-based affinitypurifications showed that arginine 58 mutation to glutamate (R58E) and threonine 94 mutation toaspartate (T94D) prevent hNME1 from binding to CoA. Overall, our results reveal a unique modeby which hNME1 binds CoA, which differs significantly from that of ADP binding: the α- andβ-phosphates of CoA are oriented away from the nucleotide-binding site, while 3 0 -phosphate facescatalytic histidine 118 (H118). The interactions formed by the CoA adenine ring and phosphategroups contribute to the specific mode of CoA binding to hNME1.