A novel redox-active switch in Fructosamine-3-kinases expands the regulatory repertoire of the protein kinase superfamily

Abstract

words)Aberrant regulation of metabolic kinases by altered redox homeostasis is a major contributing factor in aging and disease such as diabetes. However, the biochemical mechanisms by which metabolic kinases are regulated under oxidative stress is poorly understood. In this study, we demonstrate that the catalytic activity of a conserved family of Fructosamine-3-kinases (FN3Ks), which are evolutionarily related to eukaryotic protein kinases (ePKs), are regulated by redox active cysteines in the kinase domain. By solving the crystal structure of FN3K homolog from Arabidopsis thaliana (AtFN3K), we demonstrate that it forms an unexpected strandexchange dimer in which the ATP binding P-loop and adjoining beta strands are swapped between two chains in the dimer. This dimeric configuration is characterized by strained inter-chain disulfide bonds that stabilize the P-loop in an extended conformation and tethers the substratebinding lobes in a unique inactive conformation. Mutational analysis and solution studies confirm that the strained disulfides function as redox "switches" to reversibly regulate FN3K activity and dimerization. Consistently, we find that human FN3K, which contains an equivalent P-loop Cys, is also redox sensitive, whereas ancestral bacterial FN3K homologs, which lack a P-loop Cys, are not. Furthermore, CRISPR knockout of FN3K in human HepG2 cells results in significant upregulation of redox metabolites including glutathione. We propose that redox regulation evolved progressively in FN3Ks in response to changing cellular redox conditions. Our studies provide important new insights into the origin and evolution of redox regulation in the protein kinase superfamily and open new avenues for targeting human FN3K in diabetic complications.

Illuminating the functions of the understudied Fructosamine-3-kinase (FN3K) using a multi-omics approach reveals new links to lipid, carbon, and co-factor metabolic pathways

“…Since NADH exhibited the highest stabilization effect in our DSF assays, we proceeded to dock the NADH molecule onto the homology model of HsFN3K. Leveraging the crystal structure of the plant homolog from A. thaliana FN3K (AtFN3K) (PDB ID: 6OID) as a template, which we had previously demonstrated forms a disulfide-linked dimer 14 , we aimed to shed light on the binding mode of HsFN3K. Additionally, we introduced a magnesium ion into the homology model using the Metal Ion-Binding webserver 37 , given that our DSF assays indicated magnesium dependency for NADH binding.…”

“…7c), while the adenine moiety engages in π-π interaction with F39. Notably, F39 in HsFN3K corresponds to F47 in AtFN3K, which partakes in a π-π interaction with the adenine ring of the ADP molecule within the crystal structure (PDB ID: 6OID) 14 .…”

“…We previously identified differentially abundant metabolites by comparing the metabolic profiles of FN3K KO and WT HepG2 cells 14 . Metabolite set enrichment analysis (MSEA) of the differentially abundant metabolites revealed an enrichment in several pathways related to amino acid metabolism, in addition to pathways such as "Glutathione metabolism", "Pyruvate metabolism", and "Pantothenate and CoA Biosynthesis" (Supplementary Fig.…”

“…Our previous research demonstrated that under oxidative stress, HsFN3K forms disulfidelinked dimers through the P-loop cysteine (C24) 14 . Additionally, other studies have observed sulfenylation at the cysteine residue in both HsFN3K and HsFN3KRP 45 .…”

“…Previously, we reported the crystal structure of FN3K ortholog from Arabidopsis thaliana (AtFN3K), revealing an evolutionarily conserved redox regulation mechanism involving conserved cysteines in the ATP binding P-loop 14 . Specifically, we demonstrated that AtFN3K adopts a disulfide-linked dimer that can be reversed under reducing conditions and that the equivalent cysteine in HsFN3K (C24 HsFN3K ) performs an analogous role conferring redox sensitivity and disulfide-mediated oligomerization.…”