On the quaternary structure of human D-3-phosphoglycerate dehydrogenase.

Abstract

D-3-phosphoglycerate dehydrogenase (PHGDH) catalyzes the NAD⁺-dependent conversion of D-3-phospho-glycerate to 3-phosphohydroxypyruvate, the first step in the phosphorylated pathway for L-serine (L-Ser) biosynthesis. L-Ser plays different relevant metabolic roles in eukaryotic cells: alterations in L-Ser metabolism have been linked to serious neurological disorders. The human PHGDH (hPHGDH), showing a homotetrameric state in solution, is made of four domains, among which there are two regulatory domains at the C-terminus: the aspartate kinase-chorismate mutase-tyrA prephenate dehydrogenase (ACT) and allosteric substrate-binding (ASB) domains. The structure of hPHGDH was solved only for a truncated, dimeric form harboring the N-terminal end containing the substrate and the cofactor binding domains. A model ensemble of the tetrameric hPHGDH was generated using AlphaFold coupled with molecular dynamics refinement. By analyzing the inter-subunit interactions at the tetrameric interface, the residues F418, L478, P479, R454, and Y495 were selected and their role was studied by the alanine-scanning mutagenesis approach. The F418A variant modifies the putative ASB, slightly alters the activity, the fraction of protein in the tetrameric state, and the protein stability; it seems relevant in dimers' recognition to yield the tetrameric oligomer. On the contrary, the R454A, L478A, P479A, and Y495A variants (ACT domain) determine a loss of the tetrameric assembly, resulting in low stability and misfolding, triggering the aggregation and hampering the activity. The predicted tetrameric interface seems mediated by residues at the ACT domain, and the tetramer formation seems crucial for proper folding of hPHGDH, which, in turn, is essential for both stability and functionality.