Proliferating cancer cells extensively rewire central carbon and nitrogen metabolism, with heightened dependence on the malate–aspartate shuttle to maintain redox balance and supply aspartate for biosynthesis. A pivotal node in this circuitry is mitochondrial glutamate–oxaloacetate transaminase 2 (GOT2), a pyridoxal-5′-phosphate–dependent aminotransferase upregulated in aggressive malignancies, most notably pancreatic ductal adenocarcinoma. Yet its structural tractability and potential for selective modulation remain poorly defined. We therefore undertook an integrated structural and biochemical investigation of human GOT2.
Recombinant GOT2 proved recalcitrant under published conditions, so a systematic expression campaign was undertaken, delayed induction in 2YT medium, combined with periplasmic extraction and mechanical lysis, transformed a marginal 2 mg yield into reliable production of monodisperse, crystallisation-grade protein. A coupled dual-enzyme assay, reporting GOT2 turnover through malate dehydrogenase via the NAD⁺/NADH couple, provided a continuous readout for inhibitor screening.
Differential scanning fluorimetry of a mitochondrial-metabolite library identified 3,5-dinitrosalicylic acid (DNSA) as a reproducible binder, destabilising GOT2 in a manner mirroring the natural substrate L-aspartate. We then determined the highest-resolution structure of dimeric human GOT2 to date — the PLP-bound enzyme at 1.5 Å (PDB 9YB6). Soaking these crystals with DNSA yielded a 1.49 Å complex (PDB 9ZZU) in which density placed the ligand not at the active site but within a previously unrecognised pocket at the dimer interface, clamped between both protomers by Ile132, Met315 and Tyr316. Ligand engagement drove rotation of Met315 and Tyr316 to envelop the ligand — interface plasticity absent from the apo structure — indicating an allosteric mechanism distinct from substrate binding.
These findings establish GOT2 as a structurally tractable metabolic target and reveal an unanticipated, ligandable allosteric pocket at its dimer interface. Such modulators offer a rational route to disrupting GOT2-dependent metabolism, holding promise as adjuncts that enhance existing cancer therapies by exploiting the metabolic vulnerabilities underlying tumour growth.