Development of Living Cell D2HG Biosensors with a Wide Detection Range Inspired by the Structure of the D2HG-Binding Transcription Factor DhdR

Abstract

Mutant IDH1/IDH2 catalyzes the conversion of αKG to D2HG, a hallmark of many lower-grade gliomas and secondary glioblastomas. Elevated D2HG levels promote tumorigenesis through epigenetic reprogramming and immunosuppressive mechanisms, though paradoxically, D2HG can also inhibit tumor growth. To explore D2HG’s biological functions, we developed genetically encoded D2HG biosensors based on the prokaryotic transcriptional regulator DhdR. Structural analysis of DhdR, including its apo form, D2HG-bound complex, and DNA-bound complex, revealed that D2HG binding induces conformational changes that regulate DNA interaction. Leveraging these insights, we engineered biosensors (DHsers) that detect D2HG across a wide dynamic range (0.3 μM to 30 mM) with high sensitivity. We also established a standardized protocol for quantifying subcellular D2HG levels in living cells. Notably, we found STING activation promotes D2HG production, hinting at immune modulation. Our findings reveal D2HG-induced transcriptional regulation in prokaryotes and offer a platform for studying D2HG in cellular metabolism and tumorigenesis.

Keywords: D2HG, biosensor, transcriptional regulator, DhdR, Structure, allosteric regulation