Binding mechanism underlying FIH-1 suppression caused by the N-terminal disordered region of Mint3

Mint3 is known to enhance aerobic ATP production, known as the Warburg effect, by binding to FIH-1. Since this effect is considered to be beneficial for cancer cells, the interaction is a promising target for cancer therapy. However, previous research has suggested that the interacting region of Mint3 with FIH-1 is intrinsically disordered, which makes investigation of this interaction challenging. Therefore, we adopted a physicochemical approach that combined thermodynamic studies with structural analyses in solution, to clarify the binding mechanism. First, using a combination of CD, NMR, and hydrogen/deuterium exchange mass spectrometry (HDX-MS), we confirmed that the N-terminal half, which is the interacting part of Mint3, is mostly disordered. Next, using isothermal titration calorimetry (ITC), we revealed that the interaction of Mint3 and FIH-1 produces an enormous change in enthalpy and entropy. The profile is consistent with the model that the flexibility of disordered Mint3 is drastically reduced upon binding to FIH-1. Moreover, we performed a series of ITC experiments with several types of truncated Mint3s, an effective approach since the interacting part of Mint3 is disordered, and identified 78-88 as a novel core site for binding to FIH-1. The truncation study of Mint3 also revealed the thermodynamic contribution of each part to the interaction, where one contributes to the affinity ({Delta}G), while the other only affects enthalpy ({Delta}H), by forming non-covalent bonds. This insight can serve as a foothold for further investigation of IDRs and drug development for cancer therapy.