Structural and thermodynamical insights into the binding and inhibition of FIH-1 by the N-terminal disordered region of Mint3

Tensho Ten,Satoru Nagatoishi,Ryo Maeda,Masaru Hoshino,Yoshiaki Nakayama,Motoharu Seiki,Takeharu Sakamoto,Kouhei Tsumoto

doi:10.1016/j.jbc.2021.101304

Abstract

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 thermodynamic and structural studies in solution to clarify the structural and thermodynamical changes of Mint3 binding to FIH-1. First, using a combination of circular dichroism, nuclear magnetic resonance, 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, we revealed a large enthalpy and entropy change in the interaction of Mint3 using isothermal titration calorimetry (ITC). 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 amino acids 78 to 88 as a novel core site for binding to FIH-1. The truncation study of Mint3 also revealed the thermodynamic contribution of each part of Mint3 to the interaction with FIH-1, where the core sites contribute to the affinity (ΔG), while other sites only affect enthalpy (ΔH), by forming noncovalent bonds. This insight can serve as a foothold for further investigation of intrinsically disordered regions (IDRs) and drug development for cancer therapy.

Highlights

Cancer is a major cause of death worldwide
Since the glycolytic approach of energy production is less efficient in terms of ATP production than oxidative phosphorylation, which is the primary pathway of ATP production in most normal cells, the preference for aerobic glycolysis is assumed to have some implications on the differentiation between cancer cells and normal cells
The possible explanations are an adaptation to hypoxia, which is the condition that most cancer cells confront, and/or acceleration of production of building blocks required for cell proliferation, which are provided as byproducts of glycolysis

Summary

Introduction

Cancer is a major cause of death worldwide. the development of its treatment is of great interest in various research areas, including molecular targeted therapies. We performed a series of ITC experiments with several types of truncated Mint3s, an effective approach since the interacting part of Mint[3] is disordered, and identified amino acids 78 to 88 as a novel core site for binding to FIH-1. Previous research has implied that the N-terminal region of Mint[3] is the binding site of FIH-1 (3).

Results

Conclusion