Abstract

Oral Sessions

Day 3, May 19(Fri.) 9:50-10:10 Room C (101)

Application of HDX-MS for the Analysis of Protein-Protein Interaction between Mint3, an Intrinsically Disordered Protein, and FIH-1

(1Univ. Tokyo, 2Univ. Tokyo, 3Univ. Tokyo, 4Kanazawa Univ.)
oTensho Ten1, Yoshiaki Nakayama2, Satoru Nagatoishi3, Takeharu Sakamoto3, Motoharu Seiki4, Kohei Tsumoto1,2,3

In this research, we combined a series of physicochemical analysis with H/D exchange mass spectrometry (HDX-MS) to elucidate the binding mechanism of Mint3 to FIH-1.
First of all, we identified the N-terminal fragment of Mint3 (Mint3NT), the region that is capable of binding to FIH-1, as an IDP by circular dichroism (CD) spectroscopy, differential scanning calorimetry (DSC) and HDX-MS. Then, we performed isothermal titration calorimetry (ITC) analysis to characterize the interaction. The result indicates that Mint3NT has at least two core regions for binding to FIH-1, and FIH-1 needs α-keto glutaric acid (α-KG) and Fe2+ as cofactors for acquiring high affinity with Mint3NT. It is also suggested that the conformation of Mint3NT is fixed through the interaction, since -T∆S was very large. However, it is still unclear whether a secondary-structure is formed or not in the complex. Finally, we performed HDX-MS analysis to determine the binding sites of both proteins. Consequently, the region of FIH-1, which is identified to be affected by binding of Mint3NT, is consistent with the region affected by α-KG. Thus, we conclude that the binding sites of FIH-1 are located in the both ends of FIH-1 dimer.