Structural and functional study of transporters: ABCB6 and GLUT7 by using cryo-EM

Abstract

Over the past decade, remarkable advancements in cryo-electron microscopy (cryo-EM) have revolutionized the field of structural biology. Among various innovations, my focus has been on the structural analysis of membrane proteins. Due to their intrinsic characteristics, such as requiring diverse membrane reconstitution methods during the purification process, membrane proteins have posed significant challenges in structure determination using X-ray crystallography, which relies on obtaining high-quality crystals for diffraction pattern analysis. However, with the development of cryo-EM, a new avenue has emerged for determining membrane protein structures. Leveraging these advancements, I conducted structural studies on membrane proteins, particularly transporters. In the first part of this study, I targeted ATP Binding Cassette (ABC) transporters, which hydrolyze ATP and exhibit a broad substrate spectrum. Specifically, I determined the structure of ABCB6, the sixth protein in the B family of ABC transporters. Despite ABCB6 being relatively well-studied, I elucidated the previously unresolved transition state, the outward-facing open conformation, at a resolution of 3.8 Å. Additionally, I revealed that lipids play a structural role in stabilizing the protein. In the second part, I determined the structure of glucose transporter 7 (GLUT7), a protein known to transport glucose, one of the most fundamental energy sources in biological systems. Given its small molecular weight of 55 kDa, structure determination using cryo-EM was considered challenging. However, by employing nanodiscs and implementing a reference-based filtering process during data processing to eliminate bad particles, I achieved a final resolution of 3.3 Å. This study provides insights into the structural analysis of membrane proteins and the characterization of protein transition states using cryo-EM. The newly determined structures serve as foundational data for future research in this field.