THE STRUCTURAL STUDY OF THE TNF¿- ADALIMUMAB COMPLEX AND DAM1 COMPLEX USING CRYO-EM

Abstract

Cryo-EM single-particle reconstruction (SPR) has become a powerful technique to determine the structure of biological macromolecular complexes at near-atomic resolution. Based on this technique, I studied the structure of two protein complexes in this thesis. The first one is TNFα - Adalimumab complex. Tumor necrosis factor-alpha (TNFα) is a cytokine secreted by white blood cells in response to bacterial infection and is the major cause of chronic inflammation leading to rheumatoid arthritis. I determined the three-dimensional structure of 3:2 Adalimumab: TNF complex at 4.7 Å using Cryo-EM SPR. The Cryo-EM structure explains how Adalimumab can inhibit TNF trimers in solution from binding to the TNF receptor. The other one is the study of the Dam1 complex and its interaction with MTs in vitro. Dam1 is part of the ‘outer’ kinetochore and it has been shown to form a ring around the microtubules in vitro. However, the mechanism by which the Dam1 complex binds to microtubules and tracks the depolymerizing microtubules in chromosome segregation is elusive. In this study, it was found that the purified Dam1 complex is highly heterogeneous in the configuration without Microtubules (MTs). And the ring formation is biased and dynamic.