인간 인터루킨-2 와 항체 복합체들 및 원핵생물의 비상동말단연결 기작에 대한 구조 연구

Abstract

Chapter 1. Abstract Immunotherapy via interleukin-2 (IL-2) mediated activation of anti-tumor immune response is a promising approach for cancer treatment. The multi-potent cytokine, IL-2 has a central role in immune cell activation and homeostasis. Since IL-2 preferentially activates immunosuppressive T regulatory cells in an IL-2Rα dependent manner, blocking IL-2:IL-2Rα interaction is a key to amplify the IL-2 activity in effector T cells toward anti-tumor response. Anti-IL-2 monoclonal antibodies are good candidates to control the IL-2:IL-2Rα interaction. In a previous study, a new IL-2Rα mimetic antibody called TCB2 was developed, and showed that the human IL-2(hIL-2):TCB2 complex can stimulate T effector cells specifically and elicit potent anti-cancer immunotherapeutic effect, especially when administered in combination with immune checkpoint inhibitors. To understand the molecular mechanism, I determined the crystal structure of TCB2-Fab in a complex with hIL-2 at 2.5 Å resolution. My structural analysis reveals that TCB2 binds to the central area of the hIL-2Rα binding region on hIL-2. This binding mode differs from that of the previously known hIL-2Rα mimicking antibody NARA1, which recognizes the top part of hIL-2, in terms of both binding angle and epitope. TCB2 binding to hIL-2 also induces an allosteric effect that increases the affinity for the hetero-dimeric hIL-2 receptor, IL-2R(β+γ), on effector T cells. I elucidated the structure of the humanized TCB2 and hIL-2 complex and compared it to the TCB2 complex, revealing that the humanized TCB2 shares a similar structure and recognizes the same epitope as TCB2. These structural findings suggest that the humanized TCB2 antibody may retain its IL-2 binding properties and functional effects when administered in the human body, potentially leading to similar therapeutic outcomes as observed with the original TCB2 antibody. Additionally, I determined the structure of the JY7 antibody and hIL-2 complex, in contrast to TCB2, which can selectively induce the expression of regulatory T cells (Tregs). The structural analysis showed that JY7 precisely recognizes the IL-2R(β+γ) binding site on hIL-2, thereby competitively inhibiting IL-2R(β+γ) and increasing the dependence on IL-2Rα, which enables the selective induction of Tregs. These findings provide the molecular mechanism underlying the selective expression of regulatory T cells by the JY7 and hIL-2 complex.

Chapter 2. Abstract Double-strand breaks (DSBs) are one of the most lethal form of DNA damage. Cells have evolved two major mechanisms for their repair: homologous recombination (HR) and non-homologous end joining (NHEJ). While HR utilizes homology with sister chromatids to accurately repair damaged DNA, NHEJ can directly join DSB ends without requiring a template, making it the primary DSB repair mechanism in cell cycle phases where templates are unavailable. In contrast to the complex eukaryotic NHEJ involving multiple protein components, bacterial NHEJ can be performed by just two enzymes: Ku, which recognizes DNA ends, and LigD, which ligates DNA. To maintain DNA ends in close proximity for efficient joining, eukaryotes rely on a complex array of proteins, while bacterial Ku can form DNA synapsis independently. In eukaryotes, the Ku70/80 heterodimer, the homolog of bacterial Ku, possesses structural features that allow it to differentiate between the DNA inward face and the surface interacting with DNA-PKcs. In contrast to eukaryotic counterpart, two of the most salient distinguishing features of the bacterial Ku are its existence as a homodimeric form and smaller size, approximately half that of the eukaryotic Ku heterodimer. This homodimeric nature of bacterial Ku raises intriguing questions about the molecular mechanisms underlying its interactions with DNA ends and its role in facilitating the NHEJ process. Furthermore, bacterial Ku plays a crucial role in recruiting LigD to the synapse and stimulating its activity, highlighting the importance of understanding the structural basis of these interactions. To address these questions and gain insights into the molecular mechanisms of bacterial Ku-mediated NHEJ, I determined the Cryo-EM structure of the bacterial Ku-DNA complex providing a foundation for elucidating the molecular details of DNA synapsis formation and the unique features of the bacterial NHEJ machinery. LigD, the multifunctional enzyme responsible for the final ligation step of repair, consists of an ATP-dependent ligase domain, a polymerase domain, and a phosphoesterase domain. This unique domain organization endows LigD with the self-sufficiency to carry out both ligation and DNA end processing without the need for additional enzymatic activities. To better understand the mechanism of action of the LigD, we determined the crystal structure of the adenylated ligase domain of Bacillus subtilis LigD in this study.