Structural studies of nicotinic acetylcholine receptors and their regulatory complexes

Nicotinic acetylcholine receptors (nAChRs) play a key role in neuronal communication by sending electric signals upon binding of neurotransmitters, acetylcholines. nAChRs relate closely to various neuronal diseases including Alzheimer’s disease and schizophrenia. Understanding the mechanism of nAChRs would lead to better understanding of those diseases and eventually to new treatments. This dissertation covers six different projects, four of which are related to a nAChRs α1 subunit. The other two projects involve nAChR subunits, α7 and α9. For many of our studies, the X-ray crystallography technique is utilized for structural determination, which provides useful information to develop drugs or deeper understandings of molecular mechanisms. ❧ The main objective of this dissertation is to understand how a disease called myasthenia gravis (MG) is triggered at molecular level. It is an autoimmune neuromuscular disease, and it is known to be caused by autoantibodies targeting nAChRs. Tremendous researches have been done, but it is still not known how the autoantibodies actually bind the receptors. The first part of discussion is the structural determination of nAChR α1 subunit bound by one of MG mAbs named mAb35, which is a prototypical MG mAb. The first X-ray crystal structure of the complex was determined at 2.6 Å, and detailed interactions of the interface were revealed. The study was then extended to the determination of another complex structure of nAChR α1/mAb210. Various types of MG mAbs have been generated, and they have shown different characteristics. Thus, it is important to learn the differences of these mAbs, specifically mAb35 and mAb210, to understand MG better. Furthermore, this knowledge could be applied for drug developments in the future. The crystal structures of these two distinct complexes provide, for the first time, molecular details for understanding the disease mechanisms and for structure-guided drug design of MG. As the third aim, a new detection method of MG antibody was developed by utilizing all materials and knowledge obtained in the first section of MG study. The current diagnosis of MG has several limitations due to the usage of radiolabeled materials. Even though additional experiments are required for further improvement, our newly developed assay system is more time efficient and cost effective and could contribute to a MG diagnosis in the feature. The last aim related to MG is to discover proteins which can specifically target the nAChR α1 subunit at the interface between the receptor and the autoantibody, and also in near the future, those which target the binding site of MG antibodies. This project is a collaborative work with Dr. Rorberts’ group at USC and is in progress. It has already been achieved to develop a binder protein which targets the specific subunit. Structural information would enable us to design a variety of binder proteins (e.g. weaker binders or stronger binders), which could be used as therapeutic molecules. Then, the topic is moved onto other nAChR subunits. The 5th project is the structural determination of a nAChR α9 subunit. The focus of this project is a sugar chain on the α9, which is highly conserved on the cys-loop of nAChR α subunit and might have important roles in the gating mechanism of nAChRs. The X-ray crystal structure of nAChR α9 bound by α-bungarotoxin was solved at 3.0 Å. Our structure provides more insights into the possible role of the sugar chain. Lastly, this dissertation ends with purification of another nAChR subunit, α7. Due to the difficulty in expressing of the wild type α7 subunit, a chimera of α7/AChBP was produced and successfully purified using purification techniques and knowledge from the nAChR α1 study. The structure determination of the α7 bound with therapeutic compounds would lead for further drug development. ❧ In summary, our structural studies on nAChRs related to MG revealed the detailed information of the binding interface between the receptor and autoantibodies, resulting in further understanding of the disease. In addition, the new diagnostic method has been developed as well as possible therapeutic protein molecules for MG. The X-ray crystal structure of another nAChR subunit, α9 was successfully solved with a clear picture of a sugar chain, which might play an important role in functions of nAChRs. Finally, a robust purification system for the nAChR subunit α7 was developed and optimized for additional structural studies in order to develop therapeutic drugs.