Molecules such as glucose, amino acids, nucleosides, and charged ions are essential for survival of cells but a large majority of them cannot penetrate the lipid bilayer. Transporters imbedded in the cellular membrane facilitate import or export of these molecules. Transporters have been studied for more than 60 years. However, the transport mechanism of most of these membrane proteins is still largely unknown due to many technical hurdles including expression, extraction, and crystallization.

To understand membrane transport processes, we use a large array of structural and biochemical approaches. The research tool we use is cryo-electron microscopy (cryo-EM). Using this technique we can determine atomic structures of transporters frozen in multiple conformational states. Several technical advances of electron microscopy in recent years revolutionized structural biology. First, the development of direct electron detection, second, the development of algorithms for data collection from imaging to classification and also for structural reconstruction, and third, the development of volta phase plate (VPP) for in-focus data collection. They solve a major challenge for structure determination of membrane proteins and have led to a rapid boom in the cryo-EM structure of various proteins ranging from 64 kDa to 150 MDa.

Our research focuses on the family of ATP-binding cassette (ABC) transporters that transport substrate across the cell membrane using ATP hydrolysis energy. The best known example of this is P-glycoprotein (P-gp).

P-gp is an ATP - dependent efflux pump that exports a structurally diverse array of hydrophobic drugs. Substrates bind to the inward-facing conformation in a cavity exposed to the cytoplasm and the membrane bilayer inner leaflet and are released in the outward-facing conformation in the presence of ATP binding. P-gp is an important target of anti-cancer drug discovery. Prolonged treatment of cancer often leads to resistance to multiple drugs. Overexpression of P-gp is commonly found multidrug resistant in cancer cells.

Therefore, the effectiveness of cancer chemotherapy treatment is often limited by overexpression of P-gp. Co-treatment of cancer chemotherapeutics with P-gp inhibitors has been shown to be effective in limiting drug resistance.
P-gp has been extensively studied for several decades, but biological questions still remain, such as how P-gp recognizes both cancer drug and P-gp inhibitor and how we can stop their outward-facing conformation changes enabling substrate transport across membranes. Our lab would like to study the substrate transport mechanism by a combination of biochemical and structural analysis including cryo-EM . These structural and functional studies will help in not only better understanding disease from transporters but also will help achieve better drug design and development for humans.

Another focus is on the group of solute carrier (SLC) transporters. Some SLC transporters are targets of drugs and they are being actively studied.
SLC transporters mediate influx or bidirectional movement of broad-ranged small substrates including inorganic ions, amino acids, lipids, sugars, neurotransmitters, and drugs. For example, neurotransmitter transporters are found in three distinct clusters: 1) The SLCl family, 2) the SLC6 and SLC32 families, and 3) the SLCl 7 and SLC18 families. The other SLC transporters, belonging to the same family, transport a different substrate and use a different mechanism (e.g. symporter vs antiporter). For many SLC transporter families, several crucial questions remain unanswered: 1) how they interact with specific substrate; and 2) how they control the cellular influx of substrates involving their conformational change.

Our lab would like to explain the molecular mechanism of SLC using the structural and biochemical tool cryo-EM.

세포막 수송체 단백질 중 ABC(ATP-binding cassette) 수송체 와 SLC(Solute Carrier) 수송체들은 인간을 포함한 거의 모든 생명체 세포에 존재하여 포도당, 아미노산, 뉴클레오사이드 및 하전된 이온등 같이 모든 생물의 생존에 필요물질들을 세포막 내외로 운반하는 역할을 하고 있고, 이들의 기능 이상은 각종 질병에 관련되어 신약개발의 표적이 됩니다.

수송체 구조 생물학 연구실에서는 세포막 수송체 단백질들이 어떻게 다양한 기질을 세포막을 통해서 수송하는지에 대한 작동 메커니즘을 규명하여, 관련 질환 해결의 실마리로 제공하는 것을 목표로 하고 있습니다.

학부생들이 참여할 수 있는 연구 주제

ABC 수송체의 구조 변화 과정을 연구
- ABC 수송체는 가장 큰 수송체패밀리로서, ATP가 결합하는 NBD(Nucleotide binding domain)이 있으며, 그 도메인에ATP 결합 또는 가수분해 에너지는 TMD(Transmembrane domain)의 구조변화를 일으켜서 물질을 세포안밖으로 이동시킵니다.
- 대표적인 ABC수송체를 하나 선정하여, ATP 결합에 의한 구조 변화와 ATP 가수분해 후 일어나는 ABC수송체의 구조 변화와 운반하는 물질과의 결합에 의한 ABC수송체의구조 변화도 관찰할 예정입니다.

학부생들이 참여할 수 있는 연구 실험내용
1) Ecoli, Insect와 mammalian system을 이용하여 타겟이 되는ABC 수송체 단백질 발현 조건 확립,
2) ABC 수송체 단백질을 가장 안정하게 추출할 수 있는 detergent 및 최적화된 정제 버퍼 조건과 thormostability(열 안정성)등을 FSEC(Fluorescence-detection size-exclusion chromatography) 에 나온 결과를 기반하여 확립,
3) 정제된 단백질을 가지고 Electron Microscopy을 위한 그리드 준비 등을 수행할 예정입니다.

• ㆍThe mechanism of membrane transporters
• ㆍCryo-electron microscopy
• ㆍX-ray crystallography

• ㆍKwon NY, Kim Yet al., (2018), Structural diversity and flexibility of diabodies. Methods, 154,136-142
• ㆍKim Yet al., (2018), Molecular structure of human P-glycoprotein in the ATP-bound, outward-facing conformation. Science, 359(6378):915-919
• ㆍEckert JK, Kim VJ et al., (2013), The Crystal Structure of Lipopolysaccharide Binding Protein Reveals the Location of a Frequent Mutation that Impairs Innate Immunity. Immunity, 39, 1-14
• ㆍAn HJ, Kim VJ, Song DH et al., (2011), Crystallographic and mutational analysis of the CD40-CD154 complex and its implications for receptor activation. J. Biol. Chem, 286(13): 11226-11235

• ㆍB.S., Sungkyunkwan University, Suwon, Korea (2003)
• ㆍM.S., KAIST, Daejeon, Korea (2005)
• ㆍPh.D., KAIST, Daejeon, Korea (2011)

• ㆍ2011-2014 : Research associate, Dept. of Chemistry, KAIST
• ㆍ2014-2018 : Postdoctoral Fellow, The Rockefeller University, NY, USA
• ㆍ2018-2019 : Research associate, Dept. of Chemistry, KAIST
• ㆍ2019-Present : Assistant Professor, Dept. of Life Sciences, POSTECH