Together with adaptive immune system, innate immune system is indispensable part of our defense mechanism against pathogenic microbial infection. The adaptive immunity is an antigen-specific immune response, which is mediated by T and B cells, whereas innate immunity is nonspecific defense mechanisms and receptors recognize many related molecular structures called PAMPs (Pathogen-Associated Molecular Patterns). Toll-Like Receptors (TLR) family proteins play an important role in innate immune response by recognizing conserved structural patterns in diverse microbial molecules. Specially, TLR4 with its co-receptor Myeloid Differentiation factor 2 (MD-2) is responsible for LipoPolySaccharide (LPS) recognition. LPS is a major component of the outer membrane of Gram-negative bacteria and a potent inducer of innate immune response. It is extracted and transferred to TLR4-MD-2 complex by two accessory proteins, LBP (LPS-Binding Protein) and CD14. Binding of LPS to the TLR4-MD-2 complex causes a rearrangement of the extracellular part of the receptor complex, which enforce aggregation of intracellular domain signaling domain called TIR domain (TIR: Toll/Interleukin-1 Resistance). In order to understand structural basis of TLR4 activation, we undertook crystallographic studies of TLR4-MD-2 in complex with various ligand molecules.
In 2007, we determined the first structure of the TLR4-MD-2 complex with bound endotoxin antagonist Eritoran. In this study, we introduced a novel technique termed “Hybrid LRR Technique” to overcome technical difficulties associated with production and crystallization of the TLR4-MD-2 complex. By using this technique, we produced a series of hybrids of human TLR4 and hagfish VLR. Among them, three hybrids named TV3, TV8 and VT3 are successfully produced and crystallized. In particular, TV3 could be crystallized together with MD-2 and Eritoran. Eritoran is a candidate drug molecule under clinical trial phase III against severe sepsis. It mimics structure of LPS and binds to TLR4-MD-2 with high affinity. However, it cannot induce aggregation of TLR4 and therefore cannot induce immune response. The structure of TV3-MD-2-Eritoran showed that Eritoran binds to the hydrophobic pocket in human MD-2, and there is no direct interaction between Eritoran and TLR4. Based on the biochemical analysis, we proposed a model of TLR4-MD-2 dimerization induced by LPS.
In early this year, we finally succeeded in crystallization and structure determination of the TLR4-MD-2-LPS complex. In this structure, LPS binding induced the formation of an 'm'- shaped symmetrical fashion of TLR4-MD-2-LPS complex. MD-2 binds to the concave surface of the TLR4 primary interface by charge interaction and TLR4 dimerization interface is formed convex surface with hydrophobic core, which is mediated by MD-2-LPS binding. LPS interacts with a large hydrophobic pocket in MD-2 and five of the six lipid chains of LPS are buried deep inside the pocket and the remaining chain is exposed to the surface of MD-2, forming a hydrophobic interaction with another TLR4-MD-2 complex. Structures of the TLR4-MD-2-Eritoran and the TLR4-MD-2-LPS complexes illustrate the remarkable versatility of the ligand recognition mechanisms employed by the TLR family, which is essential for defense against diverse microbial infection. The structural information of TLRs will provide valuable information for design of the next generation therapeutic agent against septic shock and other inflammatory diseases.
우리 몸은 외부의 병원체로부터 스스로를 보호하기 위한 특징적인 면역체계를 구성하고 있습니다. 이러한 면역체계는 병원체들이 침입 하였을 때 면역세포들의 상호 작용에 의해서 병원체의 특이적인 항체를 생성해서 병원체들을 제거하는 적응성 면역반응(adaptive immune system)과 이와는 달리 비특이적으로 어떤 일정한 형태(pattern)를 인식하여 병원체들을 즉각적으로 제거하는 선천성 면역반응(innate immune system)으로 구성되어 있습니다.
선천성 면역반응에서 중요한 역할을 하는 것으로 Toll like receptors (TLRs)가 알려져 있습니다. TLR들은 병원체의 어떤 분자적 형태를 인식하는(pattern recognition receptor)것으로 알려져 있으며 TLR들 중에서 TLR4는 보조 수용체 단백질인 MD-2와 함께 그람 음성 균의 세포벽(Gram-negative bacteria)에 있는 lipopolysaccharide(LPS)를 인지하는 중요한 역할을 수행합니다. LPS는 그람 음성 균의 내독소로 알려져 있으며 LBP(LPS binding protein) 단백질과 CD14 단백질을 통해서 TLR4-MD-2 복합체에 전달되고 TLR4의 세포 내 부분(TIR : Toll/IL-1 Resistance)을 통해서 면역반응 신호가 활성화가 이루어집니다.
이에 우리는 TLR4-MD-2-LPS 복합체 단백질 결정 구조 분석을 통하여 어떻게 리간드인 LPS를 인식하고 이들 수용체의 활성을 유도하는지에 관하여 연구하고자 하였습니다. TLR4-MD-2-LPS 이형이량체 복합체는 리간드인 LPS와 결합하여 m-모양 형태를 이루는 것을 알게 되었으며 우선적으로 MD-2는 전하에 의해서 TLR4의 오목한 표면에 상호작용을 하여 TLR4-MD-2 이형복합체를 형성합니다. MD-2-LPS의 경우, LPS는 MD-2의 부피가 있는 소수성 주머니에 결합되고 LPS의 노출된 하나의 lipid chain이 또 다른 TLR4 볼록한 표면과의 상호작용을 통해서 이량체 형성에 가교 역할을 하는 것을 알게 되었습니다.
이러한 TLR4-MD-2-LPS의 분자적인 구조 규명은 이전 연구에서 발표 되어진 논문들과 함께 TLR들이 다양한 리간드들을 인식하는 방식에 대해서 생각해볼 수 있을 것이고 TLR들과 그들의 리간드들의 복합체에 관한 구조적인 역할은 선천성 면역체계의 연구에 중요한 구조적 정보를 제공함과 동시에 여러 가지 면역질환에 대한 치료제 개발에 이로움을 제공할 것이다.
