Crystallographic Studies of CD14 and Its Implications for Lipopolysaccharide Signaling : Crystallographic Studies of CD14 and Its Implications for Lipopolysaccharide Signaling
- 발행기관 고려대학교
- 발행년도 2005
- 학위수여년월 2005. 8
- 학위명 박사
- 학과 대학원 생명공학과 식품생명공학전공
- 식별자(기타) DL:000015976597
- 서지제어번호 000045216963
초록/요약
ABSTRACT Lipopolysaccharide, the endotoxin of Gram-negative bacteria, induces extensive immune responses that can lead to fatal septic shock syndrome. The core receptors recognizing lipopolysaccharide are CD14, TLR4, and MD-2. CD14 binds to lipopolysaccharide and presents it to the TLR4/MD-2 complex, which initiates intracellular signaling. In addition to lipopolysaccharide, CD14 is capable of recognizing a few other microbial and cellular products. To clarify molecular mechanism of LPS binding and transfer among its receptors in the proinflammatory responses and to find out the molecular recognition pattern of ligands employed by CD14 that bind a wide variety of different microbial ligands, the first crystal structure of complex of CD14 and phosphatidyl serine is elucidated to 2.5 Å resolution. A large hydrophobic pocket was found on the NH2-terminal side of the horseshoe-like structure. Ligand diversity of CD14 could be explained by the generous size of the pocket, the considerable flexibility of the rim of the pocket, and the multiplicity of grooves available for ligand binding. Based on structural findings and previous biochemical investigations, It was proposed that the lipid portion of LPS binds to the NH2-terminal phospholipids-binding pocket and the carbohydrate portion of LPS binds to the region to extend further beyond the NH2-terminal pocket and include lipopolysaccharide binding region 4 and the upper side of G2 groove on the outside of pocket. This proposal is based on the following reasoning: 1) The epitopes of antibodies and mutations that block LPS binding map around the NH2-terminal pocket; and 2) the pocket is the only hydrophobic surface large enough to accommodate lipid portion of LPS; and 3) It has been confirmed that phospholipids compete with LPS for binding to CD14 using various experiments; and 4) PGN competes with LPS for binding to CD14. Deletion of region 4 reduces binding of lipopolysaccharide as well as of peptidoglycan; and 5) The epitope of the monoclonal antibody specifically reduces the affinity for peptidoglycan without affecting that for LPS maps to the upper side of the G2 groove. Mutations that interfere with lipopolysaccharide signaling but not with lipopolysaccharide binding are also clustered in a separate area, T1-T3, on the same side of CD14. It is noteworthy that they are clustered near LPS binding site of outside of the pocket. In conclusion, the structure provides evidence that different regions around the pocket contribute to LPS binding and signaling. Structural studies of CD14 provide new insights into the mechanism by which it recognizes LPS and may help in developing therapeutic agents to counteract septic shock syndrome.
more초록/요약
ABSTRACT Lipopolysaccharide, the endotoxin of Gram-negative bacteria, induces extensive immune responses that can lead to fatal septic shock syndrome. The core receptors recognizing lipopolysaccharide are CD14, TLR4, and MD-2. CD14 binds to lipopolysaccharide and presents it to the TLR4/MD-2 complex, which initiates intracellular signaling. In addition to lipopolysaccharide, CD14 is capable of recognizing a few other microbial and cellular products. To clarify molecular mechanism of LPS binding and transfer among its receptors in the proinflammatory responses and to find out the molecular recognition pattern of ligands employed by CD14 that bind a wide variety of different microbial ligands, the first crystal structure of complex of CD14 and phosphatidyl serine is elucidated to 2.5 Å resolution. A large hydrophobic pocket was found on the NH2-terminal side of the horseshoe-like structure. Ligand diversity of CD14 could be explained by the generous size of the pocket, the considerable flexibility of the rim of the pocket, and the multiplicity of grooves available for ligand binding. Based on structural findings and previous biochemical investigations, It was proposed that the lipid portion of LPS binds to the NH2-terminal phospholipids-binding pocket and the carbohydrate portion of LPS binds to the region to extend further beyond the NH2-terminal pocket and include lipopolysaccharide binding region 4 and the upper side of G2 groove on the outside of pocket. This proposal is based on the following reasoning: 1) The epitopes of antibodies and mutations that block LPS binding map around the NH2-terminal pocket; and 2) the pocket is the only hydrophobic surface large enough to accommodate lipid portion of LPS; and 3) It has been confirmed that phospholipids compete with LPS for binding to CD14 using various experiments; and 4) PGN competes with LPS for binding to CD14. Deletion of region 4 reduces binding of lipopolysaccharide as well as of peptidoglycan; and 5) The epitope of the monoclonal antibody specifically reduces the affinity for peptidoglycan without affecting that for LPS maps to the upper side of the G2 groove. Mutations that interfere with lipopolysaccharide signaling but not with lipopolysaccharide binding are also clustered in a separate area, T1-T3, on the same side of CD14. It is noteworthy that they are clustered near LPS binding site of outside of the pocket. In conclusion, the structure provides evidence that different regions around the pocket contribute to LPS binding and signaling. Structural studies of CD14 provide new insights into the mechanism by which it recognizes LPS and may help in developing therapeutic agents to counteract septic shock syndrome.
more목차
Table of Contents
Content Page
ABSTRACT
1. INTRODUCTION
2. LITERATURE REVIEWS
2.1 X-ray Crystallography
2.1.1 X-ray Diffraction Equipment
2.1.2 Crystal Growing
2.1.3 Diffraction Theory
2.1.4 Method of Phase Determination
2.1.4.1 Multiple Isomorphous Replacement (MIR)
2.1.4.2 Multiple-wavelength Anomalous Dispersion (MAD)
2.1.4.3 Molecular Replacement (MR)
2.1.5 Refinement of Protein Structure
2.1.5.1 Refinement Theory
2.1.5.2 The free R-factor (Rfree)
2.2 CD14
2.2.1 Physiological Functions of CD14
2.2.1.1 LPS Receptor Complex in Proinflammatory Mechanism
2.2.1.2 CD14 as a Common Receptor for Bacterial Compounds
2.2.1.2.1 Gram-Negative Bacteria : Lipopolysaccharide
2.2.1.2.2 Gram-Positive Bacteria : Peptidoglycan
2.2.1.3 CD14 as a Recognition Receptor for Lipopolysaccharide
2.2.2 Signaling Pathway Related to CD14 in Macrophages
2.2.3 Septic Shock
2.2.3.1 Clinical and Experimental Sepsis Therapies
2.2.3.2 Lipid A Antagonist Targeting the LPS Receptor Complex
3. MATERIALS AND MATHODS
3.1 Cloning and Expression of CD14
Content Page
3.2.Protein Purification
3.2.1 Purification of CD14
3.2.2 Purification of Phospholipids-CD14 Complexes
3.3 Crystallization and Cryo-cooling Conditions
3.4 Preparation and Screening of Heavy Metal Derivatives
3.5 Data Collection and Processing
3.6 Structure Determination and Refinement
3.6.1 SCALEPACK2MTZ
3.6.2 TRUNCATE
3.6.3 CAD.COM
3.6.4 SCALEIT.COM
3.6.5 The Determination of Heavy Atom Positions
3.6.6 The Calculation of Phases
3.6.7 Refinement
3.7 Calculation of Electron Density Maps and Model Building
3.7.1 (Fo-Fc)-map
3.7.2 (2Fo-Fc)-map
3.8 Validation of the Model
4. RESULTS AND DISCUSSION
4.1 Protein Expression and Purification of CD14
4.2 Crystallization and Data Collection
4.3 Determination of Heavy Atom Sites
4.4 Structure Determination and Refinement
4.5 Quality of CD14 Model
4.6 Crystal Structure of Recombinant Mouse CD14
4.6.1 Overall Structure of Recombinant Mouse CD14
4.6.2 The NH2-terminal Hydrophobic Pocket
4.6.3 Phospholipid Binding Pocket
4.6.4 The Pocket Area Is Responsible for LPS Binding
Content Page
4.6.5 Model of LPS Bound to CD14
4.6.6 Regions Responsible for LPS or PGN Signaling
4.6.6.1 Regions Responsible for LPS Signaling
4.6.6.2 Regions Responsible for PGN Signaling
4.6.7 Charge Complementarities of LPS-Binding Sites of CD14
and MD-
4.6.8 Model of the LPS and CD14 Complex Formation and Transfer
to TLR4/MD-2 Comple
5. REFERENCES
6. SUMMARY
7. ABSTRACT IN KOREAN
List of Tables
Table Page 1 Interaction of CD14 with different bacterial and yeast cell
components 2 Protein storage buffer and crystallization solutions for CD14
crystallization 3 Statistics from the crystallographic analysis
List of Figures
Figure Page 1 X-ray diffraction equipment
2 Schematic diagram of a hanging drop setup
3 Bragg’s Law
4 Line diagram representation of the Ewald construction
5 Harker construction for protein phase determination
6 Schematic of experimental values for ?f’(lower) and ?f’’ (upper)
as a function of X-ray energy 7 LPS from the outer membrane of Escherichia coli binds to LBP
in the serum and is delivered to CD
8 Cell wall structure of bacteria
9 Thin section of an unidentified Gram-negative bacterium found
in a freshwater biofilm in a river near laboratory 10 Schematic representation of the general architecture of bacterial
LPS 11 Chemical structure of the bacterial LPS
12 Structure of lipid A such as is found in E. coli strains
13 Selected Gram-negative lipid A and derivative structures
14 Structures of Lys-type and Dap-type PGN
15 Binding of bacterial ligands to CD14 and sCD14
16 Simplified model of lipopolysaccharide (LPS) signalling
17 Effects of LPS and secondarily-induced effector molecule
18 Secondary structure assignment of mouse CD14 and alignment of
mouse, human, rabbit, and bovine CD
19 Purification of CD14
20 Deglycosylation of purified recombinant mouse CD14
21 Orthorhombic crystal of glycosylated CD14
22 Harker sections of the difference patterson map
Figure Page 23 Ramachandran plot for CD14, created by PROCHECK
24 Overall structure of mouse CD14 dimer
25 Primary, secondary and tertiary structures of the leucine-rich
repeat (LRR) 26 The dimerization of CD14
27 Structure of the NH2-terminal pocket
28 Native PAGE analysis of phospholipid-CD14 complexes
29 Native PAGE analysis of substitution Ra-LPS bound to CD14 to
phospholipids 30 Structure of the phospholipids binding pocket
31 Regions involved in LPS binding and signaling
32 Structure of bactericidal/permeability-increasing protein (BPI)
33 Charge complementarities of LPS binding sites of CD14 and
MD-
34 Proposed model for the LPS and CD14 complex formation and
transfer to TLR4/MD-2 complex.
