Structural and ligand binding studies of toll-like receptor 4 (TLR4) associated proteins
type: CreativeWork
description: The sensing of lipopolysaccharide (LPS) represents a major mechanism of detection of Gram-negative bacterial infection by the human innate immune system. This pathogen associated molecular pattern is recognized by the host through the transmembrane pattern recognition complex comprised of Toll-like receptor 4 (TLR4) and myeloid differentiation antigen (MD-2). As described in Chapter 1, LPS-dependent complex formation of TLR4/MD-2 heterodimers results in the activation of pro-inflammatory signaling cascades. Although inflammation is usually host protective, when uncontrolled it can cause host fatality through sepsis. The function of soluble or membrane anchored human CD14 is to bind and shuttle LPS to TLR4/MD-2, thereby enhancing host sensitivity of LPS. The mechanism by which CD14 binds and transfers LPS remains unknown, and our understanding of this process would be advanced by structural information. Thus, we determined the x-ray crystal structure of human CD14, the results of which were recently published (Kelley, S.L., et al., (2013). J. Immunol. 190:1304-1311) and are presented in Chapter 2 of this dissertation. To do so, we cloned, expressed, and purified two constructs of human CD14 from a mammalian expression system. One of the two crystallized and resulted in an x-ray crystal structure solution. The structure of human CD14 reveals a bent solenoid with an N-terminal hydrophobic pocket. Compared to the mouse CD14 crystal structure, human CD14 reveals an expansion of residues comprising the rim region and an adjacent, solvent exposed hydrophobic patch, which may facilitate LPS binding. In Chapter 3 we describe the expression and purification of mouse CD14 as an additional target for ligand binding crystallization studies. After determining that purified human and mouse CD14 bind LPS and other ligands using native PAGE gel shift and ForteBio Octet assays, we confirmed the bioactivity of both purified proteins through cell-based stimulation assays. Additional sm…
abstract: The sensing of lipopolysaccharide (LPS) represents a major mechanism of detection of Gram-negative bacterial infection by the human innate immune system. This pathogen associated molecular pattern is recognized by the host through the transmembrane pattern recognition complex comprised of Toll-like receptor 4 (TLR4) and myeloid differentiation antigen (MD-2). As described in Chapter 1, LPS-dependent complex formation of TLR4/MD-2 heterodimers results in the activation of pro-inflammatory signaling cascades. Although inflammation is usually host protective, when uncontrolled it can cause host fatality through sepsis. The function of soluble or membrane anchored human CD14 is to bind and shuttle LPS to TLR4/MD-2, thereby enhancing host sensitivity of LPS. The mechanism by which CD14 binds and transfers LPS remains unknown, and our understanding of this process would be advanced by structural information. Thus, we determined the x-ray crystal structure of human CD14, the results of which were recently published (Kelley, S.L., et al., (2013). J. Immunol. 190:1304-1311) and are presented in Chapter 2 of this dissertation. To do so, we cloned, expressed, and purified two constructs of human CD14 from a mammalian expression system. One of the two crystallized and resulted in an x-ray crystal structure solution. The structure of human CD14 reveals a bent solenoid with an N-terminal hydrophobic pocket. Compared to the mouse CD14 crystal structure, human CD14 reveals an expansion of residues comprising the rim region and an adjacent, solvent exposed hydrophobic patch, which may facilitate LPS binding. In Chapter 3 we describe the expression and purification of mouse CD14 as an additional target for ligand binding crystallization studies. After determining that purified human and mouse CD14 bind LPS and other ligands using native PAGE gel shift and ForteBio Octet assays, we confirmed the bioactivity of both purified proteins through cell-based stimulation assays. Additional sm…
author: Kelley, Stacy
language: en
keywords: Cluster of differentiation antigen 14 (CD14), Lipopolysaccharide (LPS), x-ray crystallography, soluble human CD14, structure of human CD14, Der p 2, yeast display of Der p 2, IgE detection using yeast display
date published: 2013-08-22T16:56:42Z
date modified: 2026-07-22T22:25:36Z
provider: University of Illinois - Urbana-Champaign
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of Illinois at Urbana-Champaign

PropertyValue
property ID: Handle
value: http://hdl.handle.net/2142/45650