Structural and ligand binding studies of toll-like receptor 4 (TLR4) associated proteins
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 small gel filtration mixing studies (Ranoa, D.R., Kelley, S.L., et al. (2013). J.Biol.Chem. 288: 9729-9741) confirmed the ability of our purified human CD14 to shuttle triacylated ligands to the Toll-like receptor 2 complex. Chapter 3 also describes our efforts to produce a ligand bound structure of CD14. An additional project, described in Chapter 4, focuses on a predicted Toll-like receptor 4 accessory protein called Der p 2. Der p 2 is an aeroallergen from Dermatophagoides pteronyssinus house dust mites, which has been implicated as an MD-2 analogue. By binding LPS and TLR4 in place of MD-2, Der p 2 may activate pro-inflammatory signaling and stimulate IgE production and allergic reaction, most often associated with allergic asthma. In collaboration with the lab of David Kranz (University of Illinois, Urbana), we used yeast display to create Der p 2 and MD-2 full length Aga2 fusion proteins. After verifying yeast surface expression, we assessed protein folding by characterizing the ability of Der p 2 and MD-2 to bind anti-Derp2 and anti-MD-2 monoclonal antibodies, respectively, in thermal denaturation studies. Flow cytometry assays indicate that Der p 2 binds biotinylated and non-biotinylated forms of LPS without detectable binding to purified human TLR4. Clinically, we found that this system provides a novel assay that reliably and quantitatively detects anti-Der p 2 IgE in serum and plasma samples of atopic patients. Chapter 5 summarizes our findings, describes their significance, and suggests avenues for future work. Although a ligand bound structure of CD14 has not yet been determined, we have successfully determined the x-ray crystal structure of human CD14, which significantly enhances our understanding of the LPS binding pocket. The CD14 structure and our ligand binding crystallization efforts may foster additional ligand binding structural studies, virtual docking studies, and drug design efforts thus allowing therapeutic intervention at an early step in the signaling cascade. Additionally, we utilized yeast display and flow cytometry to detect binding of Der p 2 to LPS and created a novel system for the detection of anti-Der p 2 IgE antibodies in serum and plasma. Additional mutational analysis of Der p 2 could determine the contribution of individual amino acids to LPS binding and also natural epitopes of anti-Der p 2 IgE, thereby creating new targets for drug development to treat allergic asthma. In total, our efforts have advanced our understanding of host LPS detection during bacterial infection and allergy through structural and ligand binding studies of the TLR4 accessory proteins human CD14 and Der p 2.
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
University of Illinois - Urbana-Champaign
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University of Illinois at Urbana-Champaign