Date of Award
Doctor of Philosophy
Biochemistry and Cellular and Molecular Biology
Nitin U. Jain
Gladys M. Alexandre, Michael D. Best, Engin H. Serpersu, Pamela L. Small
Endotoxin recognition by the innate immune receptor CD14 is a critical part of the innate immune system’s early detection and activation of the inflammatory response during microbial invasion. The differential recognition and high affinity binding of endotoxins from gram-negative and gram-positive bacteria is performed by the innate immune receptor CD14. Upon endotoxin binding, CD14 transfers the specific endotoxins to a Toll-like receptor signaling complex, which is responsible for initiating the intracellular signaling cascade. In the presence of overwhelming infection, the effects of CD14 lead to the over-activation of the inflammatory response, which results in the life threatening condition known as sepsis.
Preparation of a 15N isotopically labeled truncated version of soluble CD14, using Pichia pastoris, allowed direct structural observation of the binding interaction between CD14 and two endotoxin ligands, lipopolysaccharide (LPS) and lipoteichoic acid (LTA), from gram-negative and gram-positive bacteria, respectively using solution NMR spectroscopy. These studies revealed that CD14 uses both a common set of residues, and endotoxin specific subsets of residues, to bind LPS and LTA.
To further investigate the structural features of each endotoxin recognized by CD14, 13C 15N isotopically labeled Kdo2–Lipid A, a fully active chemically defined gram-negative endotoxin, and LTA lipid anchor, the minimal unit of LTA, were produced. This allowed detailed NMR spectral mapping of these agonist ligands bound to sCD14 which identified, for the first time, structural regions and features in each that are strongly affected during complex formation with sCD14.
Additionally, the presence of differential dynamic behavior was seen in both CD14 and the ligands upon complexation. This behavior suggests a likely role for dynamics in the mechanism of pattern recognition by CD14, which uses the dynamic ability of specific residue combinations to differentially affect endotoxin binding. Using NMR, the dynamic behavior of CD14 was further investigated using temperature and pH-dependence studies of isotopically labeled CD14. These studies clearly demonstrated the presence of multiple conformations for several residues, and may provide a possible explanation for the broad specificity of ligand binding by CD14.
In addition, the spin-labeling of isotopically labeled lipid A enabled the collection of intermolecular distances on CD14 bound lipid A.
Albright, Seth Andrew, "NMR Structural Studies of Endotoxin Receptor CD14 in Complex with Gram-Negative and Gram-Positive Endotoxin. " PhD diss., University of Tennessee, 2011.