An Evolutionary and Biochemical Approach to Understanding the Mechanism of Activation of Innate Immune Receptor TLR4/MD-2 with Exogenous Lipopolysaccharides and Endogenous S100A9

Abstract

The innate immune system is one of our first lines of defense against infection, and also recognizes and responds to internal tissue damage. TLR4 is an important innate immune receptor shared amongst vertebrates that performs both of these roles, recognizing lipopolysaccharides from Gram-negative bacteria as well the endogenous damage-associated protein S100A9. While TLR4 is widely studied in chronic inflammation, disease, and infection, many questions remain about the history and function of this receptor. In this work, I address two fundamental questions regarding TLR4: (1) How did TLR4 evolve species-specific recognition of different lipopolysaccharide structures, and (2) What is the binding mechanism of S100A9 and TLR4? I answer these questions combining evolutionary techniques such as ancestral sequence reconstruction and dN/dS, functional cell-based assays, mutagenesis, and in silico structure prediction and molecular dynamics simulations. The results of this work aim to uncover the evolutionary history of TLR4 specificity for microbial pathogens, as well as define a direct interaction between TLR4 and an endogenous protein which is implicated in many chronic inflammatory diseases that afflict human health.