Describe the mechanism by which TLR4, CD14, and MD2 recognize Lipopolysaccharides (LPS) and how this recognition changes gene expression in macrophages. What would happen if the TIR domain was mutated? Solution LPS is a main component of the outer membrane of Gram-negative bacteria. Minute amounts of lipopolysaccharides released from infecting pathogens can initiate potent innate immune responses. But when the Lipopolysaccharides response is not properly controlled it can lead to fatal septic shock syndrome. The common structural pattern of Lipopolysaccharides in various bacterial species is recognized by LPS binding protein (LBP), CD14 and the Toll-like receptor4 (TLR4)–MD-2 complex. Lipopolysaccharide is initially extracted from bacterial membranes and vesicles released from them by LPS binding protein in serum. LPS binding protein then transfers LPS to CD14 that can be found either in soluble form or linked to the cell surface by a glycosylphosphatidylinositol anchor. CD14 splits Lipopolysaccharides aggregates into monomeric molecules and presents them to the TLR4–MD-2 complex. Aggregation of the TLR4–MD-2 complex after binding lipopolysaccharide leads to activation of multiple signaling components like NF-B and IRF3, and the subsequent production of pro-inflammatory cytokines. CD14 exists in the form of a homodimer. Unlike other LRR family members, it does not contain an LRRCT module. Instead, the C-terminus of the LRR modules of one CD14 molecule interacts with the C-terminus of another, resulting in the formation of a dimer. The lipopolysaccharide interaction pocket of CD14 is located at the boundary of the LRRNT and the first LRR module. In addition to LPS, CD14 can bind to various other microbial products, such as peptidoglycan, lipoteichoic acid, lipoarabinomannan, and lipoproteins. Hence, it has broad ligand specificity. It functions as a pattern recognition receptor by recognizing structural motifs in various microbial products. The extracellular domain of TLR4 also belongs to the LRR family and is responsible for ligand binding. The structure of TLR4 deviates substantially from the consensus LRR structures. LRR family proteins can be classified into seven subfamilies and each subfamily is defined by its conformation. Most of the LRR proteins have uniform radii and beta sheet angles. Whereas, TLRs 1, 2, 4 and 6 are divided by a structural transition into N-terminal, central and C- terminal. The structural discontinuities seem to be caused by irregular LRR sequences in the center. The domain boundaries in TLRs 1, 2 and 6 fulfill important roles in ligand binding. The N-terminal and central domains of TLR4 clearly provide charge complementarity for binding of its surface to its co-receptor MD-2 thus, forming a stable 1:1 heterodimer. In the initial phase of infection, the innate immune system generates a rapid inflammatory response which blocks the growth of the infectious agent. Such response is followed, in vertebrates, by an adapt.

1. Describe the mechanism by which TLR4, CD14, and MD2 recognize Lipopolysaccharides (LPS)
and how this recognition changes gene expression in macrophages. What would happen if the
TIR domain was mutated?
Solution
LPS is a main component of the outer membrane of Gram-negative bacteria. Minute amounts of
lipopolysaccharides released from infecting pathogens can initiate potent innate immune
responses. But when the Lipopolysaccharides response is not properly controlled it can lead to
fatal septic shock syndrome. The common structural pattern of Lipopolysaccharides in various
bacterial species is recognized by LPS binding protein (LBP), CD14 and the Toll-like receptor4
(TLR4)–MD-2 complex. Lipopolysaccharide is initially extracted from bacterial membranes and
vesicles released from them by LPS binding protein in serum. LPS binding protein then transfers
LPS to CD14 that can be found either in soluble form or linked to the cell surface by a
glycosylphosphatidylinositol anchor. CD14 splits Lipopolysaccharides aggregates into
monomeric molecules and presents them to the TLR4–MD-2 complex. Aggregation of the
TLR4–MD-2 complex after binding lipopolysaccharide leads to activation of multiple signaling
components like NF-B and IRF3, and the subsequent production of pro-inflammatory cytokines.
CD14 exists in the form of a homodimer. Unlike other LRR family members, it does not contain
an LRRCT module. Instead, the C-terminus of the LRR modules of one CD14 molecule interacts
with the C-terminus of another, resulting in the formation of a dimer. The lipopolysaccharide
interaction pocket of CD14 is located at the boundary of the LRRNT and the first LRR module.
In addition to LPS, CD14 can bind to various other microbial products, such as peptidoglycan,
lipoteichoic acid, lipoarabinomannan, and lipoproteins. Hence, it has broad ligand specificity. It
functions as a pattern recognition receptor by recognizing structural motifs in various microbial
products. The extracellular domain of TLR4 also belongs to the LRR family and is responsible
for ligand binding. The structure of TLR4 deviates substantially from the consensus LRR
structures. LRR family proteins can be classified into seven subfamilies and each subfamily is
defined by its conformation. Most of the LRR proteins have uniform radii and beta sheet angles.
Whereas, TLRs 1, 2, 4 and 6 are divided by a structural transition into N-terminal, central and C-
terminal. The structural discontinuities seem to be caused by irregular LRR sequences in the
center. The domain boundaries in TLRs 1, 2 and 6 fulfill important roles in ligand binding. The
N-terminal and central domains of TLR4 clearly provide charge complementarity for binding of
its surface to its co-receptor MD-2 thus, forming a stable 1:1 heterodimer. In the initial phase of
infection, the innate immune system generates a rapid inflammatory response which blocks the
growth of the infectious agent. Such response is followed, in vertebrates, by an adaptive immune

2. response. The adaptive immune receptors are highly specific for the particular pathogen. Unlike
the adaptive response, the innate immune response mediated mainly by macrophages and
involves a limited number of germline-encoded pattern recognition receptors that recognize
common structural patterns among various molecules produced by microbes. If the TIR domain
was mutated, it leads to significantly reduced signaling activity of the receptors.