3 research outputs found
Molecular Recognition of Muramyl Dipeptide Occurs in the Leucine-rich Repeat Domain of Nod2
Genetic mutations in the innate immune
receptor nucleotide-binding oligomerization domain-containing 2 (Nod2)
have demonstrated increased susceptibility to Crohn’s disease,
an inflammatory bowel disease that is hypothesized to be accompanied
by changes in the gut microbiota. Nod2 responds to the presence of
bacteria, specifically a fragment of the bacterial cell wall, muramyl
dipeptide (MDP). The proposed site of this interaction is the leucine-rich
repeat (LRR) domain. Surface plasmon resonance and molecular modeling
were used to investigate the interaction of the LRR domain with MDP.
A functional and pure LRR domain was obtained from Escherichia coli expression in high yield. The LRR
domain binds to MDP with high affinity, with a <i>K</i><sub>D</sub> of 212 ± 24 nM. Critical portions of the receptor were
determined by mutagenesis of putative binding residues. Fragment analysis
of MDP revealed that both the peptide and carbohydrate portion contribute
to the binding interaction
Molecular Recognition of Muramyl Dipeptide Occurs in the Leucine-rich Repeat Domain of Nod2
Genetic mutations in the innate immune
receptor nucleotide-binding oligomerization domain-containing 2 (Nod2)
have demonstrated increased susceptibility to Crohn’s disease,
an inflammatory bowel disease that is hypothesized to be accompanied
by changes in the gut microbiota. Nod2 responds to the presence of
bacteria, specifically a fragment of the bacterial cell wall, muramyl
dipeptide (MDP). The proposed site of this interaction is the leucine-rich
repeat (LRR) domain. Surface plasmon resonance and molecular modeling
were used to investigate the interaction of the LRR domain with MDP.
A functional and pure LRR domain was obtained from Escherichia coli expression in high yield. The LRR
domain binds to MDP with high affinity, with a <i>K</i><sub>D</sub> of 212 ± 24 nM. Critical portions of the receptor were
determined by mutagenesis of putative binding residues. Fragment analysis
of MDP revealed that both the peptide and carbohydrate portion contribute
to the binding interaction
Crohn’s Disease Variants of Nod2 Are Stabilized by the Critical Contact Region of Hsp70
Nod2
is a cytosolic, innate immune receptor responsible for binding
to bacterial cell wall fragments such as muramyl dipeptide (MDP).
Upon binding, subsequent downstream activation of the NF-ÎşB
pathway leads to an immune response. Nod2 mutations are correlated
with an increased susceptibility to Crohn’s disease (CD) and
ultimately result in a misregulated immune response. Previous work
had demonstrated that Nod2 interacts with and is stabilized by the
molecular chaperone Hsp70. In this work, it is shown using purified
protein and <i>in vitro</i> biochemical assays that the
critical Nod2 CD mutations (G908R, R702W, and 1007fs) preserve the
ability to bind bacterial ligands. A limited proteolysis assay and
luciferase reporter assay reveal regions of Hsp70 that are capable
of stabilizing Nod2 and rescuing CD mutant activity. A minimal 71-amino
acid subset of Hsp70 that stabilizes the CD-associated variants of
Nod2 and restores a proper immune response upon activation with MDP
was identified. This work suggests that CD-associated Nod2 variants
could be stabilized <i>in vivo</i> with a molecular chaperone