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1.  Recombinant expression of TLR5 proteins by ligand supplementation and a leucine-rich repeat hybrid technique 
Biochemical and biophysical research communications  2012;427(1):10.1016/j.bbrc.2012.09.021.
Vertebrate TLR5 directly binds bacterial flagellin proteins and activates innate immune responses against pathogenic flagellated bacteria. Structural and biochemical studies on the TLR5/flagellin interaction have been challenging due to the technical difficulty in obtaining active recombinant proteins of TLR5 ectodomain (TLR5-ECD). We recently succeeded in production of the N-terminal leucine rich repeats (LRRs) of Danio rerio (dr) TLR5-ECD in a hybrid with another LRR protein, hagfish variable lymphocyte receptor (VLR), and determined the crystal structure of its complex with flagellin D1–D2–D3 domains. Although the structure provides valuable information about the interaction, it remains to be revealed how the C-terminal region of TLR5-ECD contributes to the interaction. Here, we present two methods to obtain recombinant TLR5 proteins that contain the C-terminal region in a baculovirus expression system. First, production of biologically active full-length drTLR5-ECD was substantially enhanced by supplementation of expression culture with purified flagellin proteins. Second, we designed TLR5-VLR hybrids using an LRR hybrid technology by single and double LRR fusions and were able to express diverse regions of drTLR5-ECD, allowing us to detect a previously unidentified TLR5/flagellin interaction. The drTLR5-VLR hybrid technique was also successfully applied to human TLR5-ECD whose expression has been highly problematic. These alternative TLR5 expression strategies provide an opportunity to obtain a complete view of the TLR5/flagellin interaction and can be applied to other LRR proteins.
doi:10.1016/j.bbrc.2012.09.021
PMCID: PMC3823237  PMID: 22989748
Toll-like receptor 5; Flagellin; Leucine-rich repeat; LRR hybrid; Variable lymphocyte receptor; Innate immunity
2.  Structure and functional characterization of the RNA-binding element of the NLRX1 innate immune modulator 
Immunity  2012;36(3):337-347.
SUMMARY
Mitochondrial NLRX1 is a member of the family of nucleotide-binding domain and leucine-rich-repeat–containing proteins (NLRs) that mediate host innate immunity as intracellular surveillance sensors against common molecular patterns of invading pathogens. NLRX1 functions in antiviral immunity, but the molecular mechanism of its ligand-induced activation is largely unknown. The crystal structure of the C-terminal fragment (residues 629-975) of human NLRX1 (cNLRX1) at 2.65 Å resolution reveals that cNLRX1 consists of an N-terminal helical (LRRNT) domain, central leucine-rich repeat modules (LRRM) and a C-terminal three-helix bundle (LRRCT). cNLRX1 assembles into a compact hexameric architecture that is stabilized by inter-subunit and inter-domain interactions of LRRNT and LRRCT in the trimer and dimer components of the hexamer, respectively. Furthermore, we find that cNLRX1 interacts directly with RNA and supports a role for NLRX1 in recognition of intracellular viral RNA in antiviral immunity.
doi:10.1016/j.immuni.2011.12.018
PMCID: PMC3368889  PMID: 22386589
3.  Structural basis of TLR5-flagellin recognition and signaling 
Science (New York, N.Y.)  2012;335(6070):859-864.
Toll-like receptor 5 (TLR5) binding to bacterial flagellin activates NF-κB signaling and triggers an innate immune response to the invading pathogen. To elucidate the structural basis and mechanistic implications of TLR5-flagellin recognition, we determined the crystal structure of zebrafish TLR5, as a VLR-hybrid protein, in complex with the D1/D2 fragment of Salmonella flagellin, FliC, at 2.47 Å resolution. TLR5 interacts primarily with the three helices of the FliC D1 domain using its lateral side. Two TLR5-FliC 1:1 heterodimers assemble into a 2:2 tail-to-tail signaling complex that is stabilized by quaternary contacts of the FliC D1 domain with the convex surface of the opposing TLR5. The proposed signaling mechanism is supported by structure-guided mutagenesis and deletion analysis on CBLB502, a therapeutic protein derived from FliC.
doi:10.1126/science.1215584
PMCID: PMC3406927  PMID: 22344444
4.  Synthesis and Toll-Like Receptor 4 (TLR4) Activity of Phosphatidylinositol Dimannoside Analogues 
Journal of Medicinal Chemistry  2011;54(20):7268-7279.
A series of five PIM2 analogues were synthesized and tested for their ability to activate primary macrophages and modulate LPS signaling. Structural changes included replacement of the fatty acid esters of the phosphatidyl moiety of PIM2 with the corresponding ether or amide. An AcPIM2 analogue possessing an ether linkage was also prepared. The synthetic methodology utilized an orthogonally protected chiral myo-inositol starting material that was conveniently prepared from myo-inositol in just two steps. Important steps in the synthetic protocols included the regio- and α-selective glycosylation of inositol O-6 and introduction of the phosphodiester utilizing phosphoramidite chemistry. Replacement of the inositol core with a glycerol moiety gave compounds described as phosphatidylglycerol dimannosides (PGM2). Biological testing of these PIM compounds indicated that the agonist activity was TLR4 dependent. An ether linkage increased agonist activity, removal of the inositol ring enhanced antagonist activity and the presence of an additional lipid chain enhanced LPS-induced cytokine production in primary macrophages. Furthermore, the interruption of the LPS-induced TLR4/MD-2 2:2 signaling complex formation by PIM2 represents a previously unidentified mechanism involved in the bioactivity of PIM molecules.
doi:10.1021/jm2008419
PMCID: PMC3280216  PMID: 21936536
5.  An unusual dimeric structure and assembly for RP105–MD-1, a regulator for the TLR4 response to LPS 
RP105–MD-1 modulates the TLR4–MD-2-mediated, innate immune response against bacterial lipopolysaccharide (LPS). The crystal structure of the bovine 1:1 RP105–MD-1 complex bound to a putative endogenous lipid at 2.9 Å resolution shares a similar overall architecture to its homologue TLR4–MD-2, but assembles into an unusual 2:2 homodimer which differs from any other known TLR-ligand assembly. The homodimer is assembled in a head-to-head orientation that juxtaposes the N-terminal leucine-rich repeats (LRRs) of the two RP105 chains, rather than the usual tail-to-tail configuration of C-terminal LRRs in ligand-activated TLR dimers, such as TLR1–2, 3 and 4. Another novel interaction is mediated by an RP105-specific Asn-linked glycan, which wedges MD-1 into the co-receptor binding concavity on RP105. This unique mode of assembly in RP105–MD-1 represents a new paradigm for TLR complexes and suggests a potential molecular mechanism for regulating LPS responses.
doi:10.1038/nsmb.2106
PMCID: PMC3362203  PMID: 21857663
6.  Structure and Mechanism of Receptor Sharing by the IL-10R2 Common Chain 
Summary
IL-10R2 is a shared cell surface receptor required for the activation of five class 2 cytokines (IL-10, IL-22, IL-26, IL-28, and IL-29) that play critical roles in host defense. To define the molecular mechanisms that regulate its promiscuous binding, we have determined the crystal structure of the IL-10R2 ecto-domain at 2.14Ǻ resolution. IL-10R2 residues required for binding were identified by alanine scanning and used to derive computational models of IL-10/IL-10R1/IL-10R2 and IL-22/IL-22R1/IL-10R2 ternary complexes. The models reveal a conserved binding epitope that is surrounded by two clefts that accommodate the structural and chemical diversity of the cytokines. These results provide a structural framework for interpreting IL-10R2 single nucleotide polymorphisms associated with human disease.
doi:10.1016/j.str.2010.02.009
PMCID: PMC2879597  PMID: 20462497
7.  Identification and Characterization of a +1 Frameshift Observed during the Expression of Epstein-Barr Virus IL-10 in Escherichia coli 
Epstein-Barr virus IL-10 (ebvIL-10) mimics the biological functions of cellular IL-10 including a number of immunoinhibitory activities on diverse immune cells. Characterization of ebvIL-10 and several mutants, expressed in Escherichia coli, by gel filtration chromatography and mass spectrometry revealed a +1 frameshift of ebvIL-10 expression. The frameshift is caused by the rare AGG codon at ebvIL-10 Arg159, which is preceded by the most inefficient stop signal, UGAC. The frameshift was corrected by substituting the rare AGG codon with an abundant arginine codon, CGU, or by enhancing the level of tRNA that decodes the AGG codon. As a result, ebvIL-10 expression levels increased by ~3-fold and the purity of the protein improved from 85–95% to 98–99%. The correction of the frameshift has been essential for continuing structural and biophysical studies of ebvIL-10.
doi:10.1016/j.pep.2006.12.001
PMCID: PMC1910691  PMID: 17224278

Results 1-7 (7)