TLR4/MD-2 activation by a synthetic agonist with nosimilarity to LPSYing Wanga,1, Lijing Sub,1, Matthew D. Morinc,1, Brian T. Jonesc, Landon R. Whitbyc,2, Murali M. R. P. Surakattulac,Hua Huangd,3, Hexin Shia, Jin Huk Choia, Kuan-wen Wanga, Eva Marie Y. Morescoa, Michael Bergerd,4, Xiaoming Zhana,Hong Zhangb,5, Dale L. Bogerc,5, and Bruce Beutlera,5

aCenter for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390; bDepartment of Biophysics, University of TexasSouthwestern Medical Center, Dallas, TX 75390; cDepartment of Chemistry, The Scripps Research Institute, La Jolla, CA 92037; and dDepartment of Genetics,The Scripps Research Institute, La Jolla, CA 92037

Contributed by Bruce Beutler, December 30, 2015 (sent for review December 16, 2015; reviewed by Jean-Marc Reichhart and Stephen R. Sprang)

Structurally disparate molecules reportedly engage and activateToll-like receptor (TLR) 4 and other TLRs, yet the interactions thatmediate binding and activation by dissimilar ligands remain unknown.We describe Neoseptins, chemically synthesized peptidomimetics thatbear no structural similarity to the established TLR4 ligand, lipopoly-saccharide (LPS), but productively engage the mouse TLR4 (mTLR4)/myeloid differentiation factor 2 (MD-2) complex. Neoseptin-3 activatesmTLR4/MD-2 independently of CD14 and triggers canonical myeloiddifferentiation primary response gene 88 (MyD88)- and Toll-interleukin1 receptor (TIR) domain-containing adaptor inducing IFN-beta (TRIF)-dependent signaling. The crystal structure mTLR4/MD-2/Neoseptin-3at 2.57-Å resolution reveals that Neoseptin-3 binds as an asymmet-rical dimer within the hydrophobic pocket of MD-2, inducing anactive receptor complex similar to that induced by lipid A. However,Neoseptin-3 and lipid A form dissimilar molecular contacts toachieve receptor activation; hence strong TLR4/MD-2 agonistsneed not mimic LPS.

neoseptins | peptidomimetic compounds | innate immunity |proinflammatory response | crystal structure

Toll-like receptors (TLRs) are innate immune receptors thatserve as sensors of microbial molecules including lipopoly-

saccharide (LPS) or its precursor lipid A, lipopeptides, flagellin,and nucleic acids. In response to TLR engagement, rapid inductionof proinflammatory signaling ensues, beginning with myeloid dif-ferentiation primary response gene 88 (MyD88)- or Toll-interleukin1 receptor (TIR) domain-containing adaptor inducing IFN-beta(TRIF)-dependent recruitment of kinases and ubiquitin ligasesthat activate MAP kinases (MAPKs), NF-κB, and IFN regulatoryfactors (IRF) (1). These transcriptional regulators induce cyto-kines, chemokines, and costimulatory molecules that activate otherreceptors to promote the innate immune response.Numerous microbial ligands have been implicated as activators

of TLR4, TLR9, TLR2, and other TLRs (2). A wide variety ofmolecules of endogenous origin have also been reported to engageTLRs, particularly TLR4 and TLR2, and activate them in theabsence of microbial challenge (3–18). These latter reports haveengendered speculation that host-derived molecules, by directlystimulating TLRs, might sometimes trigger sterile inflammation(19). Concerns as to the purity and hence the true identity of theligands notwithstanding, the reported promiscuity of TLRs raisesquestions concerning the manner in which molecules structurallyunrelated to the bona fide microbial ligands might productivelyengage a signaling receptor.To address whether and how structurally disparate molecules

might trigger biological responses through known innate immunereceptors while minimizing the possibility of microbial ligand con-tamination, we screened a library of synthetic peptidomimeticcompounds (20) for stimulatory activity in primary mouse peri-toneal macrophage cultures, measuring tumor necrosis factor(TNF) secretion as an indicator of activation. Here we describe a

molecule identified through this screen and its interaction withthe TLR4/MD-2 complex of mice.

ResultsNeoseptin-3 Induces TNFα, IL-6, and IFN-β Production.Among ∼90,000compounds tested for their ability to activate TNFα biosynthesis inwild-type mouse peritoneal macrophages, we identified only two20-compound mixtures exhibiting weak stimulatory activity (Fig.1A). Individual compound testing of the strongest of these poolsassigned activity to a single molecule, termed Neoseptin-1.Chemical modification of Neoseptin-1 combined with struc-ture–activity relationship (SAR) studies produced structurally

Significance

The Toll-like receptor 4 (TLR4)/myeloid differentiation factor 2(MD-2) complex recognizes lipopolysaccharide (LPS) on Gram-negative bacteria to induce an innate immune response. Neo-septins, chemically synthesized peptidomimetics that bind andactivate the mouse TLR4 (mTLR4)/MD-2 complex independentof LPS, were discovered through unbiased screening and re-verse genetic studies, and improved by chemical modification.NMR and X-ray crystallography of the TLR4/MD-2/Neoseptin-3complex determined the mechanism by which Neoseptin-3 ac-tivates mTLR4/MD-2 and triggers myeloid differentiation pri-mary response gene 88- and Toll-interleukin 1 receptor domain-containing adaptor inducing IFN-beta-dependent signaling.Neoseptin-3 binds as a dimer within the hydrophobic pocket ofMD-2, contacting residues distinct from those contacted by LPSor lipid A, yet triggering a conformational change very similarto that elicited by LPS or lipid A. Natural peptides might con-ceivably produce similar effects.

Author contributions: Y.W., L.S., M.D.M., M.B., H.Z., D.L.B., and B.B. designed research;Y.W., L.S., M.D.M., B.T.J., L.R.W., M.M.R.P.S., H.H., H.S., J.H.C., K.-w.W., M.B., and X.Z.performed research; Y.W. and L.S. analyzed data; E.M.Y.M., H.Z., and B.B. wrote thepaper; B.T.J., L.R.W., M.M.R.P.S., H.S., J.H.C., K.-w.W., and X.Z. assisted with experiments;and H.H. and M.B. performed the compound library screen.

Reviewers: J.-M.R., The University of Strasbourg; and S.R.S., University of Montana.

The authors declare no conflict of interest.

Data deposition: The atomic coordinates and structure factors have been deposited in theProtein Data Bank, www.pdb.org [PDB ID codes 5HG3 (mTLR4/MD2), 5HG4 (mTLR4/MD2/Neoseptin-3), and 5HG6 (mTLR4/MD2/lipid A)].1Y.W., L.S., and M.D.M. contributed equally to this work.2Present address: Department of Chemical Physiology, The Scripps Research Institute, LaJolla, CA 92037.

3Present address: Department of Immunology and Microbial Sciences, The Scripps Re-search Institute, La Jolla, CA 92037.

4Present address: Lautenberg Center for Immunology and Cancer Research, The HebrewUniversity of Jerusalem, Ein Kerem, Jerusalem, 91120 Israel.

5To whom correspondence may be addressed. Email: Bruce.Beutler@UTSouthwestern.edu, boger@scripps.edu, or zhang@chop.swmed.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1525639113/-/DCSupplemental.

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simpler, much stronger and approximately equipotent agonists,respectively designated Neoseptin-3 and Neoseptin-4 (Fig. 1B).Neoseptin-3, which induced TNFα production by macrophages ina concentration-dependent manner (Fig. 1C), was selected formore detailed studies.Further SAR analysis indicated that few chemical substitutions

were compatible with retention of biological activity (Fig. S1).Even subtle modifications such as the substitution of fluorine forhydrogen at the para position of the phenyl ring, or transfer of

the amino group of the aniline ring to an adjacent position led toa dramatic loss of activity. However, some of the modified com-pounds could antagonize Neoseptin-3.In vitro dose–response experiments demonstrated an EC50 of

18.5 μM for Neoseptin-3. Despite lower potency, Neoseptin-3efficacy approximated that of LPS in promoting macrophageTNFα production (Fig. 1C). Neoseptin-3 also activated IL-6and IFN-β production in a dose-dependent manner (Fig. 1 Dand E). Responses to Neoseptin-3 were similar for mouse bone

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Fig. 1. Neoseptin-3 induces TNFα, IL-6, and IFN-β secretion in different mouse cells. (A) Screen of peptidomimetic molecules (400 wells, 20 compounds perwell; ref. 40) for stimulation of TNFα production by mouse peritoneal macrophages, a subset of the full set of compounds examined. (B) Chemical structures ofNeoseptin-1, -3, and -4. (C–E) TNFα (C), IL-6 (D), or IFN-β (E) in the supernatants of mouse peritoneal macrophages after treatment with Neoseptin-3 or LPS for4 h. (F and G) TNFα in the supernatants of mouse BMDM (F) or BMDC (G) after treatment with Neoseptin-3 for 4 h. In C–G, the means of triplicate samples areplotted; P values were determined by Student’s t test and represent the significance of differences between responses of unstimulated cells and stimulatedcells. Results in C–G are representative of two independent experiments (error bars represent SEM). *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

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marrow-derived macrophages (BMDM) and bone marrow-de-rived dendritic cells (BMDC) (Fig. 1 F and G). These dataindicate that Neoseptin-3 stimulates production of type I IFNand proinflammatory cytokines.

Neoseptin-3 Activates NF-κB, MAPK, and TANK-Binding Kinase 1Signaling. TLR signaling induces type I IFN and proinflammatorycytokine production dependent on NF-κB, MAPKs, and IRFs,and we evaluated these signaling pathways after Neoseptin-3stimulation of macrophages. Neoseptin-3 induced phosphorylationof IκB kinases α (IKKα), IKKβ, p38, c-Jun N-terminal kinase(JNK), and ERK, and degradation of IκBα, consistent with acti-vation of MAPK and canonical NF-κB signaling (Fig. 2A). TANK-binding kinase 1 (TBK1) and IRF3 phosphorylation also increasedin response to Neoseptin-3 (Fig. 2C). Notably, the time course ofthese signaling events was similar when activated by Neoseptin-3 orby LPS (Fig. 2 B and D).

Neoseptin-3 Targets the Mouse TLR4/MD-2 Complex. To determinethe molecular target of Neoseptin-3, we analyzed its effects onperitoneal macrophages from wild-type C57BL/6J mice and micedeficient in TLR signaling components. LPS served as a controland was compared with Neoseptin-3 in its ability to induce TNFαand IFN-β production (Fig. 3 A–D). Induction of TNFα byNeoseptin-3 was completely abrogated in TLR4- or MD-2–deficientmacrophages, and dramatically reduced in macrophages from

MyD88, TRIF, interleukin-1 receptor-associated kinase 4(IRAK4), and IKKγmutant mice (Fig. 3A). Only CD14-deficientmacrophages exhibited distinct responses to the two molecules,producing TNFα in response to Neoseptin-3 but not LPS. Nei-ther Neoseptin-3 nor LPS required TLR2, TLR3, TLR6, TLR7,or TLR9 to induce TNFα. Similarly, IFN-β production in re-sponse to either Neoseptin-3 or LPS was dependent on TLR4,MD-2, and TRIF (Fig. 3C), whereas LPS, but not Neoseptin-3,additionally required CD14 (Fig. 3D). These data suggested thatNeoseptin-3 targets the TLR4/MD-2 complex. Pretreatment withEritoran, a pharmacological antagonist of TLR4, blocked pro-duction of TNFα by macrophages stimulated with either LPS orNeoseptin-3 (Fig. 3E), strongly supporting the interpretationthat TLR4/MD-2 is the direct target of Neoseptin-3.In contrast to mouse macrophages, human macrophage-like cells

generated by phorbol 12-myristate 13-acetate (PMA) treatment ofthe THP-1 monocyte line failed to respond to Neoseptin-3 (Fig. 3F).Moreover, unlike lipid IVa, an antagonist of LPS in human cells (21,22), Neoseptin-3 failed to antagonize LPS stimulation of THP-1 cells(Fig. 3F). An NF-κB–dependent luciferase reporter in HEK293Tcells could only be activated by Neoseptin-3 when mouse TLR4(mTLR4)/mouse MD-2 (mMD-2) were coexpressed, whereas LPSactivated the reporter in cells coexpressing mTLR4/mMD-2 or hu-man TLR4/humanMD-2 (hMD-2) (Fig. 3G). These data suggest thatNeoseptin-3 specifically engages and activates mouse TLR4/MD-2,but cannot activate human TLR4/MD-2 or mixed heterodimers.

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Fig. 2. Neoseptin-3 activates NF-κB, MAPK, and TBK1. Mouse peritoneal macrophages were treated with Neoseptin-3 (A and C) or LPS (B and D). Lysateswere collected at the indicated times after treatment for immunoblot analysis with the indicated antibodies. Results are representative of two independentexperiments.

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Direct interaction between Neoseptin-3 and highly purified TLR4/MD-2 complexes was demonstrated in vitro by NMR spectroscopy.In Carr Purcell MeiboomGill (CPMG) experiments (23), Neoseptin-3alone showed a relaxation time greater than 100 ms, which was re-duced upon addition of mMD-2, hMD-2, or mTLR4/mMD-2, con-sistent with binding of Neoseptin-3 to h- or mMD-2 or the mTLR4/MD-2 complex (Fig. 4). We concluded that the biologically relevantmolecular target for Neoseptin-3 is the TLR4/MD-2 complex.

Two Neoseptin-3 Molecules Bind to the Hydrophobic Pocket of MD-2and Induce Agonistic Dimerization of Two mTLR4/MD-2 Complexes.We determined the crystal structure of the mTLR4/MD-2 complex in

apo form, in complex with lipid A, and in complex with Neoseptin-3(Table S1). The overall conformation of the 1:1 mTLR4/MD-2heterodimer is similar in all three structures [the root meansquare deviations (rmsd) between the Cα atoms of these struc-tures are <0.5 Å]. As expected, the apo form of mTLR4/MD-2is a monomeric 1:1 complex in solution and does not adopt theactive dimer conformation in the crystal. Both lipid A andNeoseptin-3 induce formation of a dimer consisting of twomTLR4/MD-2 heterodimers arranged symmetrically in an “m”

shape as observed in the previously reported structures of TLR4/MD-2 bound to LPS (24, 25). Despite completely differentchemical structures, Neoseptin-3 and lipid A induce similar local

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Fig. 3. Neoseptin-3 activates mouse TLR4/MD-2. (A–D) TNFα (A and B) or IFN-β (C and D) in the supernatants of mouse peritoneal macrophages of the in-dicated genotypes after treatment with Neoseptin-3 (A and C) or LPS (B and D) (n = 3 mice per genotype). Cytokine levels were normalized to those ofstimulated C57BL/6J cells. P values were determined by Student’s t test and represent the significance of differences between responses of stimulated C57BL/6J cells and stimulated cells of mutant genotypes; red bars indicate those with statistically significant differences. (E) TNFα in the supernatants of mouseperitoneal macrophages pretreated with the TLR4 antagonist Eritoran for 1 h, followed by addition of vehicle, Neoseptin-3 (25 μM), or LPS (1 ng/mL) foranother 4 h. (F) TNFα in the supernatants of PMA-differentiated human THP-1 cells pretreated with Neoseptin-3 for 1 h, followed by addition of vehicle or LPS(1 ng/mL) for another 4 h. (G) NF-κB–dependent luciferase activity in HEK293T cells transiently expressing mouse or human TLR4 plus mouse or human MD-2and stimulated with Neoseptin-3 (50 μM) or LPS (1 μg/mL). Data were normalized to luciferase activity measured in cells treated with vehicle. P values weredetermined by Student’s t test. In E–G, the means of triplicate samples are plotted. All results are representative of two independent experiments. In A–G,error bars represent SEM. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

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conformational changes in MD-2 around the bound ligand, and anearly identical dimerization interface between the two mTLR4/MD-2 heterodimers (Fig. S2). In particular, the MD-2 Phe126 loopregion undergoes a conformational change similar to that observedin the lipid A complexed structure (Fig. S2A). Similar conforma-tional changes occur in LPS- or lipid IVa-bound mouse TLR4/MD-2structures and in the LPS-bound human TLR4-MD-2 structure (24,25). By convention, we distinguish the second TLR4 and MD-2subunits of the active heterotetrameric complex with an asterisk.Unexpectedly, the electron density map of the mTLR4/MD-2/

Neoseptin-3 structure revealed that two Neoseptin-3 molecules,designated Neo-3A and Neo-3B, bound to each 1:1 mTLR4/MD-2heterodimer. The configurations of the two bound Neoseptin-3molecules were resolved unambiguously in the clear electrondensity map (Fig. 5A). The two Neoseptin-3 molecules are packedtightly against each other at the central aniline rings and amidebond regions (Fig. 5B). Two hydrogen bonds are also formedbetween the NH group of the amide bond of one Neoseptin-3molecule and the ester carbonyl group of the other (Fig. 5B).Each Neoseptin-3 molecule interacts with different regions of

MD-2 and TLR4* and adopts a distinct conformation. TheNeoseptin-3 molecules are bound to MD-2 close to the entranceof the hydrophobic pocket and facilitate the formation of theagonistic dimerization interface with mTLR4* (Fig. 5C). At thisdimerization interface, Neo-3A forms two specific hydrogen bondswith mTLR4*: one between the phenol hydroxyl group and theside chain of residue Ser439*, and the other between the aminogroup of the central aniline and the main chain carbonyl ofSer413* (Fig. 5D). Neo-3B forms one hydrogen bond between itsphenol hydroxyl group and Glu437* of mTLR4*, and anotherhydrogen bond between its amide carbonyl group and MD-2residue Arg90 (Fig. 5D). The phenol ring of Neo-3A is nearlycompletely buried and forms part of the hydrophobic core of thedimerization interface. The terminal phenol group of Neo-3B isalso an essential part of the active dimerization interface and issandwiched between the planar guanidinium groups of Arg434*of mTLR4* and Arg90 of MD-2. The resulting dimerizationinterface also includes two hydrogen bonds between MD-2Leu125 backbone atoms and the side chain of mTLR4* Asn415,as is observed in the crystal structures of all active TLR4/MD2dimers (24, 25).Structure-based site-directed mutagenesis of mTLR4 and mMD-2

confirmed the importance of specific interactions observed in themTLR4/MD-2/Neoseptin-3 complex (Fig. 5F and Fig. S3). Inparticular, mutations of mTLR4 Ser439 and Arg434 severelyreduced NF-κB–dependent reporter activation in response toNeoseptin-3. Although the mTLR4 Ser439Ala mutation did notaffect lipid A-induced activation significantly, the Arg434Alamutation reduced the response to lipid A by ∼30% (Fig. 5F).These results are consistent with the observation that mTLR4Ser439 and Arg434 interact intimately with Neoseptin-3 (Fig. 5D)

but do not have close contacts with lipid A (Fig. 5E). Mutations ofmTLR4 Glu437 and Lys263 unexpectedly increased responsivenessto Neoseptin-3, but did not affect lipid A responsiveness. Thesetwo residues form hydrogen bonds with Neoseptin-3 and lipid A,respectively (Fig. 5 D and E), but the mutagenesis data suggest thatthese hydrogen bonds might not be critical for ligand binding. Tworesidues, Asn415 of mTLR4 and Arg90 of mMD-2 are importantfor both Neoseptin-3 and lipid-A (Fig. 5 D and E), as mutations ofthese two residues essentially abolished the responsiveness to bothNeoseptin-3 and lipid A (Fig. 5F).

Neoseptin-3 Activates mTLR4/MD-2 Through a Structural MechanismDifferent From That of LPS. The different chemical structures andsizes of Neoseptin-3 and LPS (or lipid A) translate to distinctmodes of receptor binding. The t-butyl ester group and thebenzene ring of both Neoseptin-3 molecules reside within thehydrophobic pocket of MD-2, occupying less than half the totalvolume of the pocket (Fig. 6A). The t-butyl group of Neo-3B isdeeply buried and, together with the two terminal benzene ringsof Neo-3A and Neo-3B, forms many hydrophobic contacts withMD-2 (Fig. 5D). Comparison of the bound Neoseptin-3 and lipidA revealed that the t-butyl group of Neo-3B overlaps with the R2′′chain of lipid A, whereas the two terminal benzene rings fromthe two Neoseptin-3 molecules overlap with the R3 chains (Fig.6B); these groups anchor the two Neoseptin-3 molecules to MD-2near the dimerization interface. On the other hand, the twophenol rings of Neo-3A and Neo-3B are outside the hydrophobicpocket of MD-2 but become largely buried upon dimerizationwith mTLR4*, providing the core interface between MD-2 andmTLR4*. Whereas the Neo-3A phenol ring overlaps with thelipid A R2 chain, the Neo-3B phenol ring shows no overlap withlipid A. The phenol ring of Neo-3B is sandwiched between theside chains of mMD-2 Arg90 and mTLR4* Arg434*, creating akey contact area between MD-2 and TLR4* that is not present inthe TLR4/MD-2/lipid A complex. The sites occupied by theother three acyl chains (R3′, R2′, and R3′′) and the two phos-phoglucosamine moieties of lipid A remain vacant in the mTLR4/MD-2/Neoseptin-3 structure. Therefore, a number of hydrogenbonds between lipid A phosphoglucosamine moieties and TLR4/MD-2 amino acids are not present in the Neoseptin-3 complex.Overall, through electrostatic and hydrophobic interactions largelydistinct from those induced by LPS or lipid A, Neoseptin-3 in-duced an active 2:2 mTLR4/MD-2 dimer conformation virtuallyidentical to that induced by LPS or lipid A.

DiscussionUsing an unbiased approach, we identified a weak inducer ofmacrophage TNF biosynthesis, optimized it through SAR stud-ies, and identified it as a highly efficacious and specific agonistfor the mouse TLR4/MD-2 complex. NMR and crystallographicdata indicate that binding of the ligand, Neoseptin-3, modulates

Neo-3Neo-3+

Neo-3+ Protein A Protein AmTLR4/MD2Neo-3+

mMD2-protein ANeo-3+

hMD2-protein A

CPMG 100ms1.43 ppm

CPMG 100ms1.43 ppm

1H-NMR CPMG 100ms CPMG 100ms CPMG 100ms CPMG 100ms1.43 ppm 1.43 ppm 1.43 ppm 1.43 ppm 1.43 ppm

Fig. 4. NMR spectroscopy of Neoseptin-3 with mTLR4/MD-2. One-dimensional 1H-NMR spectra of the methyl regions of Neoseptin-3 alone, with mTLR4/MD-2,with mouse MD-2/protein A, or with human MD-2/protein A. Controls were Neoseptin-3 plus protein A and protein A alone. A CPMG sequence was applied for100 ms (CPMG 100ms) as indicated.

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the conformation of MD-2 and facilitates active TLR4/MD-2dimer formation, presumably initiating a transmembrane confor-mational change that triggers adapter recruitment and signaling.

Neoseptin-3 exhibits no structural similarity to lipid A, the activemoiety of LPS molecules. Nonetheless, it closely mimics the actionof lipid A, eliciting nearly the same conformational change in the

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Fig. 5. Structure of mTLR4/MD-2/Neoseptin-3 complex. (A) 2Fo-Fc electron density map of one Neoseptin-3 molecule (Neo-3A) in the complex. The contourlevel of the density is 1.0σ. (B) Stick (Left) and atomic sphere representations (Right) of Neo-3A and Neo-3B bound to mTLR4/MD-2 complex. (C) Orthogonalviews of the overall structure of mTLR4/MD-2/Neoseptin-3. (D) Enlarged view of the dimerization interface showing interactions of Neoseptin-3 with MD-2and mTLR4*. (E) Enlarged view of the dimerization interface showing interactions of lipid A with MD-2 and mTLR4*. In B, D, and E, dashed lines representhydrogen bonds. (F) NF-κB–dependent luciferase activity in HEK293T cells transiently expressing mTLR4 and mMD-2 bearing the indicated mutations andstimulated with Neoseptin-3 (50 μM) or lipid A (10 μg/mL). Data were normalized to luciferase activity measured in stimulated cells expressing wild-type (wt)mTLR4 and mMD-2. P values were determined by Student’s t test. P values represent the significance of differences between responses of cells expressing thetwo wild-type proteins versus cells expressing a given mutant protein and stimulated with the same ligand (blue asterisks); or the significance of differencesbetween responses to stimulation with lipid A versus Neoseptin-3 for cells expressing a given mutant protein (red asterisks). The means of triplicate samplesare plotted. Results are representative of two independent experiments (error bars represent SEM). *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

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receptor complex. This fact in itself suggests the likelihood thatnon-LPS ligands of natural origin, including polypeptides, mightbe capable of activating the TLR4/MD-2 complex, renderingLPS-mimetic effects without an LPS-like structure.Our structural data reveal how activation of mTLR4/MD-2 is

achieved by Neoseptin-3. Remarkably, two ligand molecules bindto the receptor in conjunction with one another in an asymmetricalmanner. To our knowledge, such a ligand binding mode is uniqueamong all ligand–protein interactions that have been character-ized so far. The Neoseptins are endowed with structural featuresthat promote their own noncovalent dimerization when bound tothe TLR4/MD-2 receptor complex, and structural features thatpromote an activating conformational change on the part of theTLR4/MD-2 receptor complex itself. We noticed that Neo-3Ahas a lower average crystallographic B-factor relative to Neo-3B(14.4 vs. 22.9 Å2), suggesting that Neo-3A may be more orderedand binds more tightly in the receptor complex. This may bear onthe temporal sequence of binding, with Neo-3A binding firstbefore Neo-3B and helping to stabilize Neo-3B through π-stackinginteractions as well as hydrogen bonding. The requirement forbinding of two Neoseptin-3 molecules along with an overallsmaller molecular size compared with lipid A may account for thefailure of Neoseptin-3 to antagonize LPS-mediated activation ofhuman TLR4/MD-2. Although our in vitro binding assay showsthat Neoseptin-3 binds to human MD-2 and possibly to the hu-man TLR4/MD-2 complex as well, we do not yet understandwhy Neoseptin-3 fails to elicit cytokine responses in THP-1cells. One possibility is that Neoseptin-3 fails to induce agonisticdimerization of human TLR4/MD-2 in a manner analogous tothat of lipid IVa (25, 26).

The crystal structure of the mTLR4/MD-2/Neoseptin-3 com-plex explains thoroughly the sensitivity to chemical substitutionsobserved during SAR analysis of Neoseptin-3 (Fig. S1). For ex-ample, substitutions at the amine group on the aniline ring, theamide carbonyl group, or the hydroxyl group on the phenol ringall resulted in a loss of activity because they abrogated hydrogenbonding between these groups of Neoseptin-3 and mTLR4/MD-2.Modification of the t-butyl ester group to an isopropyl, ethyl,methyl ester, or carboxylic acid resulted in progressive loss ofactivity. This observation may reflect the fact that the hydro-phobic cavity of MD-2 is sufficiently large to accommodate at-butyl group at this position and a smaller or polar group at thesame position would be much less favorable. Unlike lipid A,whose six acyl chains and a number of carbonyl groups fill thehydrophobic pocket of MD-2, the two Neoseptin-3 moleculesoccupy less than half of the hydrophobic pocket of MD-2. Nev-ertheless the interactions between Neoseptin-3 and MD-2, pri-marily hydrophobic but also including a specific hydrogen bondbetween Neo-3B and Arg90 of MD-2, are sufficiently strong andspecific to anchor the ligand near the entrance of the hydro-phobic pocket of MD-2 for dimerization with mTLR4*. Likelipid A, Neoseptin-3 does not merely induce conformationalchange of the receptor, but participates in creating the di-merization interface. However, the details of this interface differsubstantially for Neoseptin-3 versus lipid A at the atomic level.The interactions between Neoseptin-3 and mTLR4* are primarilymediated by the two phenol groups of the two Neoseptin-3 mol-ecules, through both hydrophobic and π-stacking interactions aswell as specific hydrogen bonds. In the case of LPS or lipid A, theinterface between the ligand and TLR4* is primarily mediated by

A

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Fig. 6. The different binding modes of Neoseptin-3 and lipid A to mTLR4/MD-2. (A) Stereoview of the bound Neoseptin-3 and lipid A within the hydrophobicpocket of MD-2 (gray) at the dimerization interface with mTLR4* (cyan). The MD-2 in the two complex structures have been superimposed to give the relativepositioning of the bound Neoseptin-3 and lipid A. The molecular surfaces of MD-2 and mTLR4* in the Neoseptin-3 complex are shown. Lipid A is shown as thinlines with carbon atoms colored green, whereas Neoseptin-3 molecules are shown as thick sticks. (B) Stereoview of Neoseptin-3 and lipid A as bound tomTLR4/MD-2 showing the overlapping of different chemical groups in the two molecules.

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the R2 acyl chain, and hydrogen bonds between TLR4* and theR2-OH and 1-PO4 groups.The acyl chains of LPS are believed to bind to the hydrophobic

pocket of CD14 (27), which aids in the delivery of LPS to TLR4/MD-2 (28, 29). The requirement of CD14 for cytokine responsesto LPS but not Neoseptin-3 is consistent with this view. It hasalso been proposed that CD14 prompts the internalization of theTLR4/MD-2 complex and thus permits activation of intracellularsignaling, including the activation of the TRIF/TRAM pathway(30). However, insofar as Neoseptin-3 is CD14 independent, thismodel might be reexamined. Indeed, recent reports similarlydocumented dissociation between CD14, TLR4 internalization,and TRIF-dependent signaling in response to stimulation withchemically synthesized substituted pyrimido[5,4-b]indoles or amouse monoclonal TLR4/MD-2 antibody (31–33).The determination of the crystal structures of two chemically

unrelated TLR4 agonists bound to TLR4/MD-2 revealed certainshared characteristics of such ligands. First, the agonist mustcontain groups capable of mediating the formation of the activatingdimerization interface between MD-2 and mTLR4*. Neoseptin-3and lipid A achieve this via distinct functional/chemical groups anddifferent ligand-protein contacts. Second, tight and specific in-teraction with the hydrophobic pocket of MD-2 is needed toanchor the ligand properly, though it is not necessary for theligand to fill the entire pocket of MD-2. These two aspectsshould be the main target areas for future development of noveland more potent ligands as potential modulators of innate im-munity. As for Neoseptin-3, further optimization will be basedon structure guided modification of Neo-3A and Neo-3B, andcovalently linked derivatives thereof.We identified two mutations in mTLR4 (Ser413Ala and

Glu437Ala) that enhance mTLR4/MD-2 responses to a ligandthat does not exist in nature. We infer that naturally occurringmutations of TLR4 or MD-2 could allow normally noninteract-ing or nonstimulatory molecules to become receptor agonists.Conversely, it is possible that mutations within host genes mightcreate “neo-ligands” for TLRs (34). Such mutations might generallybe disfavored, manifested as lethality or as autoinflammatory orautoimmune diseases. However, some peptides or other moleculesof endogenous origin might act as agonists for the TLR4/MD-2complex in conformity with the rules elaborated above, perhapshelping to initiate sterile inflammation to contribute to the repair ofdamaged tissues (35). As our results demonstrate, two peptido-mimetic molecules, each of a small molecular size (<500 Da),can jointly elicit activation of a TLR4/MD-2 complex. High-resolution structural characterization of proposed endogenousagonists, similar to those detailed here, will be essential to determinewhether and how they are capable of activating individual TLRs.

Materials and MethodsMice. C57BL/6J, Tlr2−/−, and Tlr3−/− mice were purchased from The JacksonLaboratory. Ly96−/− (MD-2−/−) mice were from RIKEN BioResource Center.Tlr4lps3/lps3, Tlr2lngd/lngd, Tlr6int/int, Tlr7rsq1/rsq1, Tlr9CpG3/CpG3, Cd14hdl/hdl,Myd88poc/poc, Ticam1Lps2/Lps2, Irak4otiose/otiose, and Ikbkgpanr2/Y mice weregenerated on a pure C57BL/6J background by ENU mutagenesis and aredescribed at mutagenetix.utsouthwestern.edu. All experimental proceduresusing mice were approved by the Institutional Animal Care and Use Com-mittee (IACUC) of the University of Texas Southwestern Medical Center, andwere conducted in accordance with institutionally approved protocols andguidelines for animal care and use. All of the mice were maintained at theUniversity of Texas Southwestern Medical Center in accordance with in-stitutionally approved protocols.

Synthesis of Neoseptin-3. Fisher esterification of 3-hydroxy-4-nitrobenzoicacid (cat. H2SO4, MeOH, reflux, 18 h, 99% yield) afforded methyl 3-hydroxy-4-nitrobenzoate, from which the aryl triflate was prepared (Tf2O, Et3N,CH2Cl2, 0 °C, 18 h, 79% yield). Sonogashira cross-coupling of the triflate with[(4-triisopropylsilyloxy)phenyl]acetylene (PdCl2(PPh3)2, CuI, Et3N/DMF, Bu4NI,70 °C), silyl ether cleavage, and concurrent methyl ester hydrolysis

(LiOH·H2O, THF/MeOH/H2O 4:1:1, 25 °C, 12 h, 81% yield over two steps) gave3-((4-hydroxyphenyl)ethynyl)-4-nitrobenzoic acid. Carbodiimide-mediated cou-pling of the benzoic acid with L-HoPhe-OtBu (EDCI·HCl, HOAt, 2,6-lutidine, DMF,25 °C, 12 h, 72% yield) provided (S)-tert-butyl 2-(3-((4-hydroxyphenyl)ethynyl)-4-nitrobenzamido)-4-phenylbutanoate. Alkyne hydrogenation with concurrent nitrogroup reduction via Pearlman’s catalyst (H2, Pd(OH)2/C, EtOAc, 25 °C, 12 h, 94%yield) yielded Neoseptin-3.

Isolation of Peritoneal Macrophages, BMDM, BMDC, and Cell Culture. Thio-glycollate-elicitedmacrophages were recovered 4 d after i.p. injection of 2mLof BBL thioglycollate medium, brewer modified [4% (wt/vol); BD Biosciences]by peritoneal lavage with 5 mL of PBS. The peritoneal macrophages werecultured in DMEM cell culture medium [DMEM containing 10% (vol/vol) FBS(Gemini Bio Products), 1% penicillin and streptomycin (Life Technologies)] at37 °C and 95% air/5% CO2. Murine BMDMs were collected by flushing bonemarrow cells from femurs and tibiae of mice. These cells were cultured for 7 din DMEM cell culture medium containing 10% (vol/vol) conditioned mediumfrom L929 cells. For BMDCs, bone morrow cells were cultured in Petri dishes in10 mL of DMEM cell culture medium containing 10 ng/mL murine GM-CSF(R&D Systems). On day 3 of culture, this was replaced with fresh GM-CSFmedium. Loosely adherent cells were transferred to a fresh Petri dish andcultured for an additional 4 d.

THP-1 (ATCC) cells were differentiated by treatment with 100 nM PMA(Sigma) in RPMI cell culture medium [RPMI containing 10% (vol/vol) FBS(Gemini Bio Products), 1% penicillin and streptomycin (Life Technologies)] for24 h. After that, cells were washed with PBS and cultured in fresh RPMI cellculture medium for 24 h before use in experiments. HEK293T cells (ATCC)were cultured in DMEM cell culture medium.

Measurement of Cytokine Production. Cells were seeded onto 96-well plates at1 × 105 cells per well and stimulated with Neoseptin-3 [dissolved in DMSO;final DMSO concentrations (≤0.2%) were kept constant in all experiments]or ultra-pure LPS (dissolved in H2O, Enzo Life Sciences) for 4 h. Mouse TNFα,IL-6, or IFN-β, or human TNFα in the supernatants were measured by ELISAkits according to the manufacturer’s instructions (eBioscience and PBL AssayScience). Pretreatment with Eritoran (Eisai) or Neoseptin-3 was for 1 h at theindicated concentrations. Unless otherwise indicated, mouse cells were fromwild-type C57BL/6J mice.

Luciferease Assay. HEK293T cells were transfected with an NF-κB–dependentluciferase reporter plasmid (Clontech) using Lipofectamine 2000 (Life Tech-nologies) and clones with stable expression were selected by culture inDMEM containing puromycin (Life Technologies). Cells were cotransfectedwith constructs for mouse or human TLR4 plus mouse or human MD-2, and2 d later were stimulated with 50 μM Neoseptin-3 or 1 μg/mL LPS for 6 h. Cellswere lysed, and luciferase activity was measured using the Steady-Glo Lucif-erase Assay System (Promega).

Constructs. cDNAs encoding TLR4 and MD-2 (human and mouse) were am-plified using standard PCR techniques and subsequently inserted intomammalian expression vector pcDNA3-C-V5 (V5 tag at the C terminus ofencoded protein) using the In-Fusion HD Cloning Kit (Clontech).

Mutagenesis. Point mutations were introduced into mTLR4 and mMD-2 bystandard site-directed mutagenesis. Luciferase assays were conducted asdescribed above except that stimulation was with 50 μM Neoseptin-3 or10 μg/mL lipid A for 6 h.

Western Blotting. Peritoneal macrophages (1 × 106 per well) were stimulatedin 12-well plates with Neoseptin-3 (50 μM) or LPS (5 ng/mL) for the indicatedtimes and lysed directly in sample buffer (Sigma). Cell lysates were separatedby SDS/PAGE and transferred to nitrocellulose membranes. Membranes wereprobed with the following antibodies: phospho-IKKα (Ser176)/IKKβ (Ser177),IκBα, phospho-p38 (Thr180/Tyr182), phospho-JNK (Thr183/Tyr185), phospho-ERK1/2 (Thr202/Tyr204), phospho-TBK1 (Ser172), phospho-IRF3 (Ser396) (CellSignaling Technology), and α-tubulin (Sigma).

Protein Expression, Purification, and Crystallization. The hybrid construct ofmouse TLR4 (residues 26–544) fused with hagfish variable lymphocyte re-ceptor (VLR) (residues 126–200) was cloned into plasmid pAcGP67a. Themouse MD-2 (residues 19–160) fused to a protein A tag was cloned into asecond pAcGP67a plasmid. The mTLR4VLR and MD-2-Protein A constructswere coexpressed in Hi5 insect cells (Invitrogen) and purified by IgG Sepharose(GE Healthcare) affinity chromatography. The eluted mTLR4VLR/MD-2-protein

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A was buffer exchanged to a buffer containing 20 mM Hepes pH 7.5, 40 mMNaCl. The protein A tag was removed by TEV cleavage and partially degly-cosylated using PNGase F (New England BioLabs) at room temperature for 3 hand then 4 °C overnight. The tag-free mTLR4VLR/MD-2 complex (termedmTLR4/MD-2 hereafter and in the main text as they are functionally in-terchangeable) was further purified by ion exchange (HiTrap Q) and gel fil-tration (Superdex 200 16/60) chromatography. The final protein buffercontained 25 mM Hepes pH 8.0, 75 mM NaCl (Buffer A). The purified mTLR4/MD-2 complex was concentrated to 24 mg/mL.

To reconstitute the mTLR4/M-D2/lipid A complex, 2 mg/mL mouse TLR4/MD-2 protein was incubated with 206 μM Escherichia coli lipid A (Re mutant)(Enzo Life Sciences) and 0.05% Triton X-100 at 37 °C for 2 h. The mTLR4/MD-2/lipid A complex was then purified by gel filtration chromatography(Superdex 200 16/60) in Buffer A and concentrated to 8 mg/mL. To re-constitute the mTLR4/MD-2/Neoseptin-3 complex, 2 mg/mL mTLR4/MD-2protein was incubated with 1 mM Neoseptin-3 dissolved in 20% (vol/vol)DMSO at room temperature for 4 h. The Neoseptin-3 precipitates werespun down. The complex was then buffer exchanged to Buffer A by a PD-10desalting column (GE Healthcare) to remove DMSO and excess Neoseptin-3.The mTLR4/MD-2/Neoseptin-3 complex was concentrated to 12 mg/mL.

mTLR4/MD-2 andmTLR4/MD-2/lipid A crystals were grownwith a hanging-drop vapor diffusion method by mixing 1 μL of protein with 1 μL of crys-tallization solution. The optimized crystallization conditions for each com-plex are summarized in Table S2. After 1 wk, the mTLR4/MD-2 and mTLR4/MD-2/lipid A crystals were flash-frozen in liquid nitrogen in different cryo-protectant solutions (Table S2). mTLR4/MD-2/Neoseptin-3 crystals weregrown with the same hanging-drop vapor diffusion method by first mixing1.5 μL of protein with 1.5 μL of crystallization solution (Table S2) and thenmicroseeded with mTLR4/MD-2/lipid A crystal seeds. The crystals appearedovernight and, after 10 d, the crystals were flash-frozen in liquid nitrogen incryoprotectant (Table S2).

Data Collection and Structure Determination. Diffraction data were collectedat Beamlines BM19 and ID19 of Advance Photon Source, Argonne NationalLaboratory. The datawere indexed, integrated, and scaled using theHKL3000package (36). The initial phases for the apo mTLR4/MD-2, mTLR4/MD-2/lipidA, and mTLR4/MD-2/Neoseptin-3 complexes were determined by the mo-lecular replacement method using the program PHASER (37). The publishedmouse TLR4/MD-2 structure [Protein Data Bank (PDB) ID code 2Z64] was usedas a search model for apo mTLR4/MD-2. Later the refined structure of apomTLR4/MD-2 was used as a search model for mTLR4/MD-2/lipid A andmTLR4/MD-2/Neoseptin-3 complex structure determinations. The electron

densities for Neoseptin-3 and lipid A were evident from the early stages ofrefinement and the atomic models of Neoseptin-3 and lipid A were built intothe electron density map unambiguously. The manual model building wasperformed with COOT (38) and the crystallographic refinement was per-formed with Refmac5 (39). The data collection and refinement statistics forall three structures are summarized in Table S1.

In Vitro Binding Assay of Neoseptin-3 to TLR4/MD-2 by NMR Spectroscopy. One-dimensional 1H-NMR spectra of the methyl regions of Neoseptin-3 wereacquired at 25 °C on a Varian INOVA 600 spectrometer equipped with a coldprobe. Neoseptin-3 (20 μM) in the absence and presence of 10 μM protein[mouse TLR4/MD-2 complex, mouse MD-2-protein A, human MD-2-proteinA, or protein A (MP Biomedicals)] or protein A alone was dissolved in abuffer containing 70% (wt/vol) PBS, 20% (wt/vol) D2O, 10% (wt/vol) d6-DMSO (Cambridge Isotope Laboratories). To each sample was applied a CarrPurcell Meiboom Gill (CPMG) sequence for 100 ms (CPMG 100ms). The CPMGsequence allows signal relaxation during the 100-ms delay and leads to al-most complete relaxation of the signal from large size molecules. The signalof Neoseptin-3 alone changed little during the 100-ms delay because of itsvery small size. Addition of large size proteins to Neoseptin-3 resulted incomplete loss of the signal indicating that Neoseptin-3 binds to the muchhigher molecular weight protein in solution.

Statistical Analyses. Data represent means ± SEM in all graphs depicting errorbars. The statistical significance of differences between experimental groupswas determined using GraphPad Prism 6 and the indicated statistical tests.P values are indicated by *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.P ≤ 0.05 was considered statistically significant.

ACKNOWLEDGMENTS. We thank Ian Wilson for the initial design of themouse TLR4VLR and MD2-protein A hybrid constructs for coexpression ofthe complex; Jose Rizo-Rey for help with the NMR experiments; DianaTomchick and Srinivasan Raghunathan for help with X-ray data collectionand structure determination; and Peter Jurek and Anne Murray forassistance with manuscript preparation. Results shown in this report arederived from work performed at Argonne National Laboratory, StructuralBiology Center at Advanced Photon Source. Argonne is operated by UChicagoArgonne, LLC, for the US Department of Energy, Office of Biological andEnvironmental Research under Contract DE-AC02-06CH11357. This work wassupported by NIH/National Institute of General Medical Sciences (NIGMS)Grant R01GM104496 (to H.Z.) and NIH/National Institute of Allergy and Infec-tious Diseases (NIAID) Grants U24 AI082657 (to B.B. and D.L.B.) and U19AI100627 (to B.B.).

1. Kawai T, Akira S (2010) The role of pattern-recognition receptors in innate immunity:

Update on Toll-like receptors. Nat Immunol 11(5):373–384.2. Uematsu S, Akira S (2008) Toll-like receptors and innate immunity. Handbook of

Experimewntal Pharmacology, eds Bauer S, Hartmann G (Springer, Berlin), Vol. 183,

pp 1–20.3. Roelofs MF, et al. (2006) Identification of small heat shock protein B8 (HSP22) as a

novel TLR4 ligand and potential involvement in the pathogenesis of rheumatoid ar-

thritis. J Immunol 176(11):7021–7027.4. Yu M, et al. (2006) HMGB1 signals through toll-like receptor (TLR) 4 and TLR2. Shock

26(2):174–179.5. Vabulas RM, et al. (2002) HSP70 as endogenous stimulus of the Toll/interleukin-1

receptor signal pathway. J Biol Chem 277(17):15107–15112.6. Vabulas RM, et al. (2002) The endoplasmic reticulum-resident heat shock protein

Gp96 activates dendritic cells via the Toll-like receptor 2/4 pathway. J Biol Chem

277(23):20847–20853.7. Vabulas RM, et al. (2001) Endocytosed HSP60s use toll-like receptor 2 (TLR2) and TLR4

to activate the toll/interleukin-1 receptor signaling pathway in innate immune cells.

J Biol Chem 276(33):31332–31339.8. Smiley ST, King JA, Hancock WW (2001) Fibrinogen stimulates macrophage chemo-

kine secretion through toll-like receptor 4. J Immunol 167(5):2887–2894.9. Termeer C, et al. (2002) Oligosaccharides of Hyaluronan activate dendritic cells via

toll-like receptor 4. J Exp Med 195(1):99–111.10. Zhang P, Cox CJ, Alvarez KM, Cunningham MW (2009) Cutting edge: Cardiac

myosin activates innate immune responses through TLRs. J Immunol 183(1):

27–31.11. Kim HS, et al. (2007) Toll-like receptor 2 senses beta-cell death and contributes to the

initiation of autoimmune diabetes. Immunity 27(2):321–333.12. Schaefer L, et al. (2005) The matrix component biglycan is proinflammatory and

signals through Toll-like receptors 4 and 2 in macrophages. J Clin Invest 115(8):

2223–2233.13. Vogl T, et al. (2007) Mrp8 and Mrp14 are endogenous activators of Toll-like receptor

4, promoting lethal, endotoxin-induced shock. Nat Med 13(9):1042–1049.14. Biragyn A, et al. (2002) Toll-like receptor 4-dependent activation of dendritic cells by

beta-defensin 2. Science 298(5595):1025–1029.

15. Johnson GB, Brunn GJ, Kodaira Y, Platt JL (2002) Receptor-mediated monitoring of

tissue well-being via detection of soluble heparan sulfate by Toll-like receptor 4.

J Immunol 168(10):5233–5239.16. Midwood K, et al. (2009) Tenascin-C is an endogenous activator of Toll-like receptor 4

that is essential for maintaining inflammation in arthritic joint disease. Nat Med 15(7):

774–780.17. Walton KA, et al. (2003) Receptors involved in the oxidized 1-palmitoyl-2-arachidonoyl-

sn-glycero-3-phosphorylcholine-mediated synthesis of interleukin-8. A role for Toll-

like receptor 4 and a glycosylphosphatidylinositol-anchored protein. J Biol Chem

278(32):29661–29666.18. Okamura Y, et al. (2001) The extra domain A of fibronectin activates toll-like receptor

4. J Biol Chem 276(13):10229–10233.19. Rifkin IR, Leadbetter EA, Busconi L, Viglianti G, Marshak-Rothstein A (2005) Toll-like

receptors, endogenous ligands, and systemic autoimmune disease. Immunol Rev 204:

27–42.20. Whitby LR, Boger DL (2012) Comprehensive peptidomimetic libraries targeting pro-

tein-protein interactions. Acc Chem Res 45(10):1698–1709.21. Saitoh S, et al. (2004) Lipid A antagonist, lipid IVa, is distinct from lipid A in interaction

with Toll-like receptor 4 (TLR4)-MD-2 and ligand-induced TLR4 oligomerization. Int

Immunol 16(7):961–969.22. Golenbock DT, Hampton RY, Qureshi N, Takayama K, Raetz CR (1991) Lipid A-like

molecules that antagonize the effects of endotoxins on human monocytes. J Biol

Chem 266(29):19490–19498.23. Rizo J, Chen X, Araç D (2006) Unraveling the mechanisms of synaptotagmin and

SNARE function in neurotransmitter release. Trends Cell Biol 16(7):339–350.24. Park BS, et al. (2009) The structural basis of lipopolysaccharide recognition by the

TLR4-MD-2 complex. Nature 458(7242):1191–1195.25. Ohto U, Fukase K, Miyake K, Shimizu T (2012) Structural basis of species-specific en-

dotoxin sensing by innate immune receptor TLR4/MD-2. Proc Natl Acad Sci USA

109(19):7421–7426.26. Ohto U, Fukase K, Miyake K, Satow Y (2007) Crystal structures of human MD-2 and its

complex with antiendotoxic lipid IVa. Science 316(5831):1632–1634.27. Kim JI, et al. (2005) Crystal structure of CD14 and its implications for lipopolysac-

charide signaling. J Biol Chem 280(12):11347–11351.

E892 | www.pnas.org/cgi/doi/10.1073/pnas.1525639113 Wang et al.

28. Jiang Q, Akashi S, Miyake K, Petty HR (2000) Lipopolysaccharide induces physical

proximity between CD14 and toll-like receptor 4 (TLR4) prior to nuclear translocation

of NF-kappa B. J Immunol 165(7):3541–3544.29. da Silva Correia J, Soldau K, Christen U, Tobias PS, Ulevitch RJ (2001) Lipopolysac-

charide is in close proximity to each of the proteins in its membrane receptor com-

plex. transfer from CD14 to TLR4 and MD-2. J Biol Chem 276(24):21129–21135.30. Zanoni I, et al. (2011) CD14 controls the LPS-induced endocytosis of Toll-like receptor

4. Cell 147(4):868–880.31. Rajaiah R, Perkins DJ, Ireland DD, Vogel SN (2015) CD14 dependence of TLR4 endo-

cytosis and TRIF signaling displays ligand specificity and is dissociable in endotoxin

tolerance. Proc Natl Acad Sci USA 112(27):8391–8396.32. Hayashi T, et al. (2014) Novel synthetic toll-like receptor 4/MD2 ligands attenuate

sterile inflammation. J Pharmacol Exp Ther 350(2):330–340.33. Chan M, et al. (2013) Identification of substituted pyrimido[5,4-b]indoles as selective

Toll-like receptor 4 ligands. J Med Chem 56(11):4206–4223.

34. Beutler B (2007) Neo-ligands for innate immune receptors and the etiology of sterileinflammatory disease. Immunol Rev 220(1):113–128.

35. Bianchi ME (2007) DAMPs, PAMPs and alarmins: All we need to know about danger.J Leukoc Biol 81(1):1–5.

36. Minor W, Cymborowski M, Otwinowski Z, Chruszcz M (2006) HKL-3000: The in-tegration of data reduction and structure solution–from diffraction images to aninitial model in minutes. Acta Crystallogr D Biol Crystallogr 62(Pt 8):859–866.

37. McCoy AJ, Grosse-Kunstleve RW, Storoni LC, Read RJ (2005) Likelihood-enhanced fasttranslation functions. Acta Crystallogr D Biol Crystallogr 61(Pt 4):458–464.

38. Emsley P, Cowtan K (2004) Coot: Model-building tools for molecular graphics. ActaCrystallogr D Biol Crystallogr 60(Pt 12 Pt 1):2126–2132.

39. Murshudov GN, Vagin AA, Dodson EJ (1997) Refinement of macromolecular struc-tures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr 53(Pt 3):240–255.

40. Shaginian A, et al. (2009) Design, synthesis, and evaluation of an alpha-helix mimeticlibrary targeting protein-protein interactions. J Am Chem Soc 131(15):5564–5572.

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