Correction for Wang Et Al., TLR4/MD-2 Activation by a Synthetic Agonist with No Similarity to LPS Downloaded by Guest on October 2, 2021 A

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IMMUNOLOGY AND INFLAMMATION Correction for “TLR4/MD-2 activation by a synthetic agonist with no similarity to LPS,” by Ying Wang, Lijing Su, Matthew D. Morin, Brian T. Jones, Landon R. Whitby, Murali M. R. P. Surakattula, Hua Huang, Hexin Shi, Jin Huk Choi, Kuan-wen Wang, Eva Marie Y. Moresco, Michael Berger, Xiaoming Zhan, Hong Zhang, Dale L. Boger, and Bruce Beutler, which was first published February 1, 2016; 10.1073/pnas.1525639113 (Proc. Natl. Acad. Sci. U.S.A. 113, E884–E893). Recently, Dr. Yibo Wang, Dr. Hongshuang Wang, and Pro- fessor Xiaohui Wang, from the Laboratory of Chemical Biology, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, brought to our attention that R-Neoseptin-3 (the in- active enantiomer) was modeled into the crystal structure of TLR4/MD-2/Neoseptin-3 obtained from co-crystallization of TLR4/MD-2 with the active enantiomer S-Neoseptin-3, published in PNAS. The electron density map does not have high enough resolution (2.57 Å) to distinguish between R- and S-enantiomers of Neoseptin-3. S-Neo-3A fits the density data as CORRECTION well as the modeled R-Neo-3A.S-Neo-3B fits the density data slightly better than R-Neo-3B at the phenyl group. The overall conformations of the two S-Neoseptin-3 molecules are ex- tremely similar to those of the two R-Neoseptin-3 molecules modeled in the structure (Fig. 5). The key interactions between S-Neoseptin-3 and TLR4/MD-2 are the same as those between the modeled R-Neoseptin-3 and TLR4/MD-2 (Fig. 6). Our conclusions as to how activation of TLR4/MD2 is induced by S-Neoseptin-3 have not changed. We have replaced the coordinates of the crystal structure of TLR4/MD-2/R-Neoseptin-3 with the coordinates of TLR4/MD2/S-Neoseptin-3 in Protein Data Bank under the same accession code 5IJC. The authors note that Figs. 5 and 6 in the main article and Fig. S2 in the SI Appendix appeared incorrectly. The corrected main figures and their legends appear below. The SI Appendix has been corrected online.

PNAS 2021 Vol. 118 No. 24 e2106360118 https://doi.org/10.1073/pnas.2106360118 | 1of3 Downloaded by guest on October 2, 2021 A B

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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 contour level 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) Orthogonal views of the overall structure of mTLR4/MD-2/Neoseptin-3. (D) Enlarged view of the dimerization interface showing interactions of Neoseptin-3 with MD-2 and 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 represent hydrogen bonds. (F)NF-κB–dependent luciferase activity in HEK293T cells transiently expressing mTLR4 and mMD-2 bearing the indicated mutations and stimulated 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 the two wild-type proteins versus cells expressing a given mutant protein and stimulated with the same ligand (blue asterisks); or the significance of differences between responses to stimulation with lipid A versus Neoseptin-3 for cells expressing a given mutant protein (red asterisks). The means of triplicate samples are plotted. Results are representative of two independent experiments (error bars represent SEM). *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

2of3 | PNAS https://doi.org/10.1073/pnas.2106360118 Correction for Wang et al., TLR4/MD-2 activation by a synthetic agonist with no similarity to LPS Downloaded by guest on October 2, 2021 A

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Published under the PNAS license. Published June 7, 2021.

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PNAS | 3of3 Correction for Wang et al., TLR4/MD-2 activation by a synthetic agonist with no similarity https://doi.org/10.1073/pnas.2106360118 to LPS Downloaded by guest on October 2, 2021 TLR4/MD-2 activation by a synthetic agonist with no similarity to LPS

Ying 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 Texas Southwestern 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 activate molecule identified through this screen and its interaction with Toll-like receptor (TLR) 4 and other TLRs, yet the interactions that the TLR4/MD-2 complex of mice. mediate binding and activation by dissimilar ligands remain unknown. We describe Neoseptins, chemically synthesized peptidomimetics that Results bear no structural similarity to the established TLR4 ligand, lipopoly- Neoseptin-3 Induces TNFα, IL-6, and IFN-β Production. Among ∼90,000 saccharide (LPS), but productively engage the mouse TLR4 (mTLR4)/ compounds tested for their ability to activate TNFα biosynthesis in myeloid differentiation factor 2 (MD-2) complex. Neoseptin-3 activates wild-type mouse peritoneal macrophages, we identified only two mTLR4/MD-2 independently of CD14 and triggers canonical myeloid 20-compound mixtures exhibiting weak stimulatory activity (Fig. differentiation primary response gene 88 (MyD88)- and Toll-interleukin 1A). Individual compound testing of the strongest of these pools 1 receptor (TIR) domain-containing adaptor inducing IFN-beta (TRIF)- dependent signaling. The crystal structure mTLR4/MD-2/Neoseptin-3 assigned activity to a single molecule, termed Neoseptin-1. at 2.57-Å resolution reveals that Neoseptin-3 binds as an asymmet- Chemical modification of Neoseptin-1 combined with struc- rical dimer within the hydrophobic pocket of MD-2, inducing an ture–activity relationship (SAR) studies produced structurally active receptor complex similar to that induced by lipid A. However, Neoseptin-3 and lipid A form dissimilar molecular contacts to Significance achieve receptor activation; hence strong TLR4/MD-2 agonists need not mimic LPS. The Toll-like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD-2) complex recognizes lipopolysaccharide (LPS) on Gram- neoseptins | peptidomimetic compounds | innate immunity | negative bacteria to induce an innate immune response. Neo- proinflammatory response | crystal structure septins, chemically synthesized peptidomimetics that bind and activate the mouse TLR4 (mTLR4)/MD-2 complex independent oll-like receptors (TLRs) are innate immune receptors that of LPS, were discovered through unbiased screening and re- Tserve as sensors of microbial molecules including lipopoly- verse genetic studies, and improved by chemical modification. saccharide (LPS) or its precursor lipid A, lipopeptides, flagellin, NMR and X-ray crystallography of the TLR4/MD-2/Neoseptin-3 and nucleic acids. In response to TLR engagement, rapid induction complex determined the mechanism by which Neoseptin-3 ac- of proinflammatory signaling ensues, beginning with myeloid dif- tivates mTLR4/MD-2 and triggers myeloid differentiation pri- ferentiation primary response gene 88 (MyD88)- or Toll-interleukin mary response gene 88- and Toll-interleukin 1 receptor domain- 1 receptor (TIR) domain-containing adaptor inducing IFN-beta containing adaptor inducing IFN-beta-dependent signaling. (TRIF)-dependent recruitment of kinases and ubiquitin ligases Neoseptin-3 binds as a dimer within the hydrophobic pocket of that activate MAP kinases (MAPKs), NF-κB, and IFN regulatory MD-2, contacting residues distinct from those contacted by LPS factors (IRF) (1). These transcriptional regulators induce cyto- or lipid A, yet triggering a conformational change very similar kines, chemokines, and costimulatory molecules that activate other to that elicited by LPS or lipid A. Natural peptides might con- receptors to promote the innate immune response. ceivably produce similar effects. Numerous microbial ligands have been implicated as activators of TLR4, TLR9, TLR2, and other TLRs (2). A wide variety of 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. molecules of endogenous origin have also been reported to engage performed research; Y.W. and L.S. analyzed data; E.M.Y.M., H.Z., and B.B. wrote the TLRs, particularly TLR4 and TLR2, and activate them in the paper; 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; absence of microbial challenge (3–18). These latter reports have and H.H. and M.B. performed the compound library screen. engendered speculation that host-derived molecules, by directly Reviewers: J.-M.R., The University of Strasbourg; and S.R.S., University of Montana. stimulating TLRs, might sometimes trigger sterile inflammation The authors declare no conflict of interest. (19). Concerns as to the purity and hence the true identity of the Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org [PDB ID codes 5HG3 (mTLR4/MD2), 5HG4 (mTLR4/MD2/ ligands notwithstanding, the reported promiscuity of TLRs raises Neoseptin-3), and 5HG6 (mTLR4/MD2/lipid A)]. questions concerning the manner in which molecules structurally 1Y.W., L.S., and M.D.M. contributed equally to this work. unrelated to the bona fide microbial ligands might productively 2Present address: Department of Chemical Physiology, The Scripps Research Institute, La engage a signaling receptor. Jolla, CA 92037. To address whether and how structurally disparate molecules 3Present address: Department of Immunology and Microbial Sciences, The Scripps Re- might trigger biological responses through known innate immune search Institute, La Jolla, CA 92037. receptors while minimizing the possibility of microbial ligand con- 4Present address: Lautenberg Center for Immunology and Cancer Research, The Hebrew tamination, we screened a library of synthetic peptidomimetic University of Jerusalem, Ein Kerem, Jerusalem, 91120 Israel. 5To whom correspondence may be addressed. Email: Bruce.Beutler@UTSouthwestern. compounds (20) for stimulatory activity in primary mouse peri- edu, [email protected], or [email protected]. toneal macrophage cultures, measuring tumor necrosis factor This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (TNF) secretion as an indicator of activation. Here we describe a 1073/pnas.1525639113/-/DCSupplemental.

E884–E893 | PNAS | Published online February 1, 2016 www.pnas.org/cgi/doi/10.1073/pnas.1525639113 A PNAS PLUS

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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 per well; ref. 40) for stimulation of TNFα production by mouse peritoneal macrophages, a subset of the full set of compounds examined. (B) Chemical structures of Neoseptin-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 for 4h.(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 are plotted; P values were determined by Student’s t test and represent the significance of differences between responses of unstimulated cells and stimulated cells. 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. INFLAMMATION

simpler, much stronger and approximately equipotent agonists, the amino group of the aniline ring to an adjacent position led to IMMUNOLOGY AND respectively designated Neoseptin-3 and Neoseptin-4 (Fig. 1B). a dramatic loss of activity. However, some of the modified com- Neoseptin-3, which induced TNFα production by macrophages in pounds could antagonize Neoseptin-3. a concentration-dependent manner (Fig. 1C), was selected for In vitro dose–response experiments demonstrated an EC50 of more detailed studies. 18.5 μM for Neoseptin-3. Despite lower potency, Neoseptin-3 Further SAR analysis indicated that few chemical substitutions efficacy approximated that of LPS in promoting macrophage were compatible with retention of biological activity (Fig. S1). TNFα production (Fig. 1C). Neoseptin-3 also activated IL-6 Even subtle modifications such as the substitution of fluorine for and IFN-β production in a dose-dependent manner (Fig. 1 D hydrogen at the para position of the phenyl ring, or transfer of and E). Responses to Neoseptin-3 were similar for mouse bone

Wang et al. PNAS | Published online February 1, 2016 | E885 AB

Fig. 2. Neoseptin-3 activates NF-κB, MAPK, and TBK1. Mouse peritoneal macrophages were treated with Neoseptin-3 (A and C)orLPS(B and D). Lysates were collected at the indicated times after treatment for immunoblot analysis with the indicated antibodies. Results are representative of two independent experiments. marrow-derived macrophages (BMDM) and bone marrow-de- MyD88, TRIF, interleukin-1 receptor-associated kinase 4 rived dendritic cells (BMDC) (Fig. 1 F and G). These data (IRAK4), and IKKγ mutant mice (Fig. 3A). Only CD14-deficient indicate that Neoseptin-3 stimulates production of type I IFN macrophages exhibited distinct responses to the two molecules, and proinflammatory cytokines. producing TNFα in response to Neoseptin-3 but not LPS. Nei- ther Neoseptin-3 nor LPS required TLR2, TLR3, TLR6, TLR7, Neoseptin-3 Activates NF-κB, MAPK, and TANK-Binding Kinase 1 or TLR9 to induce TNFα. Similarly, IFN-β production in re- Signaling. TLR signaling induces type I IFN and proinflammatory sponse to either Neoseptin-3 or LPS was dependent on TLR4, cytokine production dependent on NF-κB, MAPKs, and IRFs, MD-2, and TRIF (Fig. 3C), whereas LPS, but not Neoseptin-3, and we evaluated these signaling pathways after Neoseptin-3 additionally required CD14 (Fig. 3D). These data suggested that stimulation of macrophages. Neoseptin-3 induced phosphorylation Neoseptin-3 targets the TLR4/MD-2 complex. Pretreatment with of IκB kinases α (IKKα), IKKβ, p38, c-Jun N-terminal kinase Eritoran, a pharmacological antagonist of TLR4, blocked pro- (JNK), and ERK, and degradation of IκBα, consistent with acti- duction of TNFα by macrophages stimulated with either LPS or vation of MAPK and canonical NF-κB signaling (Fig. 2A). TANK- Neoseptin-3 (Fig. 3E), strongly supporting the interpretation binding kinase 1 (TBK1) and IRF3 phosphorylation also increased that TLR4/MD-2 is the direct target of Neoseptin-3. in response to Neoseptin-3 (Fig. 2C). Notably, the time course of In contrast to mouse macrophages, human macrophage-like cells these signaling events was similar when activated by Neoseptin-3 or generated by phorbol 12-myristate 13-acetate (PMA) treatment of by LPS (Fig. 2 B and D). the THP-1 monocyte line failed to respond to Neoseptin-3 (Fig. 3F). Moreover, unlike lipid IVa, an antagonist of LPS in human cells (21, Neoseptin-3 Targets the Mouse TLR4/MD-2 Complex. To determine 22), Neoseptin-3 failed to antagonize LPS stimulation of THP-1 cells the molecular target of Neoseptin-3, we analyzed its effects on (Fig. 3F). An NF-κB–dependent luciferase reporter in HEK293T peritoneal macrophages from wild-type C57BL/6J mice and mice cells could only be activated by Neoseptin-3 when mouse TLR4 deficient in TLR signaling components. LPS served as a control (mTLR4)/mouse MD-2 (mMD-2) were coexpressed, whereas LPS and was compared with Neoseptin-3 in its ability to induce TNFα activated the reporter in cells coexpressing mTLR4/mMD-2 or hu- and IFN-β production (Fig. 3 A–D). Induction of TNFα by man TLR4/human MD-2 (hMD-2) (Fig. 3G). These data suggest that Neoseptin-3 was completely abrogated in TLR4- or MD-2–deficient Neoseptin-3 specifically engages and activates mouse TLR4/MD-2, macrophages, and dramatically reduced in macrophages from but cannot activate human TLR4/MD-2 or mixed heterodimers.

E886 | www.pnas.org/cgi/doi/10.1073/pnas.1525639113 Wang et al. AB PNAS PLUS

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 indicated genotypes after treatment with Neoseptin-3 (A and C)orLPS(B and D)(n = 3 mice per genotype). Cytokine levels were normalized to those of stimulated 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 mouse peritoneal macrophages pretreated with the TLR4 antagonist Eritoran for 1 h, followed by addition of vehicle, Neoseptin-3 (25 μM), or LPS (1 ng/mL) for another 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-2 and 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 were determined 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.

Direct interaction between Neoseptin-3 and highly purified TLR4/ apo form, in complex with lipid A, and in complex with Neoseptin-3 MD-2 complexes was demonstrated in vitro by NMR spectroscopy. (Table S1). The overall conformation of the 1:1 mTLR4/MD-2 In Carr Purcell Meiboom Gill (CPMG) experiments (23), Neoseptin-3 heterodimer is similar in all three structures [the root mean alone showed a relaxation time greater than 100 ms, which was re- square deviations (rmsd) between the Cα atoms of these struc- INFLAMMATION IMMUNOLOGY AND duceduponadditionofmMD-2,hMD-2,ormTLR4/mMD-2,con- tures are <0.5 Å]. As expected, the apo form of mTLR4/MD-2 sistent with binding of Neoseptin-3 to h- or mMD-2 or the mTLR4/ is a monomeric 1:1 complex in solution and does not adopt the MD-2 complex (Fig. 4). We concluded that the biologically relevant active dimer conformation in the crystal. Both lipid A and molecular target for Neoseptin-3 is the TLR4/MD-2 complex. Neoseptin-3 induce formation of a dimer consisting of two mTLR4/MD-2 heterodimers arranged symmetrically in an “m” Two Neoseptin-3 Molecules Bind to the Hydrophobic Pocket of MD-2 shape as observed in the previously reported structures of TLR4/ and Induce Agonistic Dimerization of Two mTLR4/MD-2 Complexes. MD-2 bound to LPS (24, 25). Despite completely different We determined the crystal structure of the mTLR4/MD-2 complex in chemical structures, Neoseptin-3 and lipid A induce similar local

Wang et al. PNAS | Published online February 1, 2016 | E887 Neo-3+ Neo-3+ Neo-3+ Neo-3 mTLR4/MD2 mMD2-protein A hMD2-protein A Neo-3+ Protein A Protein A

1.43 ppm 1.43 ppm 1.43 ppm 1.43 ppm 1.43 ppm 1.43 ppm 1.43 ppm 1H-NMR CPMG 100ms CPMG 100ms CPMG 100ms CPMG 100ms CPMG 100ms CPMG 100ms

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 for 100 ms (CPMG 100ms) as indicated. conformational changes in MD-2 around the bound ligand, and a but do not have close contacts with lipid A (Fig. 5E). Mutations of nearly identical dimerization interface between the two mTLR4/ mTLR4 Glu437 and Lys263 unexpectedly increased responsiveness MD-2 heterodimers (Fig. S2). In particular, the MD-2 Phe126 loop to Neoseptin-3, but did not affect lipid A responsiveness. These region undergoes a conformational change similar to that observed two residues form hydrogen bonds with Neoseptin-3 and lipid A, in the lipid A complexed structure (Fig. S2A). Similar conforma- respectively (Fig. 5 D and E), but the mutagenesis data suggest that tionalchangesoccurinLPS-orlipidIVa-boundmouseTLR4/MD-2 these hydrogen bonds might not be critical for ligand binding. Two structures and in the LPS-bound human TLR4-MD-2 structure (24, residues, Asn415 of mTLR4 and Arg90 of mMD-2 are important 25). By convention, we distinguish the second TLR4 and MD-2 for both Neoseptin-3 and lipid-A (Fig. 5 D and E), as mutations of subunits of the active heterotetrameric complex with an asterisk. these two residues essentially abolished the responsiveness to both Unexpectedly, the electron density map of the mTLR4/MD-2/ Neoseptin-3 and lipid A (Fig. 5F). Neoseptin-3 structure revealed that two Neoseptin-3 molecules, Neoseptin-3 Activates mTLR4/MD-2 Through a Structural Mechanism designated Neo-3A and Neo-3B, bound to each 1:1 mTLR4/MD-2 heterodimer. The configurations of the two bound Neoseptin-3 Different From That of LPS. The different chemical structures and molecules were resolved unambiguously in the clear electron sizes of Neoseptin-3 and LPS (or lipid A) translate to distinct density map (Fig. 5A). The two Neoseptin-3 molecules are packed modes of receptor binding. The t-butyl ester group and the tightly against each other at the central aniline rings and amide benzene ring of both Neoseptin-3 molecules reside within the bond regions (Fig. 5B). Two hydrogen bonds are also formed hydrophobic pocket of MD-2, occupying less than half the total between the NH group of the amide bond of one Neoseptin-3 volume of the pocket (Fig. 6A). The t-butyl group of Neo-3B is molecule and the ester carbonyl group of the other (Fig. 5B). deeply buried and, together with the two terminal benzene rings Each Neoseptin-3 molecule interacts with different regions of of Neo-3A and Neo-3B, forms many hydrophobic contacts with MD-2 and TLR4* and adopts a distinct conformation. The MD-2 (Fig. 5D). Comparison of the bound Neoseptin-3 and lipid Neoseptin-3 molecules are bound to MD-2 close to the entrance A revealed that the t-butyl group of Neo-3B overlaps with the R2′′ of the hydrophobic pocket and facilitate the formation of the chain of lipid A, whereas the two terminal benzene rings from agonistic dimerization interface with mTLR4* (Fig. 5C). At this the two Neoseptin-3 molecules overlap with the R3 chains (Fig. B dimerization interface, Neo-3A forms two specific hydrogen bonds 6 ); these groups anchor the two Neoseptin-3 molecules to MD-2 with mTLR4*: one between the phenol hydroxyl group and the near the dimerization interface. On the other hand, the two side chain of residue Ser439*, and the other between the amino phenol rings of Neo-3A and Neo-3B are outside the hydrophobic group of the central aniline and the main chain carbonyl of pocket of MD-2 but become largely buried upon dimerization Ser413* (Fig. 5D). Neo-3B forms one hydrogen bond between its with mTLR4*, providing the core interface between MD-2 and phenol hydroxyl group and Glu437* of mTLR4*, and another mTLR4*. Whereas the Neo-3A phenol ring overlaps with the hydrogen bond between its amide carbonyl group and MD-2 lipid A R2 chain, the Neo-3B phenol ring shows no overlap with residue Arg90 (Fig. 5D). The phenol ring of Neo-3A is nearly lipid A. The phenol ring of Neo-3B is sandwiched between the completely buried and forms part of the hydrophobic core of the side chains of mMD-2 Arg90 and mTLR4* Arg434*, creating a dimerization interface. The terminal phenol group of Neo-3B is key contact area between MD-2 and TLR4* that is not present in also an essential part of the active dimerization interface and is the TLR4/MD-2/lipid A complex. The sites occupied by the sandwiched between the planar guanidinium groups of Arg434* other three acyl chains (R3′,R2′, and R3′′) and the two phos- of mTLR4* and Arg90 of MD-2. The resulting dimerization phoglucosamine moieties of lipid A remain vacant in the mTLR4/ interface also includes two hydrogen bonds between MD-2 MD-2/Neoseptin-3 structure. Therefore, a number of hydrogen Leu125 backbone atoms and the side chain of mTLR4* Asn415, bonds between lipid A phosphoglucosamine moieties and TLR4/ as is observed in the crystal structures of all active TLR4/MD2 MD-2 amino acids are not present in the Neoseptin-3 complex. dimers (24, 25). Overall, through electrostatic and hydrophobic interactions largely Structure-based site-directed mutagenesis of mTLR4 and mMD-2 distinct from those induced by LPS or lipid A, Neoseptin-3 in- confirmed the importance of specific interactions observed in the duced an active 2:2 mTLR4/MD-2 dimer conformation virtually mTLR4/MD-2/Neoseptin-3 complex (Fig. 5F and Fig. S3). In identical to that induced by LPS or lipid A. particular, mutations of mTLR4 Ser439 and Arg434 severely reduced NF-κB–dependent reporter activation in response to Discussion Neoseptin-3. Although the mTLR4 Ser439Ala mutation did not Using an unbiased approach, we identified a weak inducer of affect lipid A-induced activation significantly, the Arg434Ala macrophage TNF biosynthesis, optimized it through SAR stud- mutation reduced the response to lipid A by ∼30% (Fig. 5F). ies, and identified it as a highly efficacious and specific agonist These results are consistent with the observation that mTLR4 for the mouse TLR4/MD-2 complex. NMR and crystallographic Ser439 and Arg434 interact intimately with Neoseptin-3 (Fig. 5D) data indicate that binding of the ligand, Neoseptin-3, modulates

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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 contour level 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) Orthogonal views of the overall structure of mTLR4/MD-2/Neoseptin-3. (D) Enlarged view of the dimerization interface showing interactions of Neoseptin-3 with MD-2 and 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 represent hydrogen bonds. (F)NF-κB–dependent luciferase activity in HEK293T cells transiently expressing mTLR4 and mMD-2 bearing the indicated mutations and stimulated 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) INFLAMMATION IMMUNOLOGY AND 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 the two wild-type proteins versus cells expressing a given mutant protein and stimulated with the same ligand (blue asterisks); or the significance of differences between responses to stimulation with lipid A versus Neoseptin-3 for cells expressing a given mutant protein (red asterisks). The means of triplicate samples are plotted. Results are representative of two independent experiments (error bars represent SEM). *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. the conformation of MD-2 and facilitates active TLR4/MD-2 Neoseptin-3 exhibits no structural similarity to lipid A, the active dimer formation, presumably initiating a transmembrane confor- moiety of LPS molecules. Nonetheless, it closely mimics the action mational change that triggers adapter recruitment and signaling. of lipid A, eliciting nearly the same conformational change in the

Wang et al. PNAS | Published online February 1, 2016 | E889 A

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 hydrophobic pocket 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 relative positioning 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 thin lines with carbon atoms colored green, whereas Neoseptin-3 molecules are shown as thick sticks. (B) Stereoview of Neoseptin-3 and lipid A as bound to mTLR4/MD-2 showing the overlapping of different chemical groups in the two molecules. receptor complex. This fact in itself suggests the likelihood that The crystal structure of the mTLR4/MD-2/Neoseptin-3 com- non-LPS ligands of natural origin, including polypeptides, might plex explains thoroughly the sensitivity to chemical substitutions be capable of activating the TLR4/MD-2 complex, rendering observed during SAR analysis of Neoseptin-3 (Fig. S1). For ex- LPS-mimetic effects without an LPS-like structure. ample, substitutions at the amine group on the aniline ring, the Our structural data reveal how activation of mTLR4/MD-2 is amide carbonyl group, or the hydroxyl group on the phenol ring achieved by Neoseptin-3. Remarkably, two ligand molecules bind all resulted in a loss of activity because they abrogated hydrogen to the receptor in conjunction with one another in an asymmetrical bonding between these groups of Neoseptin-3 and mTLR4/MD-2. manner. To our knowledge, such a ligand binding mode is unique Modification of the t-butyl ester group to an isopropyl, ethyl, among all ligand–protein interactions that have been character- methyl ester, or carboxylic acid resulted in progressive loss of ized so far. The Neoseptins are endowed with structural features activity. This observation may reflect the fact that the hydro- that promote their own noncovalent dimerization when bound to phobic cavity of MD-2 is sufficiently large to accommodate a the TLR4/MD-2 receptor complex, and structural features that t-butyl group at this position and a smaller or polar group at the promote an activating conformational change on the part of the same position would be much less favorable. Unlike lipid A, TLR4/MD-2 receptor complex itself. We noticed that Neo-3A whose six acyl chains and a number of carbonyl groups fill the has a lower average crystallographic B-factor relative to Neo-3B hydrophobic pocket of MD-2, the two Neoseptin-3 molecules 2 (14.4 vs. 22.9 Å ), suggesting that Neo-3A may be more ordered occupy less than half of the hydrophobic pocket of MD-2. Nev- and binds more tightly in the receptor complex. This may bear on ertheless the interactions between Neoseptin-3 and MD-2, pri- the temporal sequence of binding, with Neo-3A binding first marily hydrophobic but also including a specific hydrogen bond π before Neo-3B and helping to stabilize Neo-3B through -stacking between Neo-3B and Arg90 of MD-2, are sufficiently strong and interactions as well as hydrogen bonding. The requirement for specific to anchor the ligand near the entrance of the hydro- binding of two Neoseptin-3 molecules along with an overall phobic pocket of MD-2 for dimerization with mTLR4*. Like smaller molecular size compared with lipid A may account for the lipid A, Neoseptin-3 does not merely induce conformational failure of Neoseptin-3 to antagonize LPS-mediated activation of change of the receptor, but participates in creating the di- human TLR4/MD-2. Although our in vitro binding assay shows merization interface. However, the details of this interface differ that Neoseptin-3 binds to human MD-2 and possibly to the hu- substantially for Neoseptin-3 versus lipid A at the atomic level. man TLR4/MD-2 complex as well, we do not yet understand The interactions between Neoseptin-3 and mTLR4* are primarily why Neoseptin-3 fails to elicit cytokine responses in THP-1 mediated by the two phenol groups of the two Neoseptin-3 mol- cells. One possibility is that Neoseptin-3 fails to induce agonistic ecules, through both hydrophobic and π-stacking interactions as dimerization of human TLR4/MD-2 in a manner analogous to well as specific hydrogen bonds. In the case of LPS or lipid A, the that of lipid IVa (25, 26). interface between the ligand and TLR4* is primarily mediated by

E890 | www.pnas.org/cgi/doi/10.1073/pnas.1525639113 Wang et al. · PNAS PLUS the R2 acyl chain, and hydrogen bonds between TLR4* and the (LiOH H2O, THF/MeOH/H2O 4:1:1, 25 °C, 12 h, 81% yield over two steps) gave 3-((4-hydroxyphenyl)ethynyl)-4-nitrobenzoic acid. Carbodiimide-mediated cou- R2-OH and 1-PO4 groups. The acyl chains of LPS are believed to bind to the hydrophobic pling of the benzoic acid with L-HoPhe-OtBu (EDCI·HCl, HOAt, 2,6-lutidine, DMF, pocket of CD14 (27), which aids in the delivery of LPS to TLR4/ 25 °C, 12 h, 72% yield) provided (S)-tert-butyl 2-(3-((4-hydroxyphenyl)ethynyl)-4- nitrobenzamido)-4-phenylbutanoate. Alkyne hydrogenation with concurrent nitro MD-2 (28, 29). The requirement of CD14 for cytokine responses group reduction via Pearlman’scatalyst(H2,Pd(OH)2/C, EtOAc, 25 °C, 12 h, 94% to LPS but not Neoseptin-3 is consistent with this view. It has yield) yielded Neoseptin-3. also been proposed that CD14 prompts the internalization of the TLR4/MD-2 complex and thus permits activation of intracellular Isolation of Peritoneal Macrophages, BMDM, BMDC, and Cell Culture. Thio- signaling, including the activation of the TRIF/TRAM pathway glycollate-elicited macrophages were recovered 4 d after i.p. injection of 2 mL (30). However, insofar as Neoseptin-3 is CD14 independent, this of BBL thioglycollate medium, brewer modified [4% (wt/vol); BD Biosciences] model might be reexamined. Indeed, recent reports similarly by peritoneal lavage with 5 mL of PBS. The peritoneal macrophages were documented dissociation between CD14, TLR4 internalization, cultured in DMEM cell culture medium [DMEM containing 10% (vol/vol) FBS and TRIF-dependent signaling in response to stimulation with (Gemini Bio Products), 1% penicillin and streptomycin (Life Technologies)] at chemically synthesized substituted pyrimido[5,4-b]indoles or a 37 °C and 95% air/5% CO2. Murine BMDMs were collected by flushing bone – marrow cells from femurs and tibiae of mice. These cells were cultured for 7 d mouse monoclonal TLR4/MD-2 antibody (31 33). in DMEM cell culture medium containing 10% (vol/vol) conditioned medium The determination of the crystal structures of two chemically from L929 cells. For BMDCs, bone morrow cells were cultured in Petri dishes in unrelated TLR4 agonists bound to TLR4/MD-2 revealed certain 10 mL of DMEM cell culture medium containing 10 ng/mL murine GM-CSF shared characteristics of such ligands. First, the agonist must (R&D Systems). On day 3 of culture, this was replaced with fresh GM-CSF contain groups capable of mediating the formation of the activating medium. Loosely adherent cells were transferred to a fresh Petri dish and dimerization interface between MD-2 and mTLR4*. Neoseptin-3 cultured for an additional 4 d. and lipid A achieve this via distinct functional/chemical groups and THP-1 (ATCC) cells were differentiated by treatment with 100 nM PMA different ligand-protein contacts. Second, tight and specific in- (Sigma) in RPMI cell culture medium [RPMI containing 10% (vol/vol) FBS teraction with the hydrophobic pocket of MD-2 is needed to (Gemini Bio Products), 1% penicillin and streptomycin (Life Technologies)] for anchor the ligand properly, though it is not necessary for the 24 h. After that, cells were washed with PBS and cultured in fresh RPMI cell culture medium for 24 h before use in experiments. HEK293T cells (ATCC) ligand to fill the entire pocket of MD-2. These two aspects were cultured in DMEM cell culture medium. should be the main target areas for future development of novel and more potent ligands as potential modulators of innate im- Measurement of Cytokine Production. Cells were seeded onto 96-well plates at munity. As for Neoseptin-3, further optimization will be based 1 × 105 cells per well and stimulated with Neoseptin-3 [dissolved in DMSO; on structure guided modification of Neo-3A and Neo-3B, and final DMSO concentrations (≤0.2%) were kept constant in all experiments] α covalently linked derivatives thereof. or ultra-pure LPS (dissolved in H2O, Enzo Life Sciences) for 4 h. Mouse TNF , We identified two mutations in mTLR4 (Ser413Ala and IL-6, or IFN-β, or human TNFα in the supernatants were measured by ELISA Glu437Ala) that enhance mTLR4/MD-2 responses to a ligand kits according to the manufacturer’s instructions (eBioscience and PBL Assay that does not exist in nature. We infer that naturally occurring Science). Pretreatment with Eritoran (Eisai) or Neoseptin-3 was for 1 h at the indicated concentrations. Unless otherwise indicated, mouse cells were from mutations of TLR4 or MD-2 could allow normally noninteract- wild-type C57BL/6J mice. ing or nonstimulatory molecules to become receptor agonists. Conversely, it is possible that mutations within host genes might Luciferease Assay. HEK293T cells were transfected with an NF-κB–dependent create “neo-ligands” for TLRs (34). Such mutations might generally luciferase reporter plasmid (Clontech) using Lipofectamine 2000 (Life Tech- be disfavored, manifested as lethality or as autoinflammatory or nologies) and clones with stable expression were selected by culture in autoimmune diseases. However, some peptides or other molecules DMEM containing puromycin (Life Technologies). Cells were cotransfected of endogenous origin might act as agonists for the TLR4/MD-2 with constructs for mouse or human TLR4 plus mouse or human MD-2, and complex in conformity with the rules elaborated above, perhaps 2 d later were stimulated with 50 μM Neoseptin-3 or 1 μg/mL LPS for 6 h. Cells helping to initiate sterile inflammation to contribute to the repair of were lysed, and luciferase activity was measured using the Steady-Glo Lucif- damaged tissues (35). As our results demonstrate, two peptido- erase Assay System (Promega). mimetic molecules, each of a small molecular size (<500 Da), Constructs. cDNAs encoding TLR4 and MD-2 (human and mouse) were am- can jointly elicit activation of a TLR4/MD-2 complex. High- plified using standard PCR techniques and subsequently inserted into resolution structural characterization of proposed endogenous mammalian expression vector pcDNA3-C-V5 (V5 tag at the C terminus of agonists, similar to those detailed here, will be essential to determine encoded protein) using the In-Fusion HD Cloning Kit (Clontech). whether and how they are capable of activating individual TLRs. Mutagenesis. Point mutations were introduced into mTLR4 and mMD-2 by Materials and Methods standard site-directed mutagenesis. Luciferase assays were conducted as Mice. C57BL/6J, Tlr2−/−, and Tlr3−/− mice were purchased from The Jackson described above except that stimulation was with 50 μMNeoseptin-3or − − − − Laboratory. Ly96 / (MD-2 / ) mice were from RIKEN BioResource Center. 10 μg/mL lipid A for 6 h. Tlr4lps3/lps3,Tlr2lngd/lngd,Tlr6int/int,Tlr7rsq1/rsq1,Tlr9CpG3/CpG3, Cd14hdl/hdl, Myd88poc/poc, Ticam1Lps2/Lps2, Irak4otiose/otiose, and Ikbkgpanr2/Y mice were Western Blotting. Peritoneal macrophages (1 × 106 per well) were stimulated generated on a pure C57BL/6J background by ENU mutagenesis and are in 12-well plates with Neoseptin-3 (50 μM) or LPS (5 ng/mL) for the indicated described at mutagenetix.utsouthwestern.edu. All experimental procedures times and lysed directly in sample buffer (Sigma). Cell lysates were separated using mice were approved by the Institutional Animal Care and Use Com- by SDS/PAGE and transferred to nitrocellulose membranes. Membranes were mittee (IACUC) of the University of Texas Southwestern Medical Center, and probed with the following antibodies: phospho-IKKα (Ser176)/IKKβ (Ser177), were conducted in accordance with institutionally approved protocols and IκBα, phospho-p38 (Thr180/Tyr182), phospho-JNK (Thr183/Tyr185), phospho- INFLAMMATION

guidelines for animal care and use. All of the mice were maintained at the ERK1/2 (Thr202/Tyr204), phospho-TBK1 (Ser172), phospho-IRF3 (Ser396) (Cell IMMUNOLOGY AND University of Texas Southwestern Medical Center in accordance with in- Signaling Technology), and α-tubulin (Sigma). stitutionally approved protocols. Protein Expression, Purification, and Crystallization. The hybrid construct of Synthesis of Neoseptin-3. Fisher esterification of 3-hydroxy-4-nitrobenzoic mouse TLR4 (residues 26–544) fused with hagfish variable lymphocyte re- acid (cat. H2SO4, MeOH, reflux, 18 h, 99% yield) afforded methyl 3-hydroxy- ceptor (VLR) (residues 126–200) was cloned into plasmid pAcGP67a. The 4-nitrobenzoate, from which the aryl triflate was prepared (Tf2O, Et3N, mouse MD-2 (residues 19–160) fused to a protein A tag was cloned into a CH2Cl2, 0 °C, 18 h, 79% yield). Sonogashira cross-coupling of the triflate with second pAcGP67a plasmid. The mTLR4VLR and MD-2-Protein A constructs [(4-triisopropylsilyloxy)phenyl]acetylene (PdCl2(PPh3)2, CuI, Et3N/DMF, Bu4NI, were coexpressed in Hi5 insect cells (Invitrogen) and purified by IgG Sepharose 70 °C), silyl ether cleavage, and concurrent methyl ester hydrolysis (GE Healthcare) affinity chromatography. The eluted mTLR4VLR/MD-2-protein

Wang et al. PNAS | Published online February 1, 2016 | E891 A was buffer exchanged to a buffer containing 20 mM Hepes pH 7.5, 40 mM densities for Neoseptin-3 and lipid A were evident from the early stages of NaCl. The protein A tag was removed by TEV cleavage and partially degly- refinement and the atomic models of Neoseptin-3 and lipid A were built into cosylated using PNGase F (New England BioLabs) at room temperature for 3 h the electron density map unambiguously. The manual model building was and then 4 °C overnight. The tag-free mTLR4VLR/MD-2 complex (termed performed with COOT (38) and the crystallographic refinement was per- mTLR4/MD-2 hereafter and in the main text as they are functionally in- formed with Refmac5 (39). The data collection and refinement statistics for terchangeable) was further purified by ion exchange (HiTrap Q) and gel fil- all three structures are summarized in Table S1. tration (Superdex 200 16/60) chromatography. The final protein buffer contained 25 mM Hepes pH 8.0, 75 mM NaCl (Buffer A). The purified mTLR4/ In Vitro Binding Assay of Neoseptin-3 to TLR4/MD-2 by NMR Spectroscopy. One- MD-2 complex was concentrated to 24 mg/mL. dimensional 1H-NMR spectra of the methyl regions of Neoseptin-3 were To reconstitute the mTLR4/M-D2/lipid A complex, 2 mg/mL mouse TLR4/ acquired at 25 °C on a Varian INOVA 600 spectrometer equipped with a cold MD-2 protein was incubated with 206 μM Escherichia coli lipid A (Re mutant) probe. Neoseptin-3 (20 μM) in the absence and presence of 10 μM protein (Enzo Life Sciences) and 0.05% Triton X-100 at 37 °C for 2 h. The mTLR4/MD-2/ [mouse TLR4/MD-2 complex, mouse MD-2-protein A, human MD-2-protein lipid A complex was then purified by gel filtration chromatography A, or protein A (MP Biomedicals)] or protein A alone was dissolved in a (Superdex 200 16/60) in Buffer A and concentrated to 8 mg/mL. To re- buffer containing 70% (wt/vol) PBS, 20% (wt/vol) D2O, 10% (wt/vol) d6- constitute the mTLR4/MD-2/Neoseptin-3 complex, 2 mg/mL mTLR4/MD-2 DMSO (Cambridge Isotope Laboratories). To each sample was applied a Carr protein was incubated with 1 mM Neoseptin-3 dissolved in 20% (vol/vol) Purcell Meiboom Gill (CPMG) sequence for 100 ms (CPMG 100ms). The CPMG DMSO at room temperature for 4 h. The Neoseptin-3 precipitates were sequence allows signal relaxation during the 100-ms delay and leads to al- spun down. The complex was then buffer exchanged to Buffer A by a PD-10 most complete relaxation of the signal from large size molecules. The signal desalting column (GE Healthcare) to remove DMSO and excess Neoseptin-3. of Neoseptin-3 alone changed little during the 100-ms delay because of its The mTLR4/MD-2/Neoseptin-3 complex was concentrated to 12 mg/mL. very small size. Addition of large size proteins to Neoseptin-3 resulted in mTLR4/MD-2 and mTLR4/MD-2/lipid A crystals were grown with a hanging- complete loss of the signal indicating that Neoseptin-3 binds to the much μ μ drop vapor diffusion method by mixing 1 L of protein with 1 L of crys- higher molecular weight protein in solution. tallization solution. The optimized crystallization conditions for each complex are summarized in Table S2. After 1 wk, the mTLR4/MD-2 and mTLR4/ Statistical Analyses. Data represent means ± SEM in all graphs depicting error MD-2/lipid A crystals were flash-frozen in liquid nitrogen in different cryo- bars. The statistical significance of differences between experimental groups protectant solutions (Table S2). mTLR4/MD-2/Neoseptin-3 crystals were was determined using GraphPad Prism 6 and the indicated statistical tests. grown with the same hanging-drop vapor diffusion method by first mixing P values are indicated by *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. 1.5 μL of protein with 1.5 μL of crystallization solution (Table S2) and then P ≤ 0.05 was considered statistically significant. microseeded with mTLR4/MD-2/lipid A crystal seeds. The crystals appeared overnight and, after 10 d, the crystals were flash-frozen in liquid nitrogen in ACKNOWLEDGMENTS. We thank Ian Wilson for the initial design of the cryoprotectant (Table S2). mouse TLR4VLR and MD2-protein A hybrid constructs for coexpression of the complex; Jose Rizo-Rey for help with the NMR experiments; Diana Data Collection and Structure Determination. Diffraction data were collected Tomchick and Srinivasan Raghunathan for help with X-ray data collection at Beamlines BM19 and ID19 of Advance Photon Source, Argonne National and structure determination; and Peter Jurek and Anne Murray for Laboratory. The data were indexed, integrated, and scaled using the HKL3000 assistance with manuscript preparation. Results shown in this report are package (36). The initial phases for the apo mTLR4/MD-2, mTLR4/MD-2/lipid derived from work performed at Argonne National Laboratory, Structural A, and mTLR4/MD-2/Neoseptin-3 complexes were determined by the mo- Biology Center at Advanced Photon Source. Argonne is operated by UChicago Argonne, LLC, for the US Department of Energy, Office of Biological and lecular replacement method using the program PHASER (37). The published Environmental Research under Contract DE-AC02-06CH11357. This work was mouse TLR4/MD-2 structure [Protein Data Bank (PDB) ID code 2Z64] was used supported by NIH/National Institute of General Medical Sciences (NIGMS) as a search model for apo mTLR4/MD-2. Later the refined structure of apo Grant R01GM104496 (to H.Z.) and NIH/National Institute of Allergy and Infec- mTLR4/MD-2 was used as a search model for mTLR4/MD-2/lipid A and tious Diseases (NIAID) Grants U24 AI082657 (to B.B. and D.L.B.) and U19 mTLR4/MD-2/Neoseptin-3 complex structure determinations. The electron AI100627 (to B.B.).

1. Kawai T, Akira S (2010) The role of pattern-recognition receptors in innate immunity: 15. Johnson GB, Brunn GJ, Kodaira Y, Platt JL (2002) Receptor-mediated monitoring of Update on Toll-like receptors. Nat Immunol 11(5):373–384. tissue well-being via detection of soluble heparan sulfate by Toll-like receptor 4. 2. Uematsu S, Akira S (2008) Toll-like receptors and innate immunity. Handbook of J Immunol 168(10):5233–5239. Experimewntal Pharmacology, eds Bauer S, Hartmann G (Springer, Berlin), Vol. 183, 16. Midwood K, et al. (2009) Tenascin-C is an endogenous activator of Toll-like receptor 4 pp 1–20. that is essential for maintaining inflammation in arthritic joint disease. Nat Med 15(7): 3. Roelofs MF, et al. (2006) Identification of small heat shock protein B8 (HSP22) as a 774–780. novel TLR4 ligand and potential involvement in the pathogenesis of rheumatoid ar- 17. Walton KA, et al. (2003) Receptors involved in the oxidized 1-palmitoyl-2-arachidonoyl- thritis. J Immunol 176(11):7021–7027. sn-glycero-3-phosphorylcholine-mediated synthesis of interleukin-8. A role for Toll- 4. Yu M, et al. (2006) HMGB1 signals through toll-like receptor (TLR) 4 and TLR2. Shock like receptor 4 and a glycosylphosphatidylinositol-anchored protein. J Biol Chem 26(2):174–179. 278(32):29661–29666. 5. Vabulas RM, et al. (2002) HSP70 as endogenous stimulus of the Toll/interleukin-1 18. Okamura Y, et al. (2001) The extra domain A of fibronectin activates toll-like receptor receptor signal pathway. J Biol Chem 277(17):15107–15112. 4. J Biol Chem 276(13):10229–10233. 6. Vabulas RM, et al. (2002) The endoplasmic reticulum-resident heat shock protein 19. Rifkin IR, Leadbetter EA, Busconi L, Viglianti G, Marshak-Rothstein A (2005) Toll-like Gp96 activates dendritic cells via the Toll-like receptor 2/4 pathway. J Biol Chem receptors, endogenous ligands, and systemic autoimmune disease. Immunol Rev 204: 277(23):20847–20853. 27–42. 7. Vabulas RM, et al. (2001) Endocytosed HSP60s use toll-like receptor 2 (TLR2) and TLR4 20. Whitby LR, Boger DL (2012) Comprehensive peptidomimetic libraries targeting pro- to activate the toll/interleukin-1 receptor signaling pathway in innate immune cells. tein-protein interactions. Acc Chem Res 45(10):1698–1709. J Biol Chem 276(33):31332–31339. 21. Saitoh S, et al. (2004) Lipid A antagonist, lipid IVa, is distinct from lipid A in interaction 8. Smiley ST, King JA, Hancock WW (2001) Fibrinogen stimulates macrophage chemo- with Toll-like receptor 4 (TLR4)-MD-2 and ligand-induced TLR4 oligomerization. Int kine secretion through toll-like receptor 4. J Immunol 167(5):2887–2894. Immunol 16(7):961–969. 9. Termeer C, et al. (2002) Oligosaccharides of Hyaluronan activate dendritic cells via 22. Golenbock DT, Hampton RY, Qureshi N, Takayama K, Raetz CR (1991) Lipid A-like toll-like receptor 4. J Exp Med 195(1):99–111. molecules that antagonize the effects of endotoxins on human monocytes. J Biol 10. Zhang P, Cox CJ, Alvarez KM, Cunningham MW (2009) Cutting edge: Cardiac Chem 266(29):19490–19498. myosin activates innate immune responses through TLRs. J Immunol 183(1): 23. Rizo J, Chen X, Araç D (2006) Unraveling the mechanisms of synaptotagmin and 27–31. SNARE function in neurotransmitter release. Trends Cell Biol 16(7):339–350. 11. Kim HS, et al. (2007) Toll-like receptor 2 senses beta-cell death and contributes to the 24. Park BS, et al. (2009) The structural basis of lipopolysaccharide recognition by the initiation of autoimmune diabetes. Immunity 27(2):321–333. TLR4-MD-2 complex. Nature 458(7242):1191–1195. 12. Schaefer L, et al. (2005) The matrix component biglycan is proinflammatory and 25. Ohto U, Fukase K, Miyake K, Shimizu T (2012) Structural basis of species-specific en- signals through Toll-like receptors 4 and 2 in macrophages. JClinInvest115(8): dotoxin sensing by innate immune receptor TLR4/MD-2. Proc Natl Acad Sci USA 2223–2233. 109(19):7421–7426. 13. Vogl T, et al. (2007) Mrp8 and Mrp14 are endogenous activators of Toll-like receptor 26. Ohto U, Fukase K, Miyake K, Satow Y (2007) Crystal structures of human MD-2 and its 4, promoting lethal, endotoxin-induced shock. Nat Med 13(9):1042–1049. complex with antiendotoxic lipid IVa. Science 316(5831):1632–1634. 14. Biragyn A, et al. (2002) Toll-like receptor 4-dependent activation of dendritic cells by 27. Kim JI, et al. (2005) Crystal structure of CD14 and its implications for lipopolysac- beta-defensin 2. Science 298(5595):1025–1029. 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 34. Beutler B (2007) Neo-ligands for innate immune receptors and the etiology of sterile PNAS PLUS proximity between CD14 and toll-like receptor 4 (TLR4) prior to nuclear translocation inflammatory disease. Immunol Rev 220(1):113–128. of NF-kappa B. J Immunol 165(7):3541–3544. 35. Bianchi ME (2007) DAMPs, PAMPs and alarmins: All we need to know about danger. – 29. da Silva Correia J, Soldau K, Christen U, Tobias PS, Ulevitch RJ (2001) Lipopolysac- J Leukoc Biol 81(1):1 5. charide is in close proximity to each of the proteins in its membrane receptor com- 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 an plex. transfer from CD14 to TLR4 and MD-2. J Biol Chem 276(24):21129–21135. initial model in minutes. Acta Crystallogr D Biol Crystallogr 62(Pt 8):859–866. 30. Zanoni I, et al. (2011) CD14 controls the LPS-induced endocytosis of Toll-like receptor 37. McCoy AJ, Grosse-Kunstleve RW, Storoni LC, Read RJ (2005) Likelihood-enhanced fast 4. Cell 147(4):868–880. translation functions. Acta Crystallogr D Biol Crystallogr 61(Pt 4):458–464. 31. Rajaiah R, Perkins DJ, Ireland DD, Vogel SN (2015) CD14 dependence of TLR4 endo- 38. Emsley P, Cowtan K (2004) Coot: Model-building tools for molecular graphics. Acta cytosis and TRIF signaling displays ligand specificity and is dissociable in endotoxin Crystallogr D Biol Crystallogr 60(Pt 12 Pt 1):2126–2132. – tolerance. Proc Natl Acad Sci USA 112(27):8391 8396. 39. Murshudov GN, Vagin AA, Dodson EJ (1997) Refinement of macromolecular struc- 32. Hayashi T, et al. (2014) Novel synthetic toll-like receptor 4/MD2 ligands attenuate tures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr 53(Pt 3): sterile inflammation. J Pharmacol Exp Ther 350(2):330–340. 240–255. 33. Chan M, et al. (2013) Identification of substituted pyrimido[5,4-b]indoles as selective 40. Shaginian A, et al. (2009) Design, synthesis, and evaluation of an alpha-helix mimetic Toll-like receptor 4 ligands. J Med Chem 56(11):4206–4223. library targeting protein-protein interactions. J Am Chem Soc 131(15):5564–5572. INFLAMMATION IMMUNOLOGY AND

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