Title: Structural and Functional Evidence for the Role of the TLR2 DD Loop in TLR1/TLR2 Heterodimerization and Signaling
Abstract: The Toll/Interleukin-1 receptor (TIR) domain of the Toll-like receptors (TLRs) plays an important role in innate host defense signaling. The TIR-TIR platform formed by the dimerization of two TLRs promotes homotypic protein-protein interactions with additional cytoplasmic adapter molecules to form an active signaling complex resulting in the expression of pro- and anti-inflammatory cytokine genes. To generate a better understanding of the functional domains of TLR2 we performed a random mutagenesis analysis of the human TLR2 TIR domain and screened for TLR2/1 signaling-deficient mutants. Based upon the random mutagenesis results, we performed an alanine scanning mutagenesis of the TLR2 DD loop and part of the αD region. This resulted in the identification of four residues crucial for TLR2/1 signaling: Arg-748, Phe-749, Leu-752, and Arg-753. Computer-assisted energy minimization and docking studies indicated three regions of interaction in the TLR2/1 TIR-docked heterodimer. In Region I, residues Arg-748 and Phe-749 in TLR2 DD loop were involved in close contacts with Gly-676 in the TLR1 BB loop. Because this model suggested that steric hindrance would significantly alter the binding interactions between DD loop of TLR2 and BB loop of TLR1, Gly-676 in TLR1 was rationally mutated to Ala and Leu. As expected, in vitro functional studies involving TLR1 G676A and TLR1 G676L resulted in reduced PAM3CSK4 mediated NF-κB activation lending support to the computerized predictions. Additionally, mutation of an amino acid residue (TLR2 Asp-730) in Region II also resulted in decreased activity in agreement with our model, providing new insights into the structure-function relationship of TLR2/1 TIR domains. The Toll/Interleukin-1 receptor (TIR) domain of the Toll-like receptors (TLRs) plays an important role in innate host defense signaling. The TIR-TIR platform formed by the dimerization of two TLRs promotes homotypic protein-protein interactions with additional cytoplasmic adapter molecules to form an active signaling complex resulting in the expression of pro- and anti-inflammatory cytokine genes. To generate a better understanding of the functional domains of TLR2 we performed a random mutagenesis analysis of the human TLR2 TIR domain and screened for TLR2/1 signaling-deficient mutants. Based upon the random mutagenesis results, we performed an alanine scanning mutagenesis of the TLR2 DD loop and part of the αD region. This resulted in the identification of four residues crucial for TLR2/1 signaling: Arg-748, Phe-749, Leu-752, and Arg-753. Computer-assisted energy minimization and docking studies indicated three regions of interaction in the TLR2/1 TIR-docked heterodimer. In Region I, residues Arg-748 and Phe-749 in TLR2 DD loop were involved in close contacts with Gly-676 in the TLR1 BB loop. Because this model suggested that steric hindrance would significantly alter the binding interactions between DD loop of TLR2 and BB loop of TLR1, Gly-676 in TLR1 was rationally mutated to Ala and Leu. As expected, in vitro functional studies involving TLR1 G676A and TLR1 G676L resulted in reduced PAM3CSK4 mediated NF-κB activation lending support to the computerized predictions. Additionally, mutation of an amino acid residue (TLR2 Asp-730) in Region II also resulted in decreased activity in agreement with our model, providing new insights into the structure-function relationship of TLR2/1 TIR domains. The first line of defense against any invading microbe is provided by the Innate Immune system. Cells of the innate immune system sense and respond to microbial products via the Toll-like receptor family. Toll-like receptors (TLR) 2The abbreviations used are: TLR, toll-like receptor; araLAM, ara-lipoarabinomannan; PBS, phosphate-buffered saline; LPS, lipopolysaccharide; PAM3CSK4, tripalmitoyl cysteinyl lipopeptide; RM, random mutant; SAPK, stress-associated protein kinase; TIR, toll/IL-1R; WT, wild type; IL, interleukin; GFP, green fluorescent protein; HA, hemagglutinin; FACS, fluorescent-activated cell sorter; RT, reverse transcriptase; FITC, fluorescein isothiocyanate; RMS, root mean squared; MFI, mean fluorescence intensity; PDB, Protein Data Bank. 2The abbreviations used are: TLR, toll-like receptor; araLAM, ara-lipoarabinomannan; PBS, phosphate-buffered saline; LPS, lipopolysaccharide; PAM3CSK4, tripalmitoyl cysteinyl lipopeptide; RM, random mutant; SAPK, stress-associated protein kinase; TIR, toll/IL-1R; WT, wild type; IL, interleukin; GFP, green fluorescent protein; HA, hemagglutinin; FACS, fluorescent-activated cell sorter; RT, reverse transcriptase; FITC, fluorescein isothiocyanate; RMS, root mean squared; MFI, mean fluorescence intensity; PDB, Protein Data Bank. are an evolutionarily conserved family of cell surface molecules that participate in innate immune recognition of pathogen-associated molecular patterns (PAMPs) (1Medzhitov R. Janeway Jr., C.A. Cell. 1997; 91: 295-298Abstract Full Text Full Text PDF PubMed Scopus (1965) Google Scholar). To date 11 mammalian homologues of these receptors have been found (TLR1-11) (2Takeda K. Akira S. Int. Immunol. 2005; 17: 1-14Crossref PubMed Scopus (2700) Google Scholar) and individual members of the TLR family recognize a diverse array of microbial components. For example, the first human toll like receptor to be identified, TLR4, senses lipopolysaccharide (LPS) while TLR2 on the other hand senses diacylated or triacylated lipopeptides after heterodimerizing with either TLR6 or TLR1, respectively. The primary function of the TLRs is to alert the immune system to the presence of pathogenic microorganisms. Upon recognition of specific microbial components these receptors turn on a complex series of signaling events leading to the production of numerous immunologically important cytokines, chemokines, and effector molecules. Additionally, microbial products also induce the production of pro-inflammatory cytokines, such as IL-1, TNF-α, and IL-12 and the expression of co-stimulatory molecules on professional antigen presenting cells that are necessary for the activation of T and B cells. Thus, in addition to directly controlling the microbial infection, the innate immune response is also instructive to the adaptive immune response (2Takeda K. Akira S. Int. Immunol. 2005; 17: 1-14Crossref PubMed Scopus (2700) Google Scholar). The conserved cytoplasmic TIR (Toll/IL-1 receptor) domains of the IL-1 and Toll-like receptors are the critical focal point for the generation of ligand-induced cytoplasmic signaling cascades. It is generally believed that the TIR domains serve to promote homotypic protein-protein interactions between receptor chains and with additional cytoplasmic adapter molecules to form an active signaling complex. For signaling all the TLRs utilize one or more of the four known TIR-containing adaptor molecules: MyD88, TIRAP/MAL, TRIF, and TRAM (reviewed in Refs. 3O'Neill L.A. Fitzgerald K.A. Bowie A.G. Trends Immunol. 2003; 24: 286-290Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar and 4Vogel S.N. Fitzgerald K.A. Fenton M.J. Mol. Interv. 2003; 3: 466-477Crossref PubMed Scopus (203) Google Scholar). Despite the critical role of the TIR domain in coordinating the initial cytoplasmic signaling events relatively little is known regarding how homotypic TIR-TIR interactions are formed. Several studies have pointed to the important role of a conserved proline within the conserved "BB loop" as being critical for the generation of downstream signals. Indeed, mutation of this proline to a histidine in TLR4 is responsible for the loss of LPS-responsiveness in the C3H/HeJ mouse (5Poltorak A. He X. Smirnova I. Liu M.Y. Van Huffel C. Du X. Birdwell D. Alejos E. Silva M. Galanos C. Freudenberg M. Ricciardi-Castagnoli P. Layton B. Beutler B. Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6421) Google Scholar) and acts as a dominant negative mutant. Mutation of the conserved residue within TLR2 (Pro-681) was demonstrated to result in an inability to recruit MyD88 (6Xu Y. Tao X. Shen B. Horng T. Medzhitov R. Manley J.L. Tong L. Nature. 2000; 408: 111-115Crossref PubMed Scopus (229) Google Scholar) but surprisingly has no effect on the ability of TLR4 to bind MyD88 (7Dunne A. Ejdeback M. Ludidi P.L. O'neill L.A. Gay N.J. J. Biol. Chem. 2003; 278: 41443-41451Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). A peptidomimetic based on the BB loop of the MyD88 (8Bartfai T. Behrens M.M. Gaidarova S. Pemberton J. Shivanyuk A. Rebek Jr., J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7971-7976Crossref PubMed Scopus (109) Google Scholar) clearly showed the role of BB loop in signaling of a TIR domain protein (IL-1) as the mimic could block downstream signaling. Recently, Loiarro et al. (9Loiarro M. Sette C. Gallo G. Ciacci A. Fanto N. Mastroianni D. Carminati P. Ruggiero V. J. Biol. Chem. 2005; 280: 15809-15814Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar) reported that an eta-peptide derived from the BB-loop region of the MyD88 resulted in the inhibition of homodimerization of MyD88 and thereby signaling. Given the obvious importance of the TIR domain in the control and coordination of innate immune responses, it is surprising that, with the exception of the BB loop, there is a relative lack of information regarding functional domains within the cytoplasmic portion of Toll receptors that are essential for signaling activity. Potential clues as to the importance of several conserved amino acids within TIR domain were provided by Slack et al. (10Slack J.L. Schooley K. Bonnert T.P. Mitcham J.L. Qwarnstrom E.E. Sims J.E. Dower S.K. J. Biol. Chem. 2000; 275: 4670-4678Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar) who performed alanine scanning mutagenesis of a number of residues within the three conserved TIR domain boxes in the type I IL-1 receptor. Of the twelve individual mutations made, four resulted in decreased cell surface expression and of those four; only two could be demonstrated to have decreased abilities to signal for NF-κB or SAPK activation. Ronni et al. (11Ronni T. Agarwal V. Haykinson M. Haberland M.E. Cheng G. Smale S.T. Mol. Cell. Biol. 2003; 23: 2543-2555Crossref PubMed Scopus (49) Google Scholar) published a detailed alanine-scanning mutagenesis study of the TLR4 TIR domain which identified two structural surfaces that were required for TLR4-dependent signaling in macrophages. However, no information was provided regarding a molecular basis for the loss of function. Finally, Tao et al. (12Tao X. Xu Y. Zheng Y. Beg A.A. Tong L. Biochem. Biophys. Res. Commun. 2002; 299: 216-221Crossref PubMed Scopus (98) Google Scholar) have provided crystallographic data demonstrating in the crystal packing the TIR domains of C713S mutant of TLR2 formed asymmetric dimers and that the critical BB loop can adopt different conformations within the structure (PDB accession: 1O77). Interestingly, of the five chains in the crystallographic asymmetric unit, two molecules showed interaction between the αD helix and DD loops of one with the αB helix and BB loop of another. Besides packing forces, the interacting surface was also held together by an interchain S-S linkage, but the authors argued for a minimal role of the latter. In all the crystal and homology-modeled structures of the TIR domains, the DD loop is located on the opposite side of the BB loops (6Xu Y. Tao X. Shen B. Horng T. Medzhitov R. Manley J.L. Tong L. Nature. 2000; 408: 111-115Crossref PubMed Scopus (229) Google Scholar, 7Dunne A. Ejdeback M. Ludidi P.L. O'neill L.A. Gay N.J. J. Biol. Chem. 2003; 278: 41443-41451Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 12Tao X. Xu Y. Zheng Y. Beg A.A. Tong L. Biochem. Biophys. Res. Commun. 2002; 299: 216-221Crossref PubMed Scopus (98) Google Scholar). Because TLR2 is not functional as a homodimer and considering the conserved homology in the BB region among TLRs, this observation and supporting functional studies led them to suggest that TLR1 or TLR6 can form similar heterodimeric structures with TLR2. It is important to note here that Xu et al. (6Xu Y. Tao X. Shen B. Horng T. Medzhitov R. Manley J.L. Tong L. Nature. 2000; 408: 111-115Crossref PubMed Scopus (229) Google Scholar) reported that gel-filtration and dynamic light-scattering experiments carried out to understand the oligomerization state of the isolated TIR domains in solution indicated a low affinity for self-association of the TIR domains. Additionally, based on molecular modeling studies, Dunne et al. (7Dunne A. Ejdeback M. Ludidi P.L. O'neill L.A. Gay N.J. J. Biol. Chem. 2003; 278: 41443-41451Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar) predicted that the DD loops of adaptor molecules (Mal and MyD88) could be involved in molecular recognition and subsequent signaling. In summary, a clear understanding of the role of the DD loop of the TLR2 molecule in innate immunity is still elusive. In this study we report a structural and functional analysis of the TLR2 TIR domain and based on computational and experimental methods propose a model for how the TLR2 DD loop may interact with the TLR1 BB loop. pcDNA5 FRT/TO-TLR2 was generated by moving the TLR2 coding region as a HindIII and BamHI fragment from pcDNA3.1-TLR2 (13Carl V.S. Brown-Steinke K. Nicklin M.J. Smith Jr., M.F. J. Biol. Chem. 2002; 277: 17448-17456Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) to the same sites in pcDNA5FRT/TO. pDHA-TLR2 was generated by PCR amplifying the TLR2 gene from pcDNA3.1 TLR2 using primers with ApaI (5′-AAGGGCCCTCTCCAAGGAAGAATCCTCC-3′) and PstI (5′-AACTGCAGCTAGGACTTTATCGCAGCTC-3′) restriction enzyme sites at N and C terminus, respectively and ligating to the same sites in pDisplay HA mouse TLR6 (14Hajjar A.M. O'Mahony D.S. Ozinsky A. Underhill D.M. Aderem A. Klebanoff S.J. Wilson C.B. J. Immunol. 2001; 166: 15-19Crossref PubMed Scopus (416) Google Scholar) (a gift from D. Underhill, Institute for Systems Biology, Seattle, WA). Wild-type TLR1 plasmid (pFLAG-CMV-TLR1) was a gift from P. Tobias (The Scripps Research Institute, Dept of Immunology, La Jolla, CA). pFLAG-CMV2 human MyD88 was made by inserting the human MyD88 coding region into the HindIII and SmaI sites of pFLAG-CMV2. The human MyD88 coding region was PCR amplified using forward primer with HindIII (5′-TTATAAGCTTGCTGCAGGAGGTCCCGGCGC-3′) and a blunt-ended reverse primer (5′-AATTTCTAGATCAGGGCAGGGACAAGGCGTTG-3′). All the point mutations were made using QuikChange Site-directed Mutagenesis kit from Stratagene. Plasmids were verified by restriction mapping and sequencing (University of Virginia Biomolecular Research Facility, Charlottesville). The MIP-3α luciferase reporter plasmid was a gift from A. C. Keates (Harvard Medical School, Boston, MA), IL-8 luciferase was a gift from N. Mukaida (Kanazawa University, Japan), NF-κB luciferase and pEGFPN1 were obtained from Clontech (Mountain View, CA). PAM3CSK4 was obtained from EMC Microcollections (Tübingen, Germany). All other regents were obtained from Sigma. Random mutations were generated using GeneMorph PCR Mutagenesis kit (BD Biosciences). Using PCR primers Forward: (5′-TGATCCTGCTCACGGGGGTC-3′) and Reverse: BamHI (5′-AAGGATCCCTAGGACTTTATCGCAGCTCTCAG-3′) a 554-bp region of TLR2 containing the TIR domain was amplified under mutagenic conditions resulting in 1 to 3 random mutations per 500 bp. The resulting PCR product was digested with PpuMI and BamHI then cloned at the same sites in pcDNA5-TLR2. Individual clones were then screened for correct restriction pattern and tested for their ability to signal in response to PAM3CSK4 in HEK 293 transient transfection system. Clones resulting in reduced activity were sequenced using a TLR2 internal primer (5′-GCAAATTACCTGTGTGACTC-3′) to identify the mutations. Total RNA was purified using the TRIzol reagent (Invitrogen). RT of 0.5 μg of total cellular RNA was performed in a final volume of 20 μl containing 1× final first-strand buffer, 1 mm each dNTPs, 20 units of placental RNase inhibitor, 5 μm random hexamers, and 9 units of Moloney murine leukemia virus RT (Invitrogen). After incubation at 37 °C for 45 min, the samples were heated for 5 min at 92 °C to end the reaction and stored at -20 °C until PCR use. cDNA (2 μl) was subjected to real-time, quantitative PCR using the MJ Research Opticon system with SYBR Green I (Molecular Probes, Eugene, OR) as a fluorescent reporter. Duplicate PCR reactions were performed for each sample, and the average threshold cycle number was determined using the Opticon software. Levels of MIP-3α and IL-8 expression normalized to HGPRT levels were determined using the formula 2(Rt-Et), where Rt is the threshold cycle for the reference gene (HGPRT), and Et is the threshold cycle for the experimental gene (ΔΔCT method). Data are thus expressed as arbitrary units. Sequences of primers used: MIP-3α (F: 5′-CTGGCCAATGAAGGCTGTGA-3′,R:5′-ACCTCCAACCCCAGCAAGGT-3′), IL-8 (F: 5′-GGCAGCCTTCCTGATTTCTG-3′,R: 5′-GGGGTGGAAAGGTTTGGAAGT-3′) and hypoxanthine guanine phosphoribosyl transferase (HGPRT) (F: 5′-TTGGAAAGGGTGTTTATTCCTCA-3′,R:5′-TCCAGCAGGTACGCAAAGAA-3′). HEK 293 cell line was obtained from American Type Culture Collection and cultured in Dulbecco's modified Eagle's medium (Mediatech, Herndon, VA) +10% fetal bovine serum (Hyclone, Logan, UT). The HEK 293/FlpIn cell line which was engineered for use with the FlpIn recombinase system was purchased from Invitrogen and maintained in RPMI +10% fetal bovine serum plus Zeocin (Invitrogen) as recommended. HEK 293 cell line was transfected using Lipofectamine 2000 (Invitrogen) per manufacturer's recommendations. Stable Cell Lines of pDHA-TLR2 clones were made by transfecting HEK 293 cell lines with respective DNA using Lipo-fectamine 2000 and selecting with G418. For making stable cell lines of pcDNA5/FRT/TO-TLR2 clones, the HEK 293/FlpIn cells were transfected in the same way as described above with the respective pcDNA5-TLR2 DNA along with recombinase expressing plasmid pOG44 (Invitrogen) in 1:9 ratio. Hygromycin-B (Invitrogen)-resistant clones were picked and screened for surface expression with FACS. To confirm the surface expression of WT or mutant TLR2 on stable cell lines, cells were washed with cold PBS, blocked with cold 0.1% bovine serum albumin in PBS and stained with PE-conjugated monoclonal antibody against TLR2 (TLR2.1 from eBiosciences, San Diego, CA) for 20 min at room temperature. After a wash with cold 0.1% bovine serum albumin in PBS cells were analyzed by FACS along with positive (WT-TLR2) and negative control (HEK 293 cells) using FACS Calibur (Becton Dickinson) flowcytometer and FLOWJO software. Procedure for checking the surface expression of the clones using transient transfection was similar except that pEGFPN1 plasmid (Clontech) was cotransfected with the TLR2 mutant or WT TLR2 plasmids (3:1 ratio) into HEK 293 cells using Lipofectamine 2000. Cells were harvested after 36 h, washed with PBS, blocked, and stained as described above. FACS was done by gating on GFP-positive cells. Mean fluorescence intensity (MFI) for the PE channel was calculated for the GFP-positive cells. HEK 293T cells (106) in a 10-cm tissue culture plate were transfected with the indicated plasmid DNA (10 μg each) using Lipofectamine 2000. 48 h after transfection cells were washed once with ice-cold 1× PBS (Mediatech, Inc., Herndon VA) and lysed in ice-cold lysis buffer (10 mm Tris, pH 7.5, 150 mm NaCl, 1mm EDTA, pH 8.0, 1% v/v Nonidet P-40, and 10% v/v glycerol), this buffer was supplemented before use with 1× (final) Halt protease inhibitor (PI) mixture (Pierce). To remove cell debris lysates were centrifuged at 12,000 × g for 15 min. at 4 °C. Clear lysates were put in a rotary mixer at 4 °C with 4 μg of a rabbit polyclonal antibody against the HA epitope tag (Y-11; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 2 h followed by the addition of 20 μl of protein A-Sepharose bead slurry (Pierce). After washing in lysis buffer, the beads were suspended in 2× SDS gel loading dye, heated at 95 °C for 10 min, followed by centrifugation at 12,000 × g for 15 min. Samples and dual color Precision plus Protein standards (Bio-Rad) were run on 10% PAGE and transferred to nitrocellulose membrane. HA-tagged TLR2 was detected using anti-HA (262K) mouse monoclonal antibody (Cell Signaling Technologies, Inc., Danvers, MA) and FLAG-MyD88 with anti-FLAG M2 mouse monoclonal antibody (Sigma). Secondary antibody was horse-radish peroxidase-conjugated anti-mouse IgG (Cell Signaling Technologies, Inc.). Blots were developed using ECL plus Western blotting detection system, scanned and analyzed by Storm 840 and using software ImageQuant 5.2, all from Amersham Biosciences (GE Health Care). Energy Minimization—All calculations were performed using the precompiled executables of Tinker molecular modeling suite of programs v. 4.0 for Windows platform (15Hodsdon M.E. Ponder J.W. Cistola D.P. J. Mol. Biol. 1996; 264: 585-602Crossref PubMed Scopus (137) Google Scholar, 16Ren P.Y. Ponder J.W. J. Phys. Chem. B. 2003; 107: 5933-5947Crossref Scopus (1210) Google Scholar). The PDB coordinate files 1FYW and 1FYV were used as the starting structures for the TIR domain of the TLR2 and TLR1 molecules, respectively (6Xu Y. Tao X. Shen B. Horng T. Medzhitov R. Manley J.L. Tong L. Nature. 2000; 408: 111-115Crossref PubMed Scopus (229) Google Scholar). Prior to energy minimization of these structures, the covalent seleniums were replaced by sulfur atoms. The corrected structures were then minimized using the Newton program and all-atom AMBER 99 force-field to a root-mean square (RMS) gradient convergence of 0.05 kcal/mol/Å. The calculations were carried out in implicit dielectric conditions of 4.0 to mimic the receptor-like conditions where the role of water and salts can be presumed minimal (17David L. Luo R. Gilson M.K. J. Comput. Aided Mol. Des. 2001; 15: 157-171Crossref PubMed Scopus (52) Google Scholar, 18Ashish A. Kishore R. Bioorg. Med. Chem. 2002; 10: 4083-4090Crossref PubMed Scopus (6) Google Scholar). The resultant coordinates were confirmed to lack any racemization of chiral centers or cis-trans isomerization during optimization. A comparative plot of the backbone dihedrals confirmed no large alterations occurred in the structures during optimization for both the molecules. The minimized coordinates were then used for graphical analysis and docking studies. Protein-Protein Docking and Co-minimization—High resolution rigid body protein-protein docking between the TIR domains of TLR1 and TLR2 was done employing Global Range Molecular Matching (GRAMM) program v. 1.03 (19Katchalski-Katzir E. Shariv I. Eisenstein M. Friesem A.A. Aflalo C. Vakser I.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2195-2199Crossref PubMed Scopus (862) Google Scholar, 20Vakser I.A. Matar O.G. Lam C.F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8477-8482Crossref PubMed Scopus (162) Google Scholar). Employing a grid-step of 1.7, repulsion factor of 30.0, atomic radius for potential range, gray mode projection and 10 degrees for angle of rotations, 500 lowest energy matches were written out. From this output, only those docked structures were sorted which satisfied two conditions: 1) the β-sheet backbone of TIR domains of TLR2 and TLR1 were in same plane, and 2) the N termini were in parallel orientation i.e. toward the membrane. The most stable low energy structure satisfying the imposed requirements (E - E0 < 2 kcal/mol) was considered for co-minimization experiments. For co-minimization, implicit water conditions were used by considering a dielectric value of 80. A cubic box size of one edge equal to 250 Å was used and the van der Waals calculations were cut-off at 15 Å. Employing Newton program and all-atom AMBER99 force field, the coordinates of the TLR2/1 complex were minimized to a RMS gradient convergence of 0.05 kcal/mol/Å. As before, the final coordinates were confirmed to lack any racemization of chiral centers or cis-trans isomerization during optimization and were used for analyzing close contacts between the molecules. Random Mutagenesis and Functional Screening of TLR2 TIR Domain—The only detailed structure/function analysis of any TLR TIR domain was done for TLR4 by Ronni et al. (11Ronni T. Agarwal V. Haykinson M. Haberland M.E. Cheng G. Smale S.T. Mol. Cell. Biol. 2003; 23: 2543-2555Crossref PubMed Scopus (49) Google Scholar) and very little is known about the amino acid residues and subdomains of the human TLR2-TIR, which are essential for function. Thus, we decided to undertake a detailed structure/function analysis of the TLR2 TIR domain. Unlike the alanine substitution approach used by Ronni et al. (11Ronni T. Agarwal V. Haykinson M. Haberland M.E. Cheng G. Smale S.T. Mol. Cell. Biol. 2003; 23: 2543-2555Crossref PubMed Scopus (49) Google Scholar) we decided to perform random mutagenesis followed by a functional screen to identify the crucial residues involved in signaling. Random mutagenesis of TIR-TLR2 (amino acids 607-784) was done using a PCR-based commercially available kit, and the resulting product was cloned back into pcDNA5-TLR2. PCR conditions were optimized to result in 2-3 mutations per 500 bp (for details see "Materials and Methods"). The resulting random mutagenic (RM) clones were then screened for their abilities to activate an NF-κB Luc reporter construct in response to PAM3CSK4 (TLR2 agonist) in the HEK 293 transient transfection assays. Clones which demonstrated reduced activity (compared with WT) were sequenced to identify the mutation(s). This screen resulted in the identification of numerous RM clones with reduced activity, 17 of which are shown in Table 1. The inactive mutants can be basically grouped in three sets: First, clones that had mutations resulting in the stop codons within the coding region which include RM 27, 44, 51, 53, 54, 60, and 61. Signaling in these mutants is blocked implying the need of complete integrity of the TIR domain in TLR2 for the signaling to occur. The second set of clones had mostly single or multiple mutations in and around the well characterized BB loop. Under this category the mutants were RM5, RM18, RM37, RM38, RM57, RM62, and RM73. These mutants also show aberrant signaling supporting the previously pointed out crucial role of the integrity of BB loop for receptor functionality (6Xu Y. Tao X. Shen B. Horng T. Medzhitov R. Manley J.L. Tong L. Nature. 2000; 408: 111-115Crossref PubMed Scopus (229) Google Scholar). The third group of clones RM6, RM30, and RM67, had single or multiple mutations outside the BB loop. Of all these random mutant clones we chose RM18, RM 30, and RM67 for a more detailed analysis. These clones were stably transfected into HEK 293 Flp-In cells and checked for surface expression by FACS using the TL2.1 antibody. All the stable clones expressed approximately equivalent levels of TLR2 on the surface (Fig. 1A). These clones were then examined by real-time RT-PCR analysis for their abilities to induce IL-8 (Fig. 1B) and MIP-3α (Fig. 1C) mRNA in response to PAM3CSK4. These data indicated that the expression of both genes was almost completely inhibited in RM18 and RM30 and significantly repressed (∼80%) in the case of RM67. RM18 contains a single mutation in the BB loop (I685F), which, based upon previous reports of the critical role of the BB loop in mediating TLR2-MyD88 interactions, would be expected it to be inactive (6Xu Y. Tao X. Shen B. Horng T. Medzhitov R. Manley J.L. Tong L. Nature. 2000; 408: 111-115Crossref PubMed Scopus (229) Google Scholar). The reasons for the loss of activity observed for the other two clones are less clear. RM30 is a double mutant (F701V and K743E) and RM67 which has a single mutation L762Q, which also decreases receptor activity significantly. Interestingly, according to the crystal structure 1FYW the amino acids in both these mutants i.e. Lys-743 and Leu-762 lie on the opposite face of the TIR domain from BB loop whereas F701V is buried deep inside the molecule, is not surface exposed, and is in fact a valine in TLR4. Leu-762 is a conserved residue among various TLRs and is present at the base of the αD region whereas Lys-743 is present in the DD loop of TLR2 (6Xu Y. Tao X. Shen B. Horng T. Medzhitov R. Manley J.L. Tong L. Nature. 2000; 408: 111-115Crossref PubMed Scopus (229) Google Scholar). In the TLR2 molecule, the less conserved DD loop is also very mobile, as evident from the high R values for that region in the crystallographic data. Though Dunne et al. (7Dunne A. Ejdeback M. Ludidi P.L. O'neill L.A. Gay N.J. J. Biol. Chem. 2003; 278: 41443-41451Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 21O'Neill L.A. Curr. Opin. Immunol. 2006; 18: 3-9Crossref PubMed Scopus (523) Google Scholar) have suggested that the adaptor molecules like Mal and MyD88 might bind/dock via their DD loop, no such argument has been reported for TLR2 molecule. As mentioned earlier, based on the contact surface among two chains in the C713S mutant TLR2 crystal, Tao et al. (12Tao X. Xu Y. Zheng Y. Beg A.A. Tong L. Biochem. Biophys. Res. Commun. 2002; 299: 216-221Crossref PubMed Scopus (98) Google Scholar) implied that DD loop of TLR2 can interact with the BB loop of the another TLR to form TLR2·TLRx heterocomplex. However, no work identifying the key residues involved in the DD loop mediated TLR2 activity has been reported to date. Given the potential important role of the DD loop in mediating TLR2-dependent responses we decided to perform an alanine scanning mutagenesis of the TLR2 TIR domain in this region.TABLE 1Phenotypes of TLR2 RM clonesGroupNameResidues mutated in TLR2Corresponding residue(s) in TLR4NF-κB Luc activity (%WT) of RM clonesI (Truncated clones)RM27Frame Shift at Gln-747Gln-7814.9RM44M756STOPLeu-7895.6RM51M620STOPLeu-6603.7RM53