Molecular mechanisms for the subversion of MyD88 signaling by TcpC from virulent uropathogenic Escherichia coli Greg A. Snydera,1, Christine Cirlb,1, Jiansheng Jianga,2, Kang Chenc,2, Anna Waldhuberb, Patrick Smitha, Franziska Römmlerb, Nathaniel Snydera, Theresa Fresqueza, Susanne Dürrb, Nico Tjandrac, Thomas Miethkeb,3, and Tsan Sam Xiaoa,3 aLaboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; bInstitut für Medizinische Mikrobiologie, Immunologie und Hygiene, Technische Universität München, D-81675 München, Germany; and cLaboratory of Molecular Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892 Edited* by William E. Paul, National Institutes of Health, Bethesda, MD, and approved March 18, 2013 (received for review September 11, 2012) The Toll/IL-1 receptor (TIR) domains are crucial signaling modules initiate the assembly of a death domain complex “Myddosome” for during innate immune responses involving the Toll-like receptors downstream signaling (9). Structures of individual TIR domains (TLRs) and IL-1 receptor (IL-1R). Myeloid differential factor 88 have been determined for a number of receptors and adapters (MyD88) is a central TIR domain-containing adapter molecule re- (10–18), and most TIR domains contain five α helices (A–E) fi β – sponsible for nearly all TLR-mediated signaling and is targeted by surrounding a central ve-stranded sheet (A E). Three regions of high sequence conservation are defined as boxes 1–3 motifs (19) a TIR domain-containing protein C (TcpC) from virulent uropatho- β α genic Escherichia coli, a common human pathogen. The mecha- and are located at the A strand, BB loop, and E helix. nism of such molecular antagonism has remained elusive. We Despite a wealth of reports on TIR domain functions, the mo- lecular mechanism of TIR:TIR domain interactions remains a cen- present the crystal structure of the MyD88 TIR domain with distinct tral unresolved issue. Genetics and mutagenesis studies have loop conformations that underscore the functional specialization of identified the conserved Pro residue and the BB loop (box 2) as the adapter, receptor, and microbial TIR domains. Our structural essential for TLR signaling (10, 20–23). However, the importance of IMMUNOLOGY analyses shed light on the genetic mutations at these loops as well the Pro residue or the BB loop was called into question by others (6, as the Poc site. We demonstrate that TcpC directly associates with 13, 24–26). Furthermore, modeling and functional studies revealed MyD88 and TLR4 through its predicted DD and BB loops to impair the that residues outside of the BB loop play vital roles in TIR domain TLR-induced cytokine induction. Furthermore, NMR titration experi- associations or TLR signaling, such as the CD, DD, and EE loops ments identify the unique CD, DE, and EE loops from MyD88 at the (11, 14, 25, 27–30). These observations make conceptualizing a co- TcpC-interacting surface, suggesting that TcpC specifically engages herent model of the TIR:TIR domain signaling a challenge, which is these MyD88 structural elements for immune suppression. These further complicated by the findings that the modes of receptor: findings thus provide a molecular basis for the subversion of TLR adapter assembly may differ among different TLR receptors (22, 24). signaling by the uropathogenic E. coli virulence factor TcpC and In addition to eukaryotic TIR domain-containing proteins, furnish a framework for the design of novel therapeutic agents that a number of bacterial proteins have been identified that mod- modulate immune activation. ulate host immune responses through their TIR domains. These include proteins from uropathogenic Escherichia coli (UPEC) Brucella melitensis innate immune cells | bacterial pathogens strain CFT073 (TcpC) (31), (TcpB) (31), Brucella abortus (TcpB/Btp1) (32, 33), Brucella ovis (TcpB) (32), Salmonella enterica (TlpA) (34), Paracoccus dentrificans (PdTLP) oll-like receptors (TLRs) are a family of innate immune (35) and Yersinia pestis (YpTdp) (36). Many of these microbial Treceptors that recognize pathogen-associated molecular pat- proteins have been reported to interact with the host TIR domain terns, such as bacterial cell wall components and microbial nucleic proteins and may promote the formation of nonproductive TIR acids. As such, TLR signaling plays critical roles in the initiation domain complexes and thus prevent immune signaling (31, 35–37). and orchestration of both innate and adaptive immune responses For example, Low et al (35) reported the association of PdTLP (1). Ligand engagement at the TLR extracellular or endosomal with TLR4 and MyD88, although the exact binding interface domains results in the association of their cytoplasmic Toll/IL-1 remains to be characterized. As a result of their ability to sup- receptor (TIR) domains, which in turn recruit downstream TIR press immune responses, these microbial TIR domain-contain- domain-containing adapter molecules, such as MyD88 (myeloid ing proteins were proposed as a novel class of virulence factors differentiation factor 88), MAL (MyD88 adapter-like)/TIRAP that subvert host immunity through molecular mimicry (38, 39). (TIR domain-containing adaptor protein), TRIF (TIR domain- containing adapter-inducing IFN-β), and TRIF-related adapter molecule. Recruitment of the TIR domain-containing adapters Author contributions: G.A.S., C.C., J.J., K.C., T.M., and T.S.X. designed research; G.A.S., elicits a signaling cascade that leads to the activation of transcrip- C.C., J.J., K.C., A.W., P.S., F.R., N.S., T.F., and S.D. performed research; G.A.S., C.C., J.J., K.C., tion factors NF-κB and activator protein-1 (AP-1) and the pro- A.W., P.S., F.R., N.S., T.F., S.D., N.T., T.M., and T.S.X. analyzed data; and G.A.S., C.C., N.T., duction of cytokines (2, 3). T.M., and T.S.X. wrote the paper. MyD88 is an essential adapter molecule for the IL-1 receptor The authors declare no conflict of interest. and most TLR family members except for TLR3 (3). Its central *This Direct Submission article had a prearranged editor. importance in innate immune responses was underscored by the Data deposition: The atomic coordinates and structure factors have been deposited in observations that mice deficient in MyD88 are nonresponsive to the Research Collaboratory for Structural Bioinformatics Protein Data Bank, www.rcsb. LPS challenge (4) and IL-1/IL-18 stimulation (5), and that muta- org (ID codes 4EO7 and 4DOM). tions in human MyD88 render pediatric patients susceptible to 1G.A.S. and C.C. contributed equally to this work. pyrogenic bacterial infections (6). Conversely a gain-of-function 2J.J. and K.C. contributed equally to this work. variant of MyD88, L252P, found in diffuse large B-cell lymphoma, 3To whom correspondence may be addressed. E-mail: [email protected]. promotes tumor survival through enhanced NF-κB and JAK tum.de or [email protected]. kinase activation (7). Mechanistically, MyD88 is thought to form This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. homodimers (8) that are recruited by the TLR TIR domains to 1073/pnas.1215770110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1215770110 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 The best-characterized bacterial TIR-containing protein is at 1.45-Å resolution (Table S1), and it reveals a typical TIR do- TcpC, which is common in the most virulent UPEC strains from main fold with five parallel β-strands surrounded by helices and children with acute pyelonephritis. TcpC suppresses TLR-me- loops (Fig. 1A) similar to a reported NMR structure (13). All res- diated cytokine production upon infection, increases bacterial idues of the TIR domain including the long loops are well resolved burden in the urinary tract, and promotes renal tissue damage by unambiguous electron density map at high resolution (Fig. S1A). (31, 37). Likewise, the Brucella protein TcpB has been shown to A comparison of the MyD88 TIR domain structure with those from interact with both TLR signaling adapters MyD88 (31) and MAL TLR2, TLR1, and Paracoccus dentrificans (PdTLP) reveals unique B (40) and inhibits TLR signaling (32, 33). The clinical relevance of loop conformations for each domain (Fig. 1 ). For example, the the above bacterial virulence factors makes it imperative to un- BB, CD, DD, DE, and EE loops adopt strikingly different con- B–F derstand the molecular basis of the interactions between the formations (Fig. S2 ), and their sequences also seem to be conserved only among the subfamilies of the adapter, receptor, and microbial TIR domain-containing proteins and their host targets. “ ” To investigate the role of MyD88 as a central adapter mole- microbial TIR domains (Fig. S2). Notably, the conserved box2 Pro residues from TLR2 and PdTLP are 16.5 Å and 22.8 Å apart cule, as well as the mechanisms by which the bacterial virulence B factors subvert MyD88-mediated signaling, we have determined from that in MyD88, respectively (Fig. S1 ). By contrast, the crystal the crystal structure of the MyD88 TIR domain and identified and NMR structures of the MyD88 TIR domain are very similar, with minimal structural changes (Fig. S1G), suggesting that crys- a TcpC-binding surface at MyD88. In addition, we show that the tallization has no significant effect on the structure of the MyD88 predicted TcpC DD and BB loop peptides directly interact with loops. Interestingly, the BB loops of most known TIR domain MyD88 and TLR4 and suppress innate immune signaling. Our structures contain a short 310 helix (MyD88 and PdTLP) or α-helix studies thus provide a molecular mechanism for the interactions (TLR1, TLR2, TLR10, and IL-1RAPL) preceding the conserved of host and pathogen TIR domains and furnish a framework Pro residues (Fig.