Folding of Toll-like receptors by the HSP90 paralogue gp96 requires a substrate-specific cochaperone

ARTICLE Received 15 Feb 2010 | Accepted 10 Aug 2010 | Published 21 Sep 2010 DOI: 10.1038/ncomms1070 Folding of Toll-like receptors by the HSP90 paralogue gp96 requires a substrate-specific cochaperone Bei Liu1,*, Yi Yang2,*, Zhijuan Qiu2, Matthew Staron2, Feng Hong2,3, Yi Li2, Shuang Wu1,2, Yunfeng Li4, Bing Hao4, Robert Bona3, David Han5 & Zihai Li1,2 Cytosolic HSP90 requires multiple cochaperones in folding client proteins. However, the function of gp96 (HSP90b1, grp94), an HSP90 paralogue in the endoplasmic reticulum (ER), is believed to be independent of cochaperones. Here, we demonstrate that gp96 chaperones multiple Toll-like receptors (TLRs), but not TLR3, in a manner that is dependent on another ER luminal protein, CNPY3. gp96 directly interacts with CNPY3, and the complex dissociates in the presence of adenosine triphosphate (ATP). Genetic disruption of gp96–CNPY3 interaction completely abolishes their TLR chaperone function. Moreover, we demonstrate that TLR9 forms a multimolecular complex with gp96 and CNPY3, and the binding of TLR9 to either molecule requires the presence of the other. We suggest that CNPY3 interacts with the ATP-sensitive conformation of gp96 to promote substrate loading. Our study has thus established CNPY3 as a TLR-specific cochaperone for gp96. Department of Immunobiology and Cancer Immunology, Division of Basic Sciences, Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina 29425, USA. 2 Department of Immunology, University of Connecticut School of Medicine, Farmington, Connecticut 06030-1601, USA. 3 Lea’s Foundation Center for Hematologic Disorders and Neag Comprehensive Cancer Center, University of Connecticut School of Medicine, Farmington, Connecticut 06030-1628, USA. 4 Department of Microbial, Molecular and Structural Biology, University of Connecticut School of Medicine, Farmington, Connecticut 06030-3305, USA. 5 Department of Cell Biology, University of Connecticut School of Medicine, Farmington, Connecticut 06030-3501, USA. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to Z.L. (email: ). 1 nature communications | 1:79 | DOI: 10.1038/ncomms1070 | www.nature.com/naturecommunications © 2010 Macmillan Publishers Limited. All rights reserved.  ARTICLE G nature communications | DOI: 10.1038/ncomms1070 p96 is the endoplasmic reticulum (ER) resident member of the HSP90 family. Known also as grp94, HSPC4 (ref. 1) or HSP90b1 (ref. 2), gp96 has an intrinsic ATPase activity3–5 and it shares ~50% homology with the cytosolic HSP90 at the amino-acid level, consisting of the N-terminal adenosine triphosphate (ATP)-binding domain (N), followed by the charged middle domain (M) and the C-terminal homodimerization domain (C). It is constitutively expressed in virtually all cell types and its expression is upregulated by stress conditions that perturb ER functions6. HSP90 can chaperone up to 10% of the proteome7,8, and its function is assisted by a number of well-characterized cochaperones such as Hop, p23, Aha1 and CDC37 (ref. 9). These cochaperones modulate the ability of HSP90 to interact with its substrates and ATP, which are critical for the proper and necessary conformational changes of HSP90, including N-domain dimerization, in order to complete the cycle of substrate binding and release. Recently, gp96 has been found to be a master chaperone for multiple Toll-like receptors (TLRs) and integrins10–13. Deletion of gp96 in either macrophages or B cells resulted in inactivation of multiple TLRs at the post-translational level. However, it remains unclear whether gp96 chaperones its client proteins alone or does so in concert with unidentified cochaperones. The prevailing idea has been that gp96 is a unique member of the HSP90 family in that it does not require a cochaperone for function14. Consistent with this notion is that gp96 does not share the common structural features with HSP90, such as the C-terminal methionine-glutamic acid-glutamic acid-valine-aspartic acid motif for binding to cochaperones. Yet, the structural study of the near full-length gp96 (NMC) clearly suggests that additional factors are necessary for N-domain dimerization of gp96, leading to optimal function5. The ‘extended-open’ form of the N and M domains of the gp96 dimer in the presence of ADP and adenylyl-imidodiphosphate (AMP-PNP) is incompatible with its chaperone function5. TLRs are a family of pattern recognition receptors for pathogens that have critical roles in innate immunity against infection15. Thirteen TLRs exist in mammals and they localize either on the cell surface or in the endolysosome compartment. Surface TLRs, including TLR1, TLR2, TLR4, TLR5, TLR6 and TLR11, primarily recognize microbial non-nucleic acid structures. In contrast, intracellular TLRs recognize nucleic acid-based molecules: TLR3 for double-stranded RNA (dsRNA), TLR7/8 for single-stranded RNA and TLR9 for DNA. All TLRs are type I transmembrane (tm) receptors sharing characteristic structural features: an N-terminal ectodomain containing multiple leucine-rich repeats (LRRs) for ligand binding, a short helix embedded in the membrane and a C-terminal cytosolic tail containing the Toll-interleukin (IL)-1 receptor domain for recruiting signalling adaptors. TLR9 resides in the ER in the steady state. It has to exit from the ER by binding to the membrane protein Unc93b (ref. 16), and is further matured by a proteolytic cleavage of the first 14 N-terminal LRRs17,18 in the endolysosomal compartment where signals originate19. TLR9 binds to a diverse range of DNA sequences20, which induces a conformational change of the ectodomain and the close apposition of the dimeric cytoplasmic signalling tails21. TLR3 is a pattern recognition receptor for dsRNA produced commonly as the viral replication intermediate22. Activation of TLR3 also occurs in the acidic endosomal location23. Unlike all other TLRs that can signal through MyD88, the signalling of TLR3 depends exclusively on the cytosolic adaptor molecule TRIF24. Murine canopy3 (CNPY3) (UniProt accession: Q9DAU1) is a ubiquitous ER luminal protein of ~35 kDa25. CNPY3 has a unique pattern of six cysteine residues26, which is characteristic of the saposin-like proteins that form a shell-like dimer27. It also has a C-terminal basic region formed by a stretch of lysine-rich repeat. Although CNPY3 was initially thought to be important for TLR4 expression and was also named PRAT4A (a protein interacting with  TLR4)28, cnpy3 knockout (KO) mice lost the function of multiple TLRs post-translationally, except TLR3 (ref. 25). In this study, we demonstrate that CNPY3 and gp96 depend on each other for post-translational maturation of multiple TLRs, except TLR3. CNPY3 and gp96 form a complex both in vitro and in vivo. The interaction between these two molecules is critical for TLR9 folding and transport to the endolysosomes. We conclude that CNPY3 is the (...truncated)