Molecular basis of Tank-binding kinase 1 activation by transautophosphorylation Xiaolei Maa,1, Elizabeth Helgasonb,1, Qui T. Phungc, Clifford L. Quanb, Rekha S. Iyera, Michelle W. Leec, Krista K. Bowmana, Melissa A. Starovasnika, and Erin C. Dueberb,2 Departments of aStructural Biology, bEarly Discovery Biochemistry, and cProtein Chemistry, Genentech, South San Francisco, CA 94080 Edited by Tony Hunter, Salk Institute for Biological Studies, La Jolla, CA, and approved April 25, 2012 (received for review December 30, 2011) Tank-binding kinase (TBK)1 plays a central role in innate immunity: it the C-terminal scaffolding/dimerization domain (SDD), a do- serves as an integrator of multiple signals induced by receptor- main arrangement that appears to be shared among the IKK mediated pathogen detection and as a modulator of IFN levels. family of kinases (3). Deletion or mutation of the ULD in TBK1 Efforts to better understand the biology of this key immunological or IKKε severely impairs kinase activation and substrate phos- factor have intensified recently as growing evidence implicates phorylation in cells (22, 23). Furthermore, the integrity of the aberrant TBK1 activity in a variety of autoimmune diseases and ULD in IKKβ is not only required for kinase activity (24) but was fi cancers. Nevertheless, key molecular details of TBK1 regulation and shown also to confer substrate speci city in conjunction with the β substrate selection remain unanswered. Here, structures of phos- adjacent SDD (25). Recent crystal structures of the IKK phorylated and unphosphorylated human TBK1 kinase and ubiq- homodimer demonstrate that the ULD and SDD form a joint, uitin-like domains, combined with biochemical studies, indicate three-way interface with the KD within each protomer of the a molecular mechanism of activation via transautophosphorylation. dimer, suggesting that the ULD and SDD serve to buttress the These TBK1 structures are consistent with the tripartite architecture KD as well as to contribute additional binding surfaces that observed recently for the related kinase IKKβ, but domain contribu- properly orient substrates (25). Here we report the crystal structure of the kinase and ubiq- tions toward target recognition appear to differ for the two uitin-like domains (KU) of TBK1 in complex with a potent small- enzymes. In particular, both TBK1 autoactivation and substrate spec- molecule inhibitor, BX795. This structure unexpectedly reveals ificity are likely driven by signal-dependent colocalization events. an activation loop-swapped TBK1 conformation: S172 from one BIOCHEMISTRY protomer is located in close proximity to the active site of the kinase activation | crystallography neighboring protomer, providing a snapshot of a potential transautoactivation reaction intermediate. Biochemical analyses nvading bacteria and viruses possess distinct molecular sig- further demonstrate that the kinase domain alone is sufficient to Inatures that are recognized by conserved host receptors, thereby fully autoactivate and is capable of phosphorylating both mac- triggering the assembly of signaling complexes that activate the romolecular and peptide substrates. A high-resolution structure inhibitor of κB kinase (IKK) family of kinases (1–3). The canonical of monophosphorylated TBK1 KD reveals that S172 phosphor- IKKs (IKKα and IKKβ) in turn induce NF-κB–dependent gene ylation reorganizes the activation segment into a conventional transcription via phosphorylation of the inhibitory κBα (IκBα) configuration that is compatible with polypeptide substrate protein. This modification marks IκBα for K48-linked poly- binding. Taken together, these results offer insights into the ubiquitination and subsequent proteasomal degradation, resulting structural basis of TBK1 transautophosphorylation, highlight the in the release of free NF-κB to up-regulate expression of proin- structural transitions accompanying TBK1 activation, and sup- flammatory cytokines (4). By contrast, activated IKK-related port a model in which TBK1 recruitment to discrete signaling kinases [Tank-binding kinase (TBK)1 and IKKε] directly phos- complexes induces TBK1 activation through proximity. phorylate IFN regulatory factors 3 and 7 (IRF3 and IRF7). Phosphorylation promotes the dimerization and nuclear trans- Results location of these transcription factors that stimulate production of Domain Organization of TBK1 Is Consistent with the IKKβ Tripartite type I interferons (IFNs) (1, 3, 5). Recent studies have identified Architecture. To understand how the KD and ULD are organized an additional role for TBK1 in the xenophagic elimination of within TBK1, we crystallized a fragment of human TBK1 con- bacteria (6–9) and better-defined how cross-talk within the IKK taining both domains and a catalytic residue mutation (KU ; family regulates innate immune response (10). D135N residues Q2–E385) in the presence of an inhibitor, BX795 (26, Under pathological conditions, IKK-mediated pathways can 27). The structure was solved to 2.6-Å resolution by single- also be activated inappropriately by endogenous signals, contrib- fl wavelength anomalous dispersion using a selenomethionine- uting to in ammatory disorders and oncogenesis (11, 12). Whereas A canonical IKKs have long been recognized as bridges between substituted sample (Fig. S1 and Table S1). Both molecules in the chronic inflammation and cancer, IKK-related kinases more re- asymmetric unit demonstrate a tandem arrangement of the kinase cently have also been implicated in cell transformation and tumor and ubiquitin-like domains, which possess the typical bilobe progression (13). TBK1 has been of particular interest, given its identification both as an activator of the oncogenic AKT kinase (14–18) and as an essential factor in KRAS-driven cancers (19). Author contributions: X.M., E.H., Q.T.P., and E.C.D. designed research; X.M., E.H., Q.T.P., R.S.I., TBK1 activity is regulated by phosphorylation on S172 within M.W.L., K.K.B., and E.C.D. performed research; E.H., C.L.Q., R.S.I., and K.K.B. contributed new reagents/analytic tools; X.M., E.H., Q.T.P., M.W.L., M.A.S., and E.C.D. analyzed data; and X.M., the classical kinase activation loop. Serine-to-alanine substitution E.H., M.A.S., and E.C.D. wrote the paper. at this position abolishes TBK1 activity, whereas the phospho- Conflict of interest statement: All authors are employees of Genentech. mimetic mutation S172E partially restores activity to within ∼200- fold of the wild-type kinase (20). Genetic and pharmacological This article is a PNAS Direct Submission. inhibition studies have indicated that TBK1 can be activated by Freely available online through the PNAS open access option. IKKβ, as well as by apparent autophosphorylation (10). Addi- Data deposition: Atomic coordinates and structure factors reported in this paper have tional posttranslational modifications of TBK1 lysine residues by been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 4EUT and 4EUU). K63-linked polyubiquitin chains have been shown to promote 1X.M. and E.H. contributed equally to this work. production of IFNs in viral infections (21). 2To whom correspondence should be addressed. E-mail: [email protected]. TBK1 contains a predicted ubiquitin-like domain (ULD) (22) This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. that is located between the N-terminal kinase domain (KD) and 1073/pnas.1121552109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1121552109 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 kinase and ubiquitin folds, respectively (Fig. 1A). In this config- occupied by the αAL-helix of the neighboring protomer in the uration, the ULD abuts the C-terminal lobe (C lobe) of the KD. TBK1 structure (Fig. S4B). Thus, although the unphosphorylated The conventional hydrophobic surface patch of the ULD, which KUD135N structure is endowed with many active kinase traits, the often serves as a protein–protein interaction surface in the case of domain-swapped form is not compatible with substrate binding ubiquitin, faces away from the kinase domain. and, therefore, would not act on a polypeptide substrate. However, A very similar domain organization is observed for IKKβ (25). S172, located in the αAL of the exchanged TBK1 activation loop, is Superposition of the TBK1 KUD135N fragment onto the full- only 6 Å away from the P0 site of the PKA substrate and less than length IKKβ structure (via alignment of the kinase domains) 11 Å from the catalytic D135 residue. Elevated temperature factors appears to poise both TBK1 domains for interaction with the in the αAL-helix underscore the dynamic nature of the TBK1 ac- SDD (Fig. 1B). Residues identified in the KD–SDD and ULD– tivation loop (Fig. S4C) and suggest that a local reorganization of SDD interfaces of the IKKβ structure show moderate conserva- this region, which would allow phosphotransfer to S172, may be tion across IKK-family members and map to comparable surfaces plausible. B of the TBK1 KUD135N fragment structure (Fig. 1 and Fig. S2); The extended interface created by this KUD135N loop swapping however, the orientation of the ULD relative to the KD differs is comparable in size and composition to bona fide protein–pro- slightly in the two kinase structures (Fig. 1A, Inset). In the full- tein complexes, burying 2,570 Å2 of surface area per molecule. β ∼ β length IKK structure, an 30° rotation in the IKK ULD relative However, despite this extensive interaction surface, KUD135N β ’ to that of TBK1 allows the IKK ULD s hydrophobic patch to was found to be monomeric in solution (Fig. 2B). KUD135N, and A B bind to the adjacent SDD (Fig. S3 and ). Similarly, an insertion a shorter construct containing just the kinase domain (KDD135N; into the KD fold specific to IKK-family members (residues 241– Q2–R308), both behaved as monomers by size-exclusion chro- 265 in TBK1) also appears to rearrange in the presence of the matography (SEC). By contrast, full-length TBK1 (FLD135N;Q2– SDD (Fig. S3 C and D). Despite these minor differences, the L729) containing the C-terminal SDD eluted as a dimer.