Structure and ubiquitination-dependent activation of TANK-binding kinase 1

Abstract

Upon stimulation by pathogen-associated inflammatory signals, TANK-binding kinase 1 (TBK1) induces type I interferon expression and modulates nuclear factor κB (NF-κB) signaling. Here, we describe the 2.4 Å-resolution crystal structure of nearly full-length TBK1 in complex with specific inhibitors. The structure reveals a dimeric assembly created by an extensive network of interactions among the kinase, ubiquitin-like, and scaffold/dimerization domains. An intact TBK1 dimer undergoes K63-linked polyubiquitination on lysines 30 and 401, and these modifications are required for TBK1 activity. The ubiquitination sites and dimer contacts are conserved in the close homolog inhibitor of κB kinase ε (IKKε) but not in IKKβ, a canonical IKK that assembles in an unrelated manner. The multidomain architecture of TBK1 provides a structural platform for integrating ubiquitination with kinase activation and IRF3 phosphorylation. The structure of TBK1 will facilitate studies of the atypical IKKs in normal and disease physiology and further the development of more specific inhibitors that may be useful as anticancer or anti-inflammatory agents.

Figure 1. TBK1 structure and interdomain interactions

(A) The domain structure of TBK1 includes the kinase domain (KD), ubiquitin-like domain (ULD), scaffold and dimerization domain (SDD) and C-terminal domain (CTD). The linker connecting the ULD and SDD domains is colored magenta and all domains are colored as in the structural representations presented here. (B) Overall structure of the TBK1 dimer in complex with MRT67307. The domains of the second subunit in the dimer are colored dark blue (kinase), orange (ULD) and tan (SDD). The compound is shown in a stick representation (yellow). (C) Interaction of the ULD with the helix α1 in the SDD. Each of the five β-strands of the ULD contributes to the mostly hydrophobic interface. (D) The linker segment (pink) that connects the ULD and SDD domains packs into the SDD (green surface) via mostly hydrophobic interactions. (E) The largely polar interface between the kinase and SDD domains includes hydrogen bonds between Tyr564 and Glu100, Lys416 and Glu99, and Arg427 and the carbonyl of Ser266. (F) The ULD associates with the Clobe of the kinase domain. Tyr325 in the ULD hydrogen bonds with Glu109 in the kinase domain, and Lys323 in the ULD is positioned to make favorable electrostatic interactions with Glu109. See also supplementary Figure S1.

Figure 2. Structure of the kinase domain and interactions with MRT67307

(A) The kinase adopts an inactive conformation with helix αC rotated out of the active site. The activation loop is disordered (dashed line) beyond the DFG motif (red). (B) Detailed view of inhibitor interactions. MRT67307 forms dual hydrogen bonds with the amide and carbonyl groups of Cys89, as well as a water-mediated hydrogen bond with Thr156. Electron density for the compound and for the structure of the BX795 complex are shown in Supplementary Figure S2.

Figure 3. Structure, conservation and function of the TBK1 dimer interface

(A) Oblique view of the TBK1 dimer interface, highlighting interactions between the kinase and ULD domains of one subunit and the SDD domain of the opposite subunit in the dimer (tan). Hydrogen bonds that stabilize the dimer are indicated by dashed lines. (B) Conservation among 43 vertebrate TBK1 and IKKε sequences mapped onto the surface of TBK1. The inward, dimer-forming surface of TBK1 subunit is shown on the left, the outward-facing surface on the right. Contact points in the dimer interface are well-conserved (yellow circles). Note the highly conserved surface on the upper end of the SDD (surrounding Glu580). Analyses were performed with the CONSURF server (Ashkenazy et al., 2010). (C) Expression of WT or mutant TBK1 in HEK-293T cells following transient transfection of the indicated dimer contact mutants. TBK1 protein levels, Ser172 phosphorylation, as well as total and Ser396 phospho-IRF3 were analyzed by immunoblotting 60 h post-transfection. The K38M mutant is a kinase-inactive positive control, K251A is a negative control mutation remote from the dimer interface. QRT-PCR for TBK1 mRNA expression was performed 48 h post-transfection. (D) EGFP control-normalized values for mRNA levels of INFB1 and RANTES (upper panel) or ISRE and NF-κB luciferase reporter activity (lower panels) 48 h post-transfection of WT or mutant TBK1 constructs. Reporter activity was measured in tandem with a control renilla luciferase vector to which values were standardized. (E) Size exclusion chromatography-multi-angle light scattering (SEC-MALS) analysis of TBK1 dimer interface mutants. Purified TBK1 dimer interface mutants and wild type protein are analyzed on a Superdex 200 gel filtration column coupled to a multi-angle light scattering detector. All proteins analyzed contain the D135N catalytic site mutation because it can be expressed abundantly. The elution profiles as measured by refractive index are shown. The labeled horizontal traces indicate the measured molar mass, ~146.3 KDa for TBK1 wild type (expected molar mass of a dimer is 152.2 KDa) and ~ 86.9 KDa for E355A/R357A and ~ 95.1 KD for R547D. D33A, E355A and H459E/N474A elute at the same position as the wild type protein, and their molar masses are also similar. (F) Immunoblot of whole cell extracts (WCE, lower panels) or FLAG IP (upper panels) following transient transfection of FLAG- and V5-tagged TBK1-WT, TBK1-E355A/R357A, or TBK1-R547D constructs as indicated. Arrow indicates the specific band representing V5-TBK1. (G) Conserved SDD patch mutants were expressed in 293T cells and analyzed for total and phospho-TBK1 levels, total and phospho-IRF3 levels, TBK1 mRNA levels, as well as IFNB1 and RANTES expression. See also Supplementary Figure S2 for further analysis of the dimer.

Figure 4. Comparison with the structure of IKKβ

(A) Superposition of TBK1 with IKKβ (magenta). Structures are superimposed using the ULD, which highlights the general similarity in the manner in which the ULD anchors the kinase domains (KD) to the SDD in both proteins. This structural alignment also superimposes a glycine residue (black dot) in the ULD/SDD interface (Gly442 in TBK1 and Gly450 in IKKβ) that is conserved in both proteins and occurs in the same position in their primary sequences, despite the overall divergence in their SDD domains (see also Supplementary Figure S4E, F). Note also that the orientation of the kinase domains in the two structures is quite different relative to the ULD and SDD domains. (B) Surface views of TBK1 (top panels) and IKKβ (lower panels), with corresponding domains of IKKβ colored as in TBK1. In TBK1, the ULD domains bridge between dimer-related SDD domains, while in IKKβ they extend away from the opposite SDD domain. Likewise, the kinase domains in IKKβ are differently oriented and do not form dimer contacts. The IKKβ structure is drawn from PDB code 3QA8 (Xu et al., 2011).

Figure 5. TBK1 is modified by K63-linked polyubiquitin chains

(A) TBK1 is K63-linked polyubiquitinated. HA-tagged wild type or K63-only ubiquitin were cotransfected with GST-TBK1 into HEK293T cells. GST immune complexes (TBK1) were isolated followed by immunoblotting with the indicated antibodies. 5% of the whole cell lysate was loaded for comparison (input). (B) Wild type and kinase dead TBK1 are ubiquitinated equally. HA-tagged K63-only ubiquitin were cotransfected with wild type (WT) and kinase dead (KD) GST-TBK1 into HEK293T cells. GST immune complexes (TBK1) were isolated followed by immunoblotting with the indicated antibodies. 5% of the whole cell lysate was loaded for comparison (input). (C) TBK1 undergoes K63-linked polyubiquitination at Lys30 and Lys401. HEK293T cells were transfected as indicated. V5 immune complexes (TBK1) were isolated followed by immunoblotting with the indicated antibodies. 5% of the whole cell lysate was loaded for comparison (input). (D) TBK1 dimer-deficient mutants do not undergo K63-linked ubiquitination. HEK293T cells were transfected as indicated. V5 immune complexes (TBK1) were isolated followed by immunoblotting with the indicated antibodies. 5% of the whole cell lysate was loaded for comparison (input).

Figure 6. K63-linked polyubiquitination is required for TBK1-induced gene expression and kinase activation

(A) mRNA levels of INFB1 and RANTES were measured using qRT-PCR 30 h following transient transfection of the indicated ubiquitination site mutants and normalized to control EGFP-transfected cells. (B) Immunoblot showing total TBK1 and Ser172 phospho-TBK1 levels 48 h post-transfection of the indicated constructs. (C) Locations of Lys30 and Lys401 ubiquitination mapped on TBK1 structure. One TBK1 monomer is in green, the other in cyan. Note that Lys401 from monomer “a” is close to Lys30 of monomer “b” across the dimer interface. (D) qRT-PCR measurement of IFNB1 and TBK1 mRNA following TBK1 reconstitution in TBK1−/− MEFs and stimulation with polyIC. Cells were nucleofected with EGFP control or the indicated TBK1 constructs, and 24h later were stimulated with 100 µg/ml polyIC for 0h, 2h, or 4h.