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PLoS Biol
2013 Jan 01;116:e1001581. doi: 10.1371/journal.pbio.1001581.
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A structural basis for IκB kinase 2 activation via oligomerization-dependent trans auto-phosphorylation.
Polley S
,
Huang DB
,
Hauenstein AV
,
Fusco AJ
,
Zhong X
,
Vu D
,
Schröfelbauer B
,
Kim Y
,
Hoffmann A
,
Verma IM
,
Ghosh G
,
Huxford T
.
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Activation of the IκB kinase (IKK) is central to NF-κB signaling. However, the precise activation mechanism by which catalytic IKK subunits gain the ability to induce NF-κB transcriptional activity is not well understood. Here we report a 4 Å x-ray crystal structure of human IKK2 (hIKK2) in its catalytically active conformation. The hIKK2 domain architecture closely resembles that of Xenopus IKK2 (xIKK2). However, whereas inactivated xIKK2 displays a closed dimeric structure, hIKK2 dimers adopt open conformations that permit higher order oligomerization within the crystal. Reversible oligomerization of hIKK2 dimers is observed in solution. Mutagenesis confirms that two of the surfaces that mediate oligomerization within the crystal are also critical for the process of hIKK2 activation in cells. We propose that IKK2 dimers transiently associate with one another through these interaction surfaces to promote trans auto-phosphorylation as part of their mechanism of activation. This structure-based model supports recently published structural data that implicate strand exchange as part of a mechanism for IKK2 activation via trans auto-phosphorylation. Moreover, oligomerization through the interfaces identified in this study and subsequent trans auto-phosphorylation account for the rapid amplification of IKK2 phosphorylation observed even in the absence of any upstream kinase.
Figure 2. Domain–domain interactions in IKK2.(A) Close-up view of the ULD–SDD interface. Coloring is consistent with Figure 1. (B) Close-up view of the KD–SDD interface. (C) In vitro kinase assays in which hIKK2 with mutations that target interdomain interfaces (lanes 3–5) is compared against the native protein (lane 2).
Figure 3. The hIKK2 dimer interface.(A) Ribbon diagram (top) and space filling (bottom) representations of six hIKK2 protomers in the asymmetric unit. The individual subunit chains are labeled A–F and rainbow colored. (B) Ribbon diagrams of three hIKK2 dimers taken from the asymmetric unit. The distances between P578 residues from each hIKK2 in the dimers are labeled. For comparison, the xIKK2 crystal structure observed is shown below in its relatively closed conformation. (C) Overlay of the SDD from six monomers in the hIKK2 asymmetric unit. The three dimers overlay perfectly at their SDD distal ends (inside dashed box). Movement about a hinge point (black arrowheads) results in the differences observed in the portions of the SDD more proximal to the KD. (D) A close-up view reveals the series of hydrophobic and ionic interactions that mediate the dimer interface. (E) Superposition of the A:F dimer SDD from the hIKK2 structure and the xIKK2 dimer. (F) Close-up view of the boxed region from panel D reveals additional interactions present within the interface of the xIKK2 structure SDD. (G) In vitro kinase assays monitoring the activity of immunoprecipitated HA-IKK2 toward IκBα. Mutations targeting the dimer interface (lanes 3–5) are compared against the wild-type protein (lane 2).
Figure 4. Oligomerization of hIKK2 dimers in the crystal.(A) Three successive asymmetric units, each composed of six protomers, are taken from the hIKK2 X-ray crystal structure and depicted with the center asymmetric unit (labeled A–F) and colored with surface rendering as in Figure 3A. Within this arrangement can be found four unique but closely related versions of the same dimer of dimers. Beginning from the left, there are four protomers (D, E, and another D′ and E′ from the symmetry-related asymmetric unit) that assemble into a tetramer. These four polypeptide chains are depicted as opaque, while the remaining protomers are rendered as semitransparent. In the second depiction of the same assembly of three asymmetric units, chains B, C, D, and E are rendered opaque and reveal themselves to assemble with a similar tetrameric arrangement. Likewise, protomers A, B, C, and F in the third panel and A, F, and the symmetry-related A″ and F″ chains assemble into renditions of the same tetramer. (B) Close-up views of the four unique assemblies viewed perpendicular to their 2-fold rotation axes reveal their close similarity.
Figure 5. Evidence for oligomerization of hIKK2 dimers in solution.(A) Analytical ultracentrifugation sedimentation velocity experiments on concentrated samples of full-length hIKK2 reveal a pattern that correlates with monomer–dimer equilibrium as well as formation of tetramers, hexamers, and octamers in solution. Arrows mark peaks and a summary of data output including calculated molecular weights is inset. (B) When full-length, ATP-treated hIKK2 is analyzed by SEC-MALLS, one observes peaks that correspond to dimer (major peak) and tetramer (minor peak). (C) The hIKK2 I413A/L414A mutant protein displays defects in its ability to undergo reversible dimer–tetramer transitions without aggregating.
Figure 6. Oligomerization of hIKK2 dimers.(A) Ribbon diagram of the interaction between two neighboring hIKK2 dimers in the crystal. Their asymmetric association gives rise to two unique intersubunit interfaces. (B) Close-up view of residues that interact between the KDs at the V-shaped interface. (C) Additional residues that mediate V-shaped interface interactions between the ULD an SDD. (D) Close-up view of interacting residues within the anti-parallel interface. (E) In vitro kinase assay reveals that catalytic activity of hIKK2 with mutations that disrupt the V-shaped interface (lanes 3–5) is drastically reduced compared to wild-type protein (WT-lane 2). (F) In vitro kinase assays with the same WT mutant proteins in which activation loop serines are mutated to glutamate. (G) Immunoblotting with anti-phospho-Ser177,181 antibody reveals that the decrease in catalytic activity observed in the V-shaped interface mutants correlates with activation loop phosphorylation status. (H) In vitro kinase assays reveal the modest effects on hIKK2 catalytic activity of mutation at the antiparallel interface.
Figure 7. Interaction between KDs of oligomeric hIKK2.(A) Within the crystal, neighboring tetrameric assemblies interact symmetrically such that they contact one another through their V-shaped interfaces and two KDs are positioned within close proximity to one another (dashed box). (B) The close packed KDs are positioned so that their activation loops (dashed box) rest directly over the active site of a neighbor. Orange spheres mark the Cα positions on V229 and H232. (C) Close-up view of the kinase activation loops (yellow and blue) with glutamic acid residues 177 and 181 mimicking activation loop serines and the catalytic base D145 labeled. (D) In vitro kinase assay on immunoprecipitated hIKK2 with mutations at key residues that mediate KD–KD interactions in the crystal (lanes 3,4) reveals their involvement in catalytic activity. (E) Mutation of activation loop serines 177 and 181 to glutamates restores activity of immunoprecipitated IKK2 in vitro. (F) Immunoblotting with anti-phospho-Ser177,181 antibody reveals that the decrease in catalytic activity observed in the KD–KD interface mutants correlates with decreased activation loop phosphorylation.
Figure 8. In vitro reconstitution of hIKK2 trans auto-phosphorylation.A catalytically inactive (D145N) and C-terminally truncated IKK2 (lanes 1–6) and mixtures of that enzyme with a catalytically active full-length version (lanes 4–6) were incubated with Mg-ATP for the time periods indicated and then probed via Western blot with anti-phosphoSer181 antibody (above) or by SDS PAGE (below).
Figure 9. Size exclusion chromatography of endogenous and transfected IKK complexes.(A) Cytosolic extracts of untreated (above) and treated (below; 10 ng/mL of TNF for 10 min) MEF-3T3 cells were loaded onto a Superose 6 HR size exclusion column and fractionated at a flow rate of 0.5 mL/min. Fraction size was 0.5 mL. Fractions, as shown in the figure, were resolved on 10% SDS-PAGE and probed with anti-IKK2 and anti-NEMO antibodies. The experiment was performed at least thrice and representative blots are shown here. (B) HEK293T cells were transfected with HA-IKK2 (above), HA-IKK2 and NEMO (middle), or NEMO alone (below) and lysates were separated by size exclusion chromatography and probed by anti-HA or anti-NEMO antibodies.
Figure 10. IKK2 oligomerization activation model.(A) The hIKK2 X-ray crystal structure in space filling representation viewed from three different angles. The four surfaces that mediate oligomerization in the X-ray crystal structure are colored purple (dimer interface), blue (antiparallel interface), orange (V-shaped interface), and green (KD–KD interface). (B) A structure-based model for IKK2 activation via trans auto-phosphorylation. IKK2 interconverts between its open and closed dimeric forms. The open dimer can further associate to form transient homooligomers, such as observed in the hIKK2 X-ray crystal structure. Phosphorylation of one IKK2 subunit by an upstream kinase activates the kinase activity of that subunit and, as a consequence of its propensity to assemble into higher order oligomers through it V-shaped and KD-KD interfaces, is rapidly amplified via trans auto-phosphorylation.
Figure 1. The hIKK2 X-ray crystal structure.(A) Domain organization schematics of full-length hIKK2 (above) and crystallized protein construct (below). The KD, ubiquitin-like domain (ULD), scaffold dimerization domain (SDD), and NEMO-binding region (N) are indicated and the domain borders are numbered. “EE” represents mutation of two active site serines 177 and 181 to glutamic acid residues in the crystallized protein. (B) Ribbon diagram representation of the hIKK2 X-ray crystal structure. Coloring and labels correspond to part A. Individual helices of the SDD are labeled as are the N and C termini (N and C, respectively). (C) Ribbon diagram representation of the hIKK2 KD with secondary structure elements and key amino acid side chain positions labeled. (D) Close-up view of the structural elements and amino acid residues immediately surrounding the activation loop (blue). (E) Superposition of hIKK2 (green) and xIKK2 (brown) KDs depicted in Cα-trace representation. Several amino acid residues that adopt significantly different positions in the two structures are rendered as sticks (yellow for hIKK2; orange for xIKK2) and labeled.
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