Mechanism of vaccinia viral protein B14-mediated inhibition of IκB kinase β activation

Abstract

Activation of IκB kinase β (IKKβ) is a central event in the NF-κB-mediated canonical pro-inflammatory pathway. Numerous studies have reported that oligomerization-mediated trans autophosphorylation of IKKβ is indispensable for its phosphorylation, leading to its activation and IKKβ-mediated phosphorylation of substrates such as IκB proteins. Moreover, IKKβ's interaction with the NF-κB essential modifier (NEMO) is necessary for IKKβ activation. Interestingly, some viruses encode virulence factors that target IKKβ to inhibit NF-κB-mediated antiviral immune responses. One of these factors is the vaccinia viral protein B14, which directly interacts with and inhibits IKKβ. Here we mapped the interaction interface on the B14 and IKKβ proteins. We observed that B14 binds to the junction of the kinase domain (KD) and scaffold and dimerization domain (SDD) of IKKβ. Molecular docking analyses identified key interface residues in both IKKβ and B14 that were further confirmed by mutational studies to promote binding of the two proteins. During trans autophosphorylation of protein kinases in the IKK complex, the activation segments of neighboring kinases need to transiently interact with each other's active sites, and we found that the B14-IKKβ interaction sterically hinders direct contact between the kinase domains of IKKβ in the IKK complex, containing IKKβ, IKKα, and NEMO in human cells. We conclude that binding of B14 to IKKβ prevents IKKβ trans autophosphorylation and activation, thereby inhibiting NF-κB signaling. Our study provides critical structural and mechanistic information for the design of potential therapeutic agents to target IKKβ activation for the management of inflammatory disorders.

Keywords: B14; IKKβ; autophosphorylation; inhibition mechanism; molecular docking; protein kinase; trans autophosphorylation; vaccinia virus; virus.

Figure 1.

The VACV protein B14 inhibits IKKβ activation but cannot impede its IκBα kinase activity. A, to elucidate the role of B14-mediated inhibition of IKKβ activation, we analyzed the kinase activity of autoactivated IKKβ in HEK293T cells co-transfected with full-length human IKKβ and varying amounts of B14 construct. The empty vector pcDNA3 was used to adjust the total DNA of 55 μg used per transfection. IP, immunoprecipitation; WB, Western blot. B, to rule out the possibility that B14 also inhibits the activated IKKβ with dual phosphorylation on Ser-177 and Ser-181 in the phosphorylation of IκBα, in vitro kinase assays with the constitutively active IKKβ-S177E/S181E (IKKβ-SE) and B14 proteins were conducted. Maltose binding protein (MBP) was used as a negative control. Each experiment was repeated three times.

Figure 2.

Map of the B14 interaction domains on human IKKβ. A, pulldown of IKKβ by GST-B14. GST protein alone was used as a negative control. IKKβ-SE1, IKKβ (1–756) S177E/S181E; IKKβ-SE2, IKKβ (1–678) S177E/S181E; IKKβ-DN, IKKβ (1–756) D145N; IKKβ-SA, IKKβ (1–756) S177A/S181A. B and C, pulldown of B14 by GST-IKKβ truncation mutant proteins. D, domain boundaries of the IKKβ constructs are shown on the right, and the binding property between each IKKβ construct and B14 is summarized on the left. Each experiment was repeated three times. M stands for protein molecular weight standard marker.

Figure 3.

The M2 and M4 segments of IKKβ are required for B14 binding. A, molecular docking models of IKKβ–B14 interaction. The KD, ULD, and SDD of human IKKβ (PDB ID 4E3C) are colored yellow, pink, and slate, respectively. The potential B14-interacting segments present on IKKβ protein are shown as M1, M2, and M4-M6 and colored cyan, blue, magenta, green, and orange, respectively. Three residues, Phe-182, Val-183, and Leu-186 on the M3 segment, are shown in stick representation and colored cyan. B, pulldown of B14 by GST-IKKβ mutants. GST protein alone was used as a negative control. GST-IKKβ-KD-ULD contains residues 1–384 of human IKKβ, and GST-IKKβ-ULD-SDD harbors residues 307–678 of human IKKβ, both of which are used as the WT positive controls (WT1 and WT2). In addition, the M1 mutant has residues 179–197substituted with GGGGSGGS, M2 has residues 235–260 replaced with GGGGSGGS, M3 contains F182K/V183K/L186K mutations, M4 has residues 408–416 replaced with GGGGS, M5 has residues 421–426 substituted with GGGGS, and M6 has residues 577–583 replaced with GGGGS. C, the B14 structure is represented as a ribbon diagram and is colored cyan, with the potential IKKβ interacting residues shown as stick representation and colored magenta. D, pulldown of human IKKβ (1–675, S177E/S181E) by GST-B14 mutants. All experiments were repeated three times and yielded similar results. M stands for protein molecular weight standard marker.

Figure 4.

B14-IKKβ binding interface. A and B, models of B14/IKKβ complex structures. Closed Xenopus IKKβ (xIKKβ, PDB code 3QA8, A) and open human IKKβ (hIKKβ, PDB code 4E3C, B) structures are shown as ribbons and colored green. VACV B14 is shown as a ribbon and colored cyan. M2, M4, and the activation segment of IKKβ (AS) are colored blue, magenta, and red, respectively. C and D, B14-IKKβ binding interface for xIKKβ and hIKKβ, respectively. The key interacting residues are shown as sticks. E, GST-B14 was pulled down by human WT and mutant IKKβ. The empty vector was expressed as a control. LY refers to the L259K and Y261A IKKβ double mutant. The M2M4 IKKβ mutant contains both the M2 and M4 substitutions described in Fig. 3. WB, Western blot. The experiment was repeated three times with consistent results.

Figure 5.

A model for B14-mediated inhibition of IKKβ activation. The KBD of NEMO interacts with the NBD of IKKβ, which cross-links IKKβ to a large oligomer. Upon activation by upstream signaling events or by high IKK concentration, the activation segments of the neighboring KDs can contact each other for trans autophosphorylation. Binding of B14 to the junction of KD and SDD of IKKβ causes a steric hindrance that impedes the optimal contact between KDs in the IKK complex, which, in turn, blocks the insertion of the activation segment of one KD to the active site of another for trans autophosphorylation and activation.