Mapping the IκB Kinase β (IKKβ)-binding Interface of the B14 Protein, a Vaccinia Virus Inhibitor of IKKβ-mediated Activation of Nuclear Factor κB

Asa P Oldring,Stephen C Graham,Daniel S Mansur,Jonathan M Grimes,David I Stuart,Mohammad W Bahar,Laura E Mccoy,Brian J Ferguson,Camilla T.O Benfield,Geoffrey L Smith

doi:10.1074/jbc.m111.231381

Abstract

The IκB kinase (IKK) complex regulates activation of NF-κB, a critical transcription factor in mediating inflammatory and immune responses. Not surprisingly, therefore, many viruses seek to inhibit NF-κB activation. The vaccinia virus B14 protein contributes to virus virulence by binding to the IKKβ subunit of the IKK complex and preventing NF-κB activation in response to pro-inflammatory stimuli. Previous crystallographic studies showed that the B14 protein has a Bcl-2-like fold and forms homodimers in the crystal. However, multi-angle light scattering indicated that B14 is in monomer-dimer equilibrium in solution. This transient self-association suggested that the hydrophobic dimerization interface of B14 might also mediate its interaction with IKKβ, and this was investigated by introducing amino acid substitutions on the dimer interface. One mutant (Y35E) was entirely monomeric but still co-immunoprecipitated with IKKβ and blocked both NF-κB nuclear translocation and NF-κB-dependent gene expression. Therefore, B14 homodimerization is nonessential for binding and inhibition of IKKβ. In contrast, a second monomeric mutant (F130K) neither bound IKKβ nor inhibited NF-κB-dependent gene expression, demonstrating that this residue is required for the B14-IKKβ interaction. Thus, the dimerization and IKKβ-binding interfaces overlap and lie on a surface used for protein-protein interactions in many viral and cellular Bcl-2-like proteins.

Highlights

NF-␬B is a critical transcription factor that regulates many important cellular processes, such as differentiation, apoptosis, and the inflammatory response to infection
The B14 protein is nonessential for virus replication in cell culture, but a deletion mutant lacking the B14R gene was attenuated in a mouse intradermal model compared with control viruses, and the attenuated phenotype was characterized by an increased local inflammatory response to infection [22]
SEC-Multi-angle light scattering (MALS) analysis confirmed that mutations Y35E, L126E, and F130K all significantly disrupted dimerization: these mutant proteins were monomeric in solution at concentrations at which significant dimerization of the wildtype protein was observed (Fig. 2D)

Summary

Introduction

NF-␬B is a critical transcription factor that regulates many important cellular processes, such as differentiation, apoptosis, and the inflammatory response to infection. These mutants were assessed for their ability to form dimers, bind IKK␤, and inhibit NF-␬B-dependent gene expression. The analysis of nuclear translocation of p65 supported the reporter assay data in showing that the F130K mutation caused functional impairment, whereas the Y35E mutation did not impair the ability of the VACV B14 protein to block NF-␬B-dependent signaling.

Results

Conclusion