Mapping the IkappaB kinase beta (IKKbeta)-binding interface of the B14 protein, a vaccinia virus inhibitor of IKKbeta-mediated activation of nuclear factor kappaB.

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 IKKß 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 IKKß, and this was investigated by introducing amino acid substitutions on the dimer interface. One mutant (Y35E) was entirely monomeric but still co-immunoprecipitated with IKKß and blocked both NF-κB nuclear translocation and NF-κB-dependent gene expression. Therefore, B14 homodimerization is nonessential for binding and inhibition of IKKß. In contrast, a second monomeric mutant (F130K) neither bound IKKß nor inhibited NF-κB-dependent gene expression, demonstrating that this residue is required for the B14-IKKß interaction. Thus, the dimerization and IKKß-binding interfaces overlap and lie on a surface used for protein-protein interactions in many viral and cellular Bcl-2-like proteins.

Autonomous tetramerization domains in the glycan-binding receptors DC-SIGN and DC-SIGNR.

Multivalent binding of glycans on pathogens and on mammalian cells by the receptors DC-SIGN (CD209) and DC-SIGNR (L-SIGN, CD299) is dependent on correct disposition of the C-type carbohydrate-recognition domains projected at the C-terminal ends of necks at the cell surface. In the work reported here, neck domains of DC-SIGN and DC-SIGNR expressed in isolation are shown to form tetramers in the absence of the CRDs. Stability analysis indicates that interactions between the neck domains account fully for the stability of the tetrameric extracellular portions of the receptors. The neck domains are approximately 40% alpha-helical based on circular dichroism analysis. However, in contrast to other glycan-binding receptors in which fully helical neck regions are intimately associated with C-terminal C-type CRDs, the neck domains in DC-SIGN and DC-SIGNR act as autonomous tetramerization domains and the neck domains and CRDs are organized independently. Neck domains from polymorphic forms of DC-SIGNR that lack some of the repeat sequences show modestly reduced stability, but differences near the C-terminal end of the neck domains lead to significantly enhanced stability of DC-SIGNR tetramers compared to DC-SIGN.