Structurally plastic NEMO and oligomerization prone IKK2 subunits define the behavior of human IKK2:NEMO complexes in solution

Abstract

The human IκB Kinase (IKK) is a multisubunit protein complex of two kinases and one scaffolding subunit that controls induction of transcription factor NF-κB activity. IKK behaves as an entity of aberrantly high apparent molecular weight in solution. Recent X-ray crystallographic and cryo-electron microscopy structures of individual catalytic subunits (IKK1/IKKα and IKK2/IKKβ) reveal that they are both stably folded dimeric proteins that engage in extensive homo-oligomerization through unique surfaces that are required for activation of their respective catalytic activities. The NEMO/IKKγ subunit is a predominantly coiled coil protein that is required for activation of IKK through the canonical NF-κB signaling pathway. Here we report size-exclusion chromatography, multi-angle light scattering, analytical centrifugation, and thermal denaturation analyses of full-length human recombinant NEMO as well as deletion and disease-linked variants. We observe that NEMO is predominantly a dimer in solution, although by virtue of its modular coiled coil regions NEMO exhibits complicated solution dynamics involving portions that are mutually antagonistic toward homodimerization. This behavior causes NEMO to behave as a significantly larger sized particle in solution. Analyses of NEMO in complex with IKK2 indicate that NEMO preserves this structurally dynamic character within the multisubuit complex and provides the complex-bound IKK2 further propensity toward homo-oligomerization. These observations provide critical information on the structural plasticity of NEMO subunit dimers which helps clarify its role in diseases and in IKK regulation through oligomerization-dependent phosphorylation of catalytic IKK2 subunit dimers.

Keywords: Analytical ultracentrifugation; Circular dichroism spectroscopy; IκB kinase; Multi-angle light scattering; NEMO; NF-κB; Size-exclusion chromatography.

Fig. 1.

Recombinant full-length human NEMO protein and deletion constructs. A) Two-dimensional structural diagram of human NEMO protein. Portions of known structure are shaded grey and known or suspected helical regions are depicted as cylinders. The linear structure is divided into seven segments (numbered above): 1-An amino-terminal segment; 2-the kinase binding domain (KBD) that interacts through noncovalent association with the C-terminal NEMO-binding domain (NBD) of either IKK1 or IKK2; 3) an enigmatic intervening domain (IVD) that is predicted to be helical (dashed cylinder); 4) the first of two colied coil regions (CC1) that has been shown to interact with viral protein vFLIP; 5) a second coiled-coil (CC2) that associates with the HOIP protein of the LUBAC complex and linear ubiquitin; 6) a linker that ends in a Pro-rich sequence; 7) a zinc finger motif that has been shown to interact with ubiquitin. Approximate amino acid numbering (1–419) is given at each segment border. B) Coomassie-stained SDS-PAGE analysis of recombinant full-length (lane 2) and deletion construct human NEMO proteins (lanes 3–10). C) Analytical SEC chromatograms of full-length NEMO (solid line) and NEMO deletion constructs NEMO^250–365 (dotted line) and NEMO^1–110 (solid line with hollow circles) overlaid on calibration standard (solid line with filled squares) with known MW of standard proteins labeled in kilodaltons (kDa).

Fig. 2.

Shape-independent size determination of full-length human NEMO in solution. A) Size-exclusion chromatography with multi-angle light scattering (SEC-MALS) analysis of 6 μM sample. The calculated MW of the His-tagged monomer is 50 kDa. Determined MW is given in kilodaltons (kDa) with percent error in measurement. The apparent MW determined previously by SEC only is given in grey. B) Analytical ultracentrifugation (AUC) sedimentation velocity measurement data for full-length NEMO. Peaks are assigned to a percentage value (fraction of total protein detected) and approximate MW.

Fig. 3.

Analysis of NEMO deletion constucts in solution. A) SEC-MALS analysis of NEMO^1–110. For all NEMO deletion construct SEC-MALS chromatograms, monomer MW is given in parentheses above the percent error. Apparent MW determined by SEC only is in grey. B) SEC-MALS analysis of NEMO^1–250, C) NEMO^250–365, D) NEMO^111–250, E) NEMO^1–365, and F) NEMO^1–130. G) NanoDFS analysis of deletion construct NEMO proteins. Each colored line represents measurement of the NEMO protein construct indicated. H) Circular dichroism (CD) spectroscopy of full-length NEMO (black line) and NEMO^1–210 (red line). I) Temperature-dependent CD signal at 222 nm of full-length NEMO (black) and NEMO^1–210 (red).

Fig. 4.

Analysis of variant NEMO proteins containing disease-linked point mutations in solution. A) SEC-MALS analysis of NEMO^D113N, B) NEMO^L153R, C) NEMO^R123W, D) NEMO^Q157P, E) NEMO^A162P, F) NEMO^R175P, and G) NEMO^L227P each at 6 μM concentration. H) NanoDFS analysis of disease-linked NEMO variants. Each colored line represents measurement of the NEMO variant protein indicated. I) CD spectroscopy of full-length wild-type NEMO (black line) and NEMO^L227P (red line).

Fig. 5.

Solution analysis of recombinant full-length IKK2 protein and NEMO:IKK2 complex. A) Domain organization of the IKK2 subunit with kinase domain (KD), ubiquitin-like domain (ULD), and scaffold-dimerization domain (SDD) labeled and human amino acid numbering indicated. B) Schematic representation of the IKK2 homodimer depicting the relative arrangement of domains and homodimerization through the portion of the SDD distal to the KD and ULD. C) SEC-MALS chromatogram of full-length human IKK2 protein. D) AUC sedimentation velocity measurement of full-length human IKK2 in solution. E) SEC-MALS chromatogram of recombinant IKK2:NEMO complex. F) AUC sedimentation velocity measurement of the IKK2:NEMO complex.

Fig. 6.

Solution analysis of IKK2 in complex with NEMO protein variants. SEC-MALS chromatograms of full-length human IKK2 protein in complex with A) NEMO^1–365, B) NEMO^1–280, C) NEMO^1–250, D) NEMO^1–210, E) NEMO^1–130, and F) NEMO^L227P.

Fig. 7.

Schematic of the proposed role of NEMO IVD in influencing dimerization of flanking coiled coil regions of NEMO, which either on their own or through association with additional factors are capable of dimerization. Abbreviations for individual NEMO structural elements are the same as in Fig. 1A and those elements of known structure are shaded grey.