The IKK-binding domain of NEMO is an irregular coiled coil with a dynamic binding interface

Abstract

NEMO is an essential component in the activation of the canonical NF-κB pathway and exerts its function by recruiting the IκB kinases (IKK) to the IKK complex. Inhibition of the NEMO/IKKs interaction is an attractive therapeutic paradigm for diseases related to NF-κB mis-regulation, but a difficult endeavor because of the extensive protein-protein interface. Here we report the high-resolution structure of the unbound IKKβ-binding domain of NEMO that will greatly facilitate the design of NEMO/IKK inhibitors. The structures of unbound NEMO show a closed conformation that partially occludes the three binding hot-spots and suggest a facile transition to an open state that can accommodate ligand binding. By fusing coiled-coil adaptors to the IKKβ-binding domain of NEMO, we succeeded in creating a protein with improved solution behavior, IKKβ-binding affinity and crystallization compatibility, which will enable the structural characterization of new NEMO/inhibitor complexes.

Figure 1

Coiled-coil heptad repeats. (a) a-g heptad repeats in a typical coiled coil. (b) coiled-coil interhelical distances: da, da’ (Cα are depicted as sphere, a’ is the start of the next heptad).

Figure 2

The coiled-coil NEMO constructs are stable and bind IKKβ with high affinity. (a) Overlay of the CD spectra for the NEMO constructs, showing the high coiled-coil content. WT NEMO(44–111) in black. (b) Temperature unfolding curves as monitored by CD at 222 nm. (c) Binding affinity of NEMO constructs for FITC-IKKβ_KKRR(701–745) by fluorescence anisotropy; lines represent the curve fitting. GST-NEMO(1–196) in black.

Figure 3

The structure of unbound NEMO is an irregular coiled coil. (a) The NEMO-EEAA dimer is shown as a ribbon, light blue = coiled-coil adaptors, blue = NEMO (51–112). a (hot pink) and d (blue) residues are shown in spheres. Residues in the discontinuity region (73–90) are not shown (except Leu79). (b) Sequence alignment of NEMO and NEMO-EEAA, the coiled-coil adaptors sequence is underlined and the mutations are highlighted in pink; the heptad repeats are marked under the sequence: the region with low coiled-coil propensity is in bold and the stutter region is in blue. (c,d) Effect of the stutter on core residue packing: the Cα of a and d residues are shown, coloring as in (a): (c) ideal coiled-coil region of NEMO (23–57), left, compared to the irregular region of NEMO (69–103), right. (d) the core packing is disrupted in the discontinuities region, shown the dimer of NEMO (72–92) with side chains of a and d residues in sticks (coloring as in (a)). (e) Superposition of apo NEMO-EEAA structure (blue) and NEMO (44–111) from the IKKβ-bound structure (grey, PDB: 3BRV, IKKβ is not displayed), shown as ribbons; the structures are aligned on chain A, region 44–111 only.

Figure 4

The three hot-spot residues binding pockets. NEMO-EEAA (blue, left panels), NEMO in complex with IKKβ (PDB: 3BRV; grey, right panels), only hot-spot residues of IKKβ are shown (deep salmon). The IKKβ is shown overlaid to the NEMO-EEAA structure in transparency in the left panels for reference. (a,b) Pocket I: L708 and V709 of IKKβ in sticks. NEMO’s Glu60, Leu61, Ala 64 (top helix) and Arg62′, Ile65′, Arg66′, Asn69′ (bottom helix), also in sticks. (c,d) Pocket II: L719 and I723 of IKKβ in sticks. NEMO’s Ile71, Arg75, Glu78, Leu79 (top helix) and Leu72′, Arg75′, Cys/Ala76′, Leu79′ (bottom helix), also in sticks. (e,f), Pocket III: F734, W739 and W741 of IKKβ in sticks, NEMO’s Phe92, Leu93, Lys96, Phe99, Ala100 (top helix) and Lys90′, Leu93′, Phe97′, Ala100′, Arg101′ (bottom helix), also in sticks; (g,h) surface rendering of pocket III of NEMO-EEAA (left) and NEMO in complex with IKKβ (IKKβ removed, right). The surface area is colored by hydrophobicity red = hydrophobic, white = polar.

Figure 5

The I65M NEMO mutant mimics the open bun conformation. NEMO-EEAA (blue), NEMO-I65M (orange) and NEMO in the IKKβ complex (PDB: 3BRV, grey). (a) Proteins displayed as transparent ribbons encompassing residues 55–81 are superimposed on chain A. The angle between the helix axis for NEMO-EEAA and NEMO-I65M is shown by the arrows in the region 57–77: 5.66 degrees. (b) Effect of the Met/Ile mutation (shown as sticks).

Figure 6

Analysis of NEMO dynamics. Left panels: normalized B-factors for chains A (a) and B (b) of the structure of NEMO-EEAA. The normalized B-factors are calculated from the average B-factor over all atoms for the residue, and then averaged over a moving window of three residues. Residues 65–92 of chain B are highlighted in green bars. Right panels: Analysis of the dynamics over the last 40 ns of the MD simulation of NEMO-EEAA. The region 65–92 is boxed. (c) Standard deviation of the average interhelical distances calculated as (da + da’)/2, using residue d in the center of each heptad as the abscissa value. (d) RMSF for Cα atoms.