Polyubiquitin Drives the Molecular Interactions of the NF-κB Essential Modulator (NEMO) by Allosteric Regulation

Abstract

The NF-κB essential modulator (NEMO) is the master regulator of NF-κB signaling, controlling the immune and nervous systems. NEMO affects the activity of IκB kinase-β (IKKβ), which relieves the inhibition of the NF-κB transcriptional regulation machinery. Despite major effort, there is only a very sparse, phenomenological understanding of how NEMO regulates IKKβ and shows specificity in its large range of molecular interactions. We explore the key molecular interactions of NEMO using a molecular biophysics approach, incorporating rapid-mixing stopped-flow, high-pressure, and CD spectroscopies. Our study demonstrates that NEMO has a significant degree of native structural disorder and that molecular flexibility and ligand-induced conformational change are at the heart of the molecular interactions of NEMO. We found that long chain length, unanchored, linear polyubiquitin drives NEMO activity, enhancing the affinity of NEMO for IKKβ and the kinase substrate IκBα and promoting membrane association. We present evidence that unanchored polyubiquitin achieves this regulation by inducing NEMO conformational change by an allosteric mechanism. We combine our quantitative findings to give a detailed molecular mechanistic model for the activity of NEMO, providing insight into the molecular mechanism of NEMO activity with broad implications for the biological role of free polyubiquitin.

Keywords: NEMO; adaptor protein; allosteric regulation; biophysics; conformational change; high pressure; polyubiquitin chain.

FIGURE 1.

Summary of NEMO structural biology and predicted disordered structure content. i, NEMO domain map. αH, α-helix; CC1 and CC2, coiled coils 1 and 2, respectively; NOA, NEMO-optineurin-ABIN; LZ, leucine zipper; ZF, zinc finger. ii, solved NEMO structures deposited in the Protein Data Bank (codes 3BRV, 3CL3, 2ZVN, and 2JVX). iii, predicted structurally disordered regions of NEMO based on consensus between PONDR-FIT, ANCHOR, IUPred, and SPINE-D. Only regions of >5 amino acids are shown for clarity. iv, binding regions for IKKβ and polyubiquitin.

FIGURE 2.

CD spectrum showing that NEMO has a large percentage of disordered structure content. A, far-UV CD spectra of NEMO. The data shown are the raw averaged data from 10 spectra, with the corresponding buffer spectra subtracted. The inset shows the temperature dependence of the signal at 222 nm, fit to Equation 1. B, results of fitting far-UV CD data (contin set 7). The variation in turn content is not shown for clarity. Conditions were 20 mm Tris-SO₄ (pH 8), 50 mm NaCl, 2 mm β-mercaptoethanol, and 6 μm NEMO. MRE, mean residue ellipticity; deg, degrees.

FIGURE 3.

Fluorescence probes of NEMO conformational state. A, fluorescence emission spectra attributable to ANS emission in the presence (solid blue line and dashed blue line (deconvolved spectrum)) and absence (solid black line) of NEMO. B, fluorescence emission spectrum of the single NEMO Trp residue (Trp⁶ (W6)). The center of spectral mass (CSM) is shown as an asterisk at the emission maxima. Conditions were 50 mm Tris-Cl (pH 8), 50 mm NaCl, 5 mm DTT, and 6 μm NEMO at 10 °C.

FIGURE 4.

Pressure and temperature dependence of NEMO ANS (A and C) and Trp⁶ (B and D) emission. The specific conditions are given under “Experimental Procedures.” The relative ANS emission reflects the relative change in the ith conformational state, as given in Equation 3. The solid lines are the fits to Equation 3. The data are the pressure dependence at 10 °C (red), 15 °C (blue), 20 °C (green), and 25 °C (black). The insets show the temperature dependence of the extracted K₀ values. The solid line is the fit to the van 't Hoff equation. Conditions were 50 mm Tris-Cl (pH 8), 50 mm NaCl, 5 mm DTT, and 6 μm NEMO.

FIGURE 5.

Ligand binding is coupled to NEMO conformational change. Shown is the concentration dependence of the ligand on either ANS or Trp⁶ emission for polyubiquitin (Poly-Ubi; A) or the NBD and IκBα peptides (B). A signal value of 1 represents the background-subtracted NEMO emission. Solid lines are fits to a weak binding isotherm (E_m = (E_max[L])/(K + L), where L is ligand) or the Hill equation (E_m = (E_max[L]ⁿ)/(Kⁿ + Lⁿ)) (B, black lines). Conditions were 50 mm Tris-Cl (pH 8), 50 mm NaCl, 5 mm DTT, 25 μg/ml polyubiquitin, 2 mm IκBα, and 6 μm NEMO at 10 °C. W6, Trp⁶.

FIGURE 6.

Deconvolved ANS emission spectra in the presence of NEMO (black line) and NEMO with a peptide derived from collagen (red line). The spectra do not show any significant numerical difference outside of standard error.

FIGURE 7.

Kinetics of ligand-induced NEMO conformational change. A, example stopped-flow transient showing the multiphasic nature of the emission change detected upon rapid mixing of NEMO versus 0.5 mm NBD (black line) or IκBα (gray line). The solid red line is the fit to Equation 4. B, concentration dependence of NBD/IκBα on the extracted observed rate constants in the absence (closed circles) and presence (open circles) of Ub₁₀. Solid lines are the fit to Equation 6. Conditions were 50 mm Tris-Cl (pH 8), 50 mm NaCl, 5 mm DTT, and 5 μm NEMO at 10 °C.

FIGURE 8.

SDS-PAGE showing the results of the liposome binding assay. The protein bands shown ran at ∼50 kDa and are indicative of full-length NEMO. The contrast has been enhanced evenly across the image to better visualize the bands. The asterisks mark those samples in which polyubiquitin (Ubi₁₀) was present and are coincident with the occurrence of significantly observable protein bands. The polyubiquitin concentration was 0.5 μm, and the NBD and IκBα concentrations were 1 mm. Lipo, liposome; +ve, positive control.

FIGURE 9.

Molecular mechanistic model for the activity of NEMO, incorporating ligand-induced conformational change. A, mechanistic scheme outlining the steps associated with ligand binding and subsequent conformational change. B, molecular model for the role of free polyubiquitin chains in mediating the recruitment of both IKKβ and IκBα but also recruitment of the ternary complex to the membrane.