Consequences of fuzziness in the NFκB/IκBα interaction

Abstract

This chapter provides a short review of various biophysical experiments that have been applied to the inhibitor of kappa B, IκBα and its binding partner, nuclear factor kappa B, or NFκB. The picture that emerges from amide hydrogen/deuterium exchange, NMR and binding kinetics experiments is one in which parts of both proteins are "fuzzy" in the free-state and some parts remain "fuzzy" in the NFκB-IκBα complex. The NFκB family of transcription factors responds to inflammatory cytokines with rapid transcriptional activation, in which NFκB enters the nucleus and binds DNA. Just as rapidly as transcription is activated, it is subsequently repressed by newly synthesized IκBα?that also enters the nucleus and removes NFκB from the DNA. Because IκBα?is an ankyrin repeat protein, it's "fuzziness" can be controlled by mutagenesis to stabilized the folded state. Experimental comparison with such stabilized mutants helps provide evidence that much of the system control depends on the "fuzziness" of IκBα.

Figure 1

(A) Schematic diagram of NF-κB(p65) one of the most abundant NF-κB family members in the cell and of IκBα, the key member of the inhibitor family. (B) LEFT: The crystal structure of IκBα (blue) bound to NF-κB (p50, green; p65, red) . RIGHT: The crystal structure of NF-κB (p50, green; p65, red) bound to κB site DNA (gold) . (Figure prepared using PyMOL ).

Figure 2

(A) Observed vs. theoretical residual dipolar couplings measured by the program PALES for IκBα(67–206) (closed symbols) and SVD (open symbols) using the crystal structure of the IκBα•NF-κB complex (PDB accession code 1IKN, ). (B) Observed vs. AMD-calculated residual dipolar couplings for IκBα(67–206). The RDCs were measured as previously described .

Figure 3

(A) PONDR analysis of the intrinsic disorder in the ankyrin repeat domain of IκBα. (B) Summary of native state amide H/D exchange results on free IκBα. IκBα(67–317) was allowed to exchange for 2 min, and after quenching, the protein was digested with pepsin and the amount of exchange in each peptide was analyzed by MALDI mass spectrometry .

Figure 3

The HSQC spectra of free and IκBα-bound p65(289–321). The secondary chemical shifts of bound p65(289–321) show the characteristic positive values characteristic of helical regions, conforming to the helical areas seen in these same residues in the crystal structure of the IκBα•NF-κB complex (PDB accession code 1NFI, ). The chemical shifts for free p65(289–321) are indicative of an unfolded peptide.

Figure 5

(A) Native state amide H/D exchange data for the region of IκBα corresponding to the β-turn of AR5 (TOP) and AR6 (bottom) in the free-state (open circles) and in the NF-κB-bound state (closed circles). (B) Structural summary of the amide H/D exchange data (red is highly exchanging and blue is slowly exchanging) for IκBα in the free state (LEFT) and in the NF-κB-bound state (RIGHT) showing that exchange is similar for most of the IκBα molecule in each state, except for the β-turns of AR5 and AR6 that are exchanging much less in the bound state.

Figure 6

Structural ensemble of IκBα(67–206) showing representative structures from the AMD simulation. The molecule is colored red where NMR experiments indicated residues were undergoing conformational exchange (R_ex) .

Figure 7

(A) Equilibrium unfolding experiments with wild type IκBα and (B) Y254L,T257A mutant IκBα. The insets show the change in fluorescence of W258, a naturally-occurring Trp258 in AR6. In the wild type protein, this residue does not change fluorescence appreciably with denaturant, however in the stabilized mutant, its fluorescence changes in a manner similar to the CD signal indicating it follows the major cooperative folding transition of the protein. (C) IκB isoforms were measured by quantitative Western blot. The plot shows the levels of IκBα(Y254L,T257A) (blue) and wild-type IκBα (black) after cyclohexamide treatment in NF-κB −/− cells.

Figure 8

(A) Real-time SPR binding experiment in which κB-site DNA was bound to the streptavidin chip at t=0, then NF-κB(p50_(19-363)/p65_1-35) was allowed to associate with the DNA, and finally varying concentrations of IκBα were injected through the second sample loopand the dissociation rate constant (k_d) was measured . A schematic of the binding events is shown below the graph. (B) Dissociation rate constants for active dissociation are plotted as a function of IκBα variant concentration. (C) Comparison of the active dissociation rate constants of IκBα folding variants: wild type IκBα (●), IκBα(C186P, A220P) (■), IκBα(Q111G) (▲), IκBα(Y254L/Q255H) (►), IκBα(Y254L/T257A) (◄) .