12 research outputs found
Disulfide-Mediated Stabilization of the IκB Kinase Binding Domain of NF-κB Essential Modulator (NEMO)
Human NEMO (NF-κB
essential modulator) is a 419 residue scaffolding
protein that, together with catalytic subunits IKKα and IKKβ,
forms the IκB kinase (IKK) complex, a key regulator of NF-κB
pathway signaling. NEMO is an elongated homodimer comprising mostly
α-helix. It has been shown that a NEMO fragment spanning residues
44–111, which contains the IKKα/β binding site,
is structurally disordered in the absence of bound IKKβ. Herein
we show that enforcing dimerization of NEMO<sub>1–120</sub> or NEMO<sub>44–111</sub> constructs through introduction
of one or two interchain disulfide bonds, through oxidation of the
native Cys54 residue and/or at position 107 through a Leu107Cys mutation,
induces a stable α-helical coiled-coil structure that is preorganized
to bind IKKβ with high affinity. Chemical and thermal denaturation
studies showed that, in the context of a covalent dimer, the ordered
structure was stabilized relative to the denatured state by up to
3 kcal/mol. A full-length NEMO-L107C protein formed covalent dimers
upon treatment of mammalian cells with H<sub>2</sub>O<sub>2</sub>.
Furthermore, NEMO-L107C bound endogenous IKKβ in A293T cells,
reconstituted TNF-induced NF-κB signaling in NEMO-deficient
cells, and interacted with TRAF6. Our results indicate that the IKKβ
binding domain of NEMO possesses an ordered structure in the unbound
state, provided that it is constrained within a dimer as is the case
in the constitutively dimeric full-length NEMO protein. The stability
of the NEMO coiled coil is maintained by strong interhelix interactions
in the region centered on residue 54. The disulfide-linked constructs
we describe herein may be useful for crystallization of NEMO’s
IKKβ binding domain in the absence of bound IKKβ, thereby
facilitating the structural characterization of small-molecule inhibitors
Mutation of Nonessential Cysteines Shows That the NF-κB Essential Modulator Forms a Constitutive Noncovalent Dimer That Binds IκB Kinase‑β with High Affinity
NEMO
(NF-κB essential modulator) associates with catalytic
subunits IKKα and IKKβ to form the IκB kinase (IKK)
complex and is a key regulator of NF-κB pathway signaling. Biochemical
and structural characterization of NEMO has been challenging, however,
leading to conflicting data about basic biochemical properties such
as the oligomeric state of active NEMO and its binding affinity for
IKKβ. We show that up to seven of NEMO’s 11 cysteine
residues can be mutated to generate recombinant full-length NEMO that
is highly soluble and active. Using a fluorescence anisotropy binding
assay, we show that full-length NEMO binds a 44-mer peptide encompassing
residues 701–745 of IKKβ with a <i>K</i><sub>D</sub> of 2.2 ± 0.8 nM. The IKKβ binding affinities of
mutants with five and seven Cys-to-Ala substitutions are indistinguishable
from that of wild-type NEMO. Moreover, when expressed in NEMO −/–
fibroblasts, the five-Ala and seven-Ala NEMO mutants can interact
with cellular IKKβ and restore NF-κB signaling to provide
protection against tumor necrosis factor α-induced cell death.
Treatment of the NEMO-reconstituted cells with H<sub>2</sub>O<sub>2</sub> led to the formation of covalent dimers for wild-type NEMO
and the five-Ala mutant, but not for the seven-Ala mutant, confirming
that Cys54 and/or Cys347 can mediate interchain disulfide bonding.
However, the IKKβ binding affinity of NEMO is unaffected by
the presence or absence of interchain disulfide bonding at Cys54,
which lies within the IKKβ binding domain of NEMO, or at Cys347,
indicating that NEMO exists as a noncovalent dimer independent of
the redox state of its cysteines. This conclusion was corroborated
by the observation that the secondary structure content of NEMO and
its thermal stability were independent of the presence or absence
of interchain disulfide bonds
ELISA.
<p>Optical density (OD) at 450 nm measured at 2.5 h of pNPP incubation. OD for the different compounds at different concentrations is given. A and B illustrate the ability of the compounds to recognize Aβ fibrils. They indicate two series of experiments performed with following compounds A: ACI-80-Kφ to ACI-86-Kφ. ACI-80 without φ-label was run as a control. B: ACI-87-Kφ to ACI-89-Kφ, as well as fluorinated derivatives. ACI-80-Kφ, ACI-82-Kφ and ACI-83-Kφ were included as controls. C and D illustrate the ability of the compounds to recognize peptide film which largely consists of Aβ monomers.</p
Surface plasmon resonance analysis of the interaction between immobilized Aβ1–42 fibrils and ACI-80-Kφ and various derivatives (Kφ presents a lysine (K) covalently linked to a fluorescein isothiocyanate (φ).
<p>ACI-80 derivatives were solved in running buffer (PBS, pH 7.4). The injected volume of ACI-80 derivatives was 10 µl of a 50 µg/ml concentration using a flow rate of 5 µl/min. The response of ACI-80-Kφ in resonance units [RU] was defined as 100%. Values >100% denote increased Aβ interaction of the ACI-80 derivative in comparison to ACI-80-Kφ. All derivatives were φ-labeled. Only the variations in comparison to ACI-80-Kφ are indicated in the figure.</p
Pyroglutamate content of ACI-80, ACI-80-Kφ and [<sup>127</sup>I]-ACI-80.
<p>Kφ presents a lysine (K) linked to a fluorescein isothiocyanate (φ).</p
ELISA: Mean binding values for compounds with concentration of 10 µg/ml.
<p>All values were compared to that of ACI-80-Kφ. Compound binding to compound film, containing predominantly monomers and to fibrils was measured. Average values of two or three experiments unless marked otherwise. *value of one single experiment only. Kφ presents a lysine (K) linked to a fluorescein isothiocyanate (φ).</p
D-enantiomeric peptide variants binding to fibrillar Aß1–42, covalently immobilized on a CM5 sensor chip via amine coupling.
<p>For each peptide variant experimental sensorgrams (black traces) obtained with injections at 2500 nM, 12500 nM and 62500 nM (ACI-80-Kφ, ACI-87-Kφ) or 500 nM, 2500 nM and 12500 nM (ACI-88-Kφ, ACI-89-Kφ) are shown. Injections were performed for 60 seconds each and dissociation phases were recorded for at least 30 seconds. The sensorgrams were globally fit (red curves) to a heterogeneous ligand model accounting for different refractive indices.</p
Results of the binding assays for ACI-80-Kφ derivatives to Aβ1–42 fibrils using surface plasmon resonance.
<p>Interaction and dissociation was measured with respect to the maximal interaction signal during injection and the response 60 s after the end of injection, respectively. ACI-80-Kφ binding was defined as 100%. Kφ presents a lysine (K) linked to a fluorescein isothiocyanate (φ).</p
<i>Ex vivo</i> staining of brain tissue sections from 13 months old male double transgenic AD mice APP (V717I) x PS1 (A246E) using different φ-labeled ACI-80 derivatives, 6E10-Aβ-antibody and DAPI.
<p>Left column: triple image overlay of respective stains reveal that the φ-compounds identify plaques. White scale bars 20 µm. Results of non-transgenic litter mate controls are not shown as no staining of φ-labeled ACI-80 derivatives and 6E10-Aβ-antibody could be detected.</p
List of investigated D-enantiomeric peptide compounds.
<p>Modifications in the original amino acid sequence of ACI-80 are printed in bold. Amino acid residues are given in the one-letter-code. All amino-acids are D-enantiomers. Kφ presents a lysine (K) linked to a fluorescein isothiocyanate (φ).</p