PDBsum entry 2v4h

Transcription

PDB id: 2v4h

Contents

Protein chains

98 a.a. *

91 a.a. *

125 a.a. *

Waters ×51

* Residue conservation analysis

PDB id:

2v4h

Name:

Transcription

Title:

Nemo cc2-lz domain - 1d5 darpin complex

Structure:

Nf-kappa-b essential modulator. Chain: a, b. Fragment: cc2-lz domain, residues 251-337. Synonym: nf-kappa-b essential modifier, inhibitor of nuclear factor kappa-b kinase subunit gamma, ikb kinase-associated protein 1, nemo. Engineered: yes. 1d5 darpin. Chain: c, d. Engineered: yes.

Source:

Mus musculus. House mouse. Organism_taxid: 10090. Expressed in: escherichia coli. Expression_system_taxid: 469008. Synthetic construct. Organism_taxid: 32630. Expression_system_taxid: 562.

Resolution:

2.90Å R-factor: 0.211 R-free: 0.268

Authors:

O.Grubisha,S.Duquerroy,F.Cordier,A.Haouz,M.Delepierre,M.Veron,F.Agou

Key ref:

O.Grubisha et al. (2010). DARPin-Assisted Crystallography of the CC2-LZ Domain of NEMO Reveals a Coupling between Dimerization and Ubiquitin Binding. J Mol Biol, 395, 89. PubMed id: 19854204 DOI: 10.1016/j.jmb.2009.10.018

Date:

22-Sep-08 Release date: 03-Nov-09

Protein chain

O88522  (NEMO_MOUSE) -  NF-kappa-B essential modulator from Mus musculus

Seq: 412 a.a.

Struc: 98 a.a.*

Protein chain

O88522  (NEMO_MOUSE) -  NF-kappa-B essential modulator from Mus musculus

Seq: 412 a.a.

Struc: 91 a.a.*

Protein chains

No UniProt id for this chain

Struc: 125 a.a.

* PDB and UniProt seqs differ at 12 residue positions (black crosses)

DOI no: 10.1016/j.jmb.2009.10.018

J Mol Biol 395:89 (2010)

PubMed id: 19854204

DARPin-Assisted Crystallography of the CC2-LZ Domain of NEMO Reveals a Coupling between Dimerization and Ubiquitin Binding.

O.Grubisha, M.Kaminska, S.Duquerroy, E.Fontan, F.Cordier, A.Haouz, B.Raynal, J.Chiaravalli, M.Delepierre, A.Israël, M.Véron, F.Agou.

ABSTRACT

NEMO is an integral part of the IkappaB kinase complex and serves as a molecular switch by which the NF-kappaB signaling pathway can be regulated. Oligomerization and polyubiquitin (poly-Ub) binding, mediated through the regulatory CC2-LZ domain, were shown to be key features governing NEMO function, but the relationship between these two activities remains unclear. In this study, we solved the structure of this domain in complex with a designed ankyrin repeat protein, which helps its crystallization. We generated several NEMO mutants in this domain, including those associated with human diseases incontinentia pigmenti and immunodeficiency with or without anhidrotic ectodermal dysplasia. Analytical ultracentrifugation and thermal denaturation experiments were used to evaluate the dimerization properties of these mutants. A fluorescence-based assay was developed, as well, to quantify the interaction to monoubiquitin and poly-Ub chains. Moreover, the effect of these mutations was investigated for the full-length protein. We show that a proper folding of the ubiquitin-binding domain, termed NOA/UBAN/NUB, into a stable coiled-coil dimer is required but not sufficient for efficient interaction with poly-Ub. In addition, we show that binding to poly-Ub and, to a lesser extent, to monoubiquitin increases the stability of the NOA coiled-coil dimer. Collectively, these data provide structural insights into how several pathological mutations within and outside of the CC2-LZ's NOA ubiquitin binding site affect IkappaB kinase activation in the NF-kappaB signaling pathway.

Selected figure(s)

Figure 2.
Fig. 2. Structural details and sequence conservation of the NOA motif. (a) Alignment of the NOA sequence from human, mouse, and Drosophila NEMO homologues and from human Optineurin and ABIN-1, -2, and -3 proteins. Numbers correspond to the first and last residue of the protein sequence. Identical and similar residues are colored red and yellow, respectively. Diamonds indicate residues linked to human pathologies: EDA-ID (blue), ID (green), and IP (red), corresponding to the following mutations (human numbering): D311N, E315A, R319Q, and A323P. The heptad position (a–g) is indicated at the bottom. (b) Close-up view of the NEMO NOA region (helices are colored as in Fig. 1). Conserved residues are shown as ball-and-stick representations, with O and N atoms in red and blue, respectively. Broken lines point to residues involved in salt bridge or H-bond formation. For clarity, only one set of conserved residues is displayed. (c) Helical wheel representation of the NOA coiled coil, starting from residue V293 at position c. Residues linked to human pathologies are colored red. Blue lines indicate intermolecular salt bridges.

Figure 4.
Fig. 4. Folding and dimeric stability of the human CC2-LZ[215–362] and its mutants. (a) Summary of molecular weight average and dimerization constant of WT and mutant CC2-LZ[215–362] from SE at 10 °C (14,000 rpm). The molecular weights deduced from SEC/LS are also indicated. (b) Measurements of the molecular weight of WT and A323P determined by SEC/LS. (c) Sedimentation coefficient distribution c(S[20]^W) of WT and E296A at 10 or 35 °C.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2010, 395, 89) copyright 2010.

Figures were selected by the author.