PDBsum entry 1my5

Transcription

PDB id: 1my5

Contents

Protein chains

107 a.a. *

101 a.a. *

Waters ×168

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Solvent exposed non-Contacting amino acids play a critical role in nf-Kappab/ikappabalpha complex formation.

Authors

T.Huxford, D.Mishler, C.B.Phelps, D.B.Huang, L.L.Sengchanthalangsy, R.Reeves, C.A.Hughes, E.A.Komives, G.Ghosh.

Ref.

J Mol Biol, 2002, 324, 587-597. [DOI no: 10.1016/S0022-2836(02)01149-X]

PubMed id

12460563

Abstract

IkappaBalpha inhibits transcription factor NF-kappaB activity by specific binding to NF-kappaB heterodimers composed of p65 and p50 subunits. It binds with slightly lower affinity to p65 homodimers and with significantly lower affinity to homodimers of p50. We have employed a structure-based mutagenesis approach coupled with protein-protein interaction assays to determine the source of this dimer selectivity exhibited by IkappaBalpha. Mutation of amino acid residues in IkappaBalpha that contact NF-kappaB only marginally affects complex binding affinity, indicating a lack of hot spots in NF-kappaB/IkappaBalpha complex formation. Conversion of the weak binding NF-kappaB p50 homodimer into a high affinity binding partner of IkappaBalpha requires transfer of both the NLS polypeptide and amino acid residues Asn202 and Ser203 from the NF-kappaB p65 subunit. Involvement of Asn202 and Ser203 in complex formation is surprising as these amino acid residues occupy solvent exposed positions at a distance of 20A from IkappaBalpha in the crystal structures. However, the same amino acid residue positions have been genetically isolated as determinants of binding specificity in a homologous system in Drosophila. X-ray crystallographic and solvent accessibility experiments suggest that these solvent-exposed amino acid residues contribute to NF-kappaB/IkappaBalpha complex formation by modulating the NF-kappaB p65 subunit NLS polypeptide.

Figure 4.
Figure 4. Mutation of Asn202 in p65 decreases IkBa binding affinity of both the NF-kB p65 homodimer and p50/p65 heterodimer. (a) Mutation of NF-kB p65(191-325) homodimer amino acid residues Asn202 and Ser203 to the corresponding p50 amino acid residues Thr258 and Ala259 results in a measurable decrease in IkBa binding affinity (right half) when compared to the native p65(191-325) homodimer (left half). (b) Mutation of NF-kB p65(191-325) homodimer Asn202 to Arg, analogous to a genetically derived mutation that disrupts binding of the Drosophila homologues Dorsal and Cactus, results in a significant loss of IkBa binding affinity (compare right and left gel halves). (c) A decrease in IkBa binding affinity also accompanies incorporation of the Asn202 to Arg mutation within the context of the NF-kB p50(245-376)/p65(191-325) heterodimer (compare left and right gel halves).

Figure 5.
Figure 5. Structural differences in the NLS polypeptides of NF-kB p65 and p65 Asn202Arg homodimers. (a) Ribbon diagram of the NF-kB p65(191-304) homodimer. A ball and stick representation of the Asn202 side-chain indicates its position near the dimer interface in this protein. (b) Similar ribbon diagram of NF-kB p65(191-304) bearing the Asn202 to Arg mutation. The mutant Arg side-chain is depicted as a ball and stick model. (c) Stereoview of electron density from a 2F[O] -F[C] difference Fourier map contoured at 2s for the region of the native p65 NLS polypeptide and a crystallographic neighboring molecule. (d) The same region in the Asn202 to Arg mutated p65 homodimer displays broken and disordered electron density for the NLS polypeptide as well as additional side-chain electron density at the site of mutation. Note the overall improved electron density for the ordered region of the mutated protein, which was determined with higher resolution diffraction data.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2002, 324, 587-597) copyright 2002.