Solvent exposed non-contacting amino acids play a critical role in NF-kappaB/IkappaBalpha complex formation.
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.
Huxford
Tom
T
Department of Chemistry and Biochemistry, University of California at San Diego, Mail Code 0359, 9500 Gilman Drive, La Jolla, CA 92093-0359, USA.
Mishler
Dennis
D
Phelps
Christopher B
CB
Huang
De-Bin
DB
Sengchanthalangsy
Lei Lei
LL
Reeves
Ryan
R
Hughes
Carrie A
CA
Komives
Elizabeth A
EA
Ghosh
Gourisankar
G
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000235:genetics / Q000502:physiology*; D000818:Animals; D001120:Arginine / Q000378:metabolism; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D020672:I-kappa B Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019913:Nuclear Localization Signals / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012997:Solvents / Q000737:chemistry; D013329:Structure-Activity Relationship
D000596:Amino Acids; D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D016328:NF-kappa B; D019913:Nuclear Localization Signals; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D012997:Solvents; D000072000:NF-KappaB Inhibitor alpha; D001120:Arginine