Huxford Tom, Mishler Dennis, Phelps Christopher B, Huang De-Bin, Sengchanthalangsy Lei Lei, Reeves Ryan, Hughes Carrie A, Komives Elizabeth A, Ghosh Gourisankar
Department of Chemistry and Biochemistry, University of California at San Diego, Mail Code 0359, 9500 Gilman Drive, La Jolla, CA 92093-0359, USA.
J Mol Biol. 2002 Dec 6;324(4):587-97. doi: 10.1016/s0022-2836(02)01149-x.
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.
IκBα通过与由p65和p50亚基组成的NF-κB异二聚体特异性结合来抑制转录因子NF-κB的活性。它与p65同二聚体的结合亲和力略低，与p50同二聚体的结合亲和力则显著更低。我们采用了基于结构的诱变方法并结合蛋白质-蛋白质相互作用分析，以确定IκBα所表现出的这种二聚体选择性的来源。IκBα中与NF-κB接触的氨基酸残基发生突变，对复合物的结合亲和力仅产生轻微影响，这表明在NF-κB/IκBα复合物形成过程中不存在热点区域。要将弱结合的NF-κB p50同二聚体转化为IκBα的高亲和力结合伴侣，需要从NF-κB p65亚基转移核定位信号（NLS）多肽以及氨基酸残基Asn202和Ser203。Asn202和Ser203参与复合物形成令人惊讶，因为在晶体结构中，这些氨基酸残基位于距离IκBα 20埃的溶剂暴露位置。然而，在果蝇的同源系统中，相同的氨基酸残基位置已被遗传分离出来作为结合特异性的决定因素。X射线晶体学和溶剂可及性实验表明，这些溶剂暴露的氨基酸残基通过调节NF-κB p65亚基的NLS多肽来促进NF-κB/IκBα复合物的形成。
