RESUMO
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.
Assuntos
Aminoácidos/fisiologia , Proteínas I-kappa B/química , Proteínas I-kappa B/metabolismo , NF-kappa B/química , NF-kappa B/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Aminoácidos/genética , Animais , Arginina/metabolismo , Sítios de Ligação , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dimerização , Proteínas I-kappa B/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Inibidor de NF-kappaB alfa , NF-kappa B/genética , Sinais de Localização Nuclear/química , Sinais de Localização Nuclear/genética , Sinais de Localização Nuclear/metabolismo , Mutação Puntual , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Solventes/química , Relação Estrutura-AtividadeRESUMO
The changes in backbone hydrogen/deuterium (H/2H) exchange in the regulatory subunit (R(I)alpha(94-244)) of cyclic AMP-dependent protein kinase A (PKA) were probed by MALDI-TOF mass spectrometry. The three naturally occurring states of the regulatory subunit were studied: (1) free R(I)alpha(94-244), which likely represents newly synthesized protein, (2) R(I)alpha(94-244) bound to the catalytic (C) subunit, or holoenzyme, and (3) R(I)alpha(94-244) bound to cAMP. Protection from amide exchange upon C-subunit binding was observed for the helical subdomain, including the A-helix and B-helix, pointing to regions adjacent to those shown to be important by mutagenesis. In addition, C-subunit binding caused changes in observed amide exchange in the distal cAMP-binding pocket. Conversely, cAMP binding caused protection in the cAMP-binding pocket and increased exchange in the helical subdomain. These results suggest that the mutually exclusive binding of either cAMP or C-subunit is controlled by binding at one site transmitting long distance changes to the other site.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Hidrogênio/química , Estrutura Terciária de Proteína , Subunidades Proteicas/metabolismo , Amidas/química , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/química , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Subunidades Proteicas/química , Solventes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The epitope of a monoclonal antibody raised against human thrombin has been determined by hydrogen/deuterium exchange coupled to MALDI mass spectrometry. The antibody epitope was identified as the surface of thrombin that retained deuterium in the presence of the monoclonal antibody compared to control experiments in its absence. Covalent attachment of the antibody to protein G beads and efficient elution of the antigen after deuterium exchange afforded the analysis of all possible epitopes in a single MALDI mass spectrum. The epitope, which was discontinuous, consisting of two peptides close to anion-binding exosite I, was readily identified. The epitope overlapped with, but was not identical to, the thrombomodulin binding site, consistent with inhibition studies. The antibody bound specifically to human thrombin and not to murine or bovine thrombin, although these proteins share 86% identity with the human protein. Interestingly, the epitope turned out to be the more structured of two surface regions in which higher sequence variation between the three species is seen.
