RESUMEN
The N-terminal domain of MDM4 binds to the N-terminal transactivation domain of the tumor suppressor p53 and is an important negative regulator of its transactivation activity. As such, inhibition of the binding of MDM4 to p53 is a target for anticancer therapy. The protein has not been crystallized satisfactorily for structural studies without the addition of an N-terminal p53 peptide. We selected a single-domain antibody (VH9) that bound to the human domain with a dissociation constant of 44 nM. We solved the structure of the complex at 2.0-A resolution. The asymmetric unit contained eight molecules of VH9 and four molecules of MDM4. A molecule of VH9 was located in each transactivation domain binding site, and the four non-MDM4-bound VH9 domains provided additional crystal contacts. There are differences between the structures of human MDM4 domain bound to VH9 and those of human and zebra fish MDM4 bound to a p53 peptide. Molecular dynamics simulations showed that the binding pocket in the three MDM4 structures converged to a common conformation after removal of the ligands, indicating that the differences are due to induced fit. The largest conformational changes were for the MDM4 molecules bound to p53. The simulated and observed structures should aid rational drug design. The use of single-domain antibodies to aid crystallization by creating a molecular scaffold may have a wider range of applications.
Asunto(s)
Anticuerpos/química , Proteínas Nucleares/química , Proteínas Proto-Oncogénicas/química , Secuencia de Aminoácidos , Sitios de Unión , Proteínas de Ciclo Celular , Simulación por Computador , Cristalografía por Rayos X , Humanos , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Proteína p53 Supresora de Tumor/químicaRESUMEN
HDM2 is a negative regulator of p53 that inhibits its transcriptional activity and subjects it to degradation by an E3 ligase activity. The primary binding site for HDM2 on p53 is located in its N-terminal domain. A second site on the p53 core domain (p53C) binds to an unidentified site in HDM2. We found that this site is in its acidic domain and part of the zinc finger domain by examining the interaction of full-length and domain constructs of p53 with the N-terminal region of HDM2 and peptide arrays derived from the full-length protein. NMR spectroscopy showed that peptides derived from this region of HDM2 bound to residues in the specific DNA-binding site of p53C. The peptides were displaced from the site by gadd45 sequence-specific DNA. Phosphorylation of single amino acids in the central domain of HDM2 did not abolish the interaction between the HDM2-derived peptides and p53C. We speculate that this second binding site helps in stabilizing the interaction between HDM2 and p53 during p53 degradation.
Asunto(s)
Genes p53 , Proteína p53 Supresora de Tumor/química , Secuencia de Aminoácidos , Área Bajo la Curva , Sitios de Unión , Unión Competitiva , Calorimetría , ADN/química , ADN Complementario/metabolismo , Escherichia coli/metabolismo , Polarización de Fluorescencia , Humanos , Immunoblotting , Péptidos y Proteínas de Señalización Intracelular/química , Espectroscopía de Resonancia Magnética , Microscopía Fluorescente , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Unión Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Proteína p53 Supresora de Tumor/metabolismo , Dedos de Zinc , Proteinas GADD45RESUMEN
HDM2 is a ubiquitin E3 ligase that is a key negative regulator of the tumor suppressor p53. Here, we report the determination of the solution structure of the C4 zinc finger domain of HDM2 using multidimensional NMR. The HDM2 C4 zinc finger domain has a fold consisting of a 3(10) helix followed by four beta-strands, which shares significant structural similarity to the zinc ribbon protein family. Family based sequence analysis identified two putative binding sites, one of which resembles an RNA binding motif.
