RESUMEN
The plant homeodomain (PHD) fingers are among the largest family of epigenetic domains, first characterized as readers of methylated H3K4. Readout of histone post-translational modifications by PHDs has been the subject of intense investigation; however, less is known about the recognition of secondary structure features within the histone tail itself. We solved the crystal structure of the PHD finger of the bromodomain adjacent to zinc finger 2A [BAZ2A, also known as TIP5 (TTF-I/interacting protein 5)] in complex with unmodified N-terminal histone H3 tail. The peptide is bound in a helical folded-back conformation after K4, induced by an acidic patch on the protein surface that prevents peptide binding in an extended conformation. Structural bioinformatics analyses identify a conserved Asp/Glu residue that we name 'acidic wall', found to be mutually exclusive with the conserved Trp for K4Me recognition. Neutralization or inversion of the charges at the acidic wall patch in BAZ2A, and homologous BAZ2B, weakened H3 binding. We identify simple mutations on H3 that strikingly enhance or reduce binding, as a result of their stabilization or destabilization of H3 helicity. Our work unravels the structural basis for binding of the helical H3 tail by PHD fingers and suggests that molecular recognition of secondary structure motifs within histone tails could represent an additional layer of regulation in epigenetic processes.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Histonas/metabolismo , Modelos Moleculares , Proteínas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Sitios de Unión , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/genética , Secuencia Conservada , Cristalografía por Rayos X , Histonas/química , Histonas/genética , Humanos , Cinética , Ligandos , Simulación de Dinámica Molecular , Mutación , Oligonucleótidos/química , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Conformación Proteica en Hélice alfa , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , Proteínas/química , Proteínas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Factores Generales de TranscripciónRESUMEN
Plant homeodomain (PHD) zinc fingers are histone reader domains that are often associated with human diseases. Despite this, they constitute a poorly targeted class of readers, suggesting low ligandability. Here, we describe a successful fragment-based campaign targeting PHD fingers from the proteins BAZ2A and BAZ2B as model systems. We validated a pool of in silico fragments both biophysically and structurally and solved the first crystal structures of PHD zinc fingers in complex with fragments bound to an anchoring pocket at the histone binding site. The best-validated hits were found to displace a histone H3 tail peptide in competition assays. This work identifies new chemical scaffolds that provide suitable starting points for future ligand optimization using structure-guided approaches. The demonstrated ligandability of the PHD reader domains could pave the way for the development of chemical probes to drug this family of epigenetic readers.