RESUMEN
Centrioles are key eukaryotic organelles that are responsible for the formation of cilia and flagella, and for organizing the microtubule network and the mitotic spindle in animals. Centriole assembly requires oligomerization of the essential protein spindle assembly abnormal 6 (SAS-6), which forms a structural scaffold templating the organization of further organelle components. A dimerization interaction between SAS-6 N-terminal "head" domains was previously shown to be essential for protein oligomerization in vitro and for function in centriole assembly. Here, we developed a pharmacophore model allowing us to assemble a library of low-molecular-weight ligands predicted to bind the SAS-6 head domain and inhibit protein oligomerization. We demonstrate using NMR spectroscopy that a ligand from this family binds at the head domain dimerization site of algae, nematode, and human SAS-6 variants, but also that another ligand specifically recognizes human SAS-6. Atomistic molecular dynamics simulations starting from SAS-6 head domain crystallographic structures, including that of the human head domain which we now resolve, suggest that ligand specificity derives from favorable Van der Waals interactions with a hydrophobic cavity at the dimerization site.
Asunto(s)
Proteínas de Caenorhabditis elegans/antagonistas & inhibidores , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/metabolismo , Centriolos/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Multimerización de Proteína , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Caenorhabditis elegans/efectos de los fármacos , Caenorhabditis elegans/crecimiento & desarrollo , Centriolos/efectos de los fármacos , Simulación de Dinámica Molecular , Conformación ProteicaRESUMEN
Centrioles are conserved organelles fundamental for the organisation of microtubules in animal cells. Oligomerisation of the spindle assembly abnormal protein 6 (SAS-6) is an essential step in the centriole assembly process and may act as trigger for the formation of these organelles. SAS-6 oligomerisation is driven by two independent interfaces, comprising an extended coiled coil and a dimeric N-terminal globular domain. However, how SAS-6 oligomerisation is controlled remains unclear. Here, we show that in the Caenorhabditis elegans SAS-6, a segment of the N-terminal globular domain, unresolved in crystallographic structures, comprises a flexible loop that assists SAS-6 oligomerisation. Atomistic molecular dynamics simulations and nuclear magnetic resonance experiments suggest that transient interactions of this loop across the N-terminal dimerisation interface stabilise the SAS-6 oligomer. We discuss the possibilities presented by such flexible SAS-6 segments for the control of centriole formation.