RESUMEN
Coiled-coils are ubiquitous protein-protein interaction motifs found in many eukaryotic proteins. The elongated, flexible and often irregular nature of coiled-coils together with their tendency to form fibrous arrangements in crystals imposes challenges on solving the phase problem by molecular replacement. Here, we report the successful combinatorial use of native and rational engineered disulfide bridges together with sulfur-SAD phasing as a powerful tool to stabilize and solve the structure of coiled-coil domains in a straightforward manner. Our study is a key example of how modern sulfur SAD combined with mutagenesis can help to advance and simplify the structural study of challenging coiled-coil domains by X-ray crystallography.
Asunto(s)
Secuencias de Aminoácidos/genética , Modelos Moleculares , Dominios y Motivos de Interacción de Proteínas/genética , Proteínas/química , Cristalografía por Rayos X , Disulfuros/química , Conformación Proteica , Ingeniería de Proteínas , Proteínas/genéticaRESUMEN
Tight regulation of kinesin activity is crucial and malfunction is linked to neurological diseases. Point mutations in the KIF21A gene cause congenital fibrosis of the extraocular muscles type 1 (CFEOM1) by disrupting the autoinhibitory interaction between the motor domain and a regulatory region in the stalk. However, the molecular mechanism underlying the misregulation of KIF21A activity in CFEOM1 is not understood. Here, we show that the KIF21A regulatory domain containing all disease-associated substitutions in the stalk forms an intramolecular antiparallel coiled coil that inhibits the kinesin. CFEOM1 mutations lead to KIF21A hyperactivation by affecting either the structural integrity of the antiparallel coiled coil or the autoinhibitory binding interface, thereby reducing its affinity for the motor domain. Interaction of the KIF21A regulatory domain with the KIF21B motor domain and sequence similarities to KIF7 and KIF27 strongly suggest a conservation of this regulatory mechanism in other kinesin-4 family members.
Asunto(s)
Enfermedades Hereditarias del Ojo/genética , Fibrosis/genética , Cinesinas/antagonistas & inhibidores , Cinesinas/genética , Trastornos de la Motilidad Ocular/genética , Dominios Proteicos/genética , Animales , Células COS , Línea Celular , Chlorocebus aethiops , Cristalografía por Rayos X , Células HEK293 , Humanos , Cinesinas/metabolismo , Simulación del Acoplamiento Molecular , Mutación/genética , Unión Proteica/genética , Pliegue de ProteínaRESUMEN
Dysfunction of cilia is associated with common genetic disorders termed ciliopathies. Knowledge on the interaction networks of ciliary proteins is therefore key for understanding the processes that are underlying these severe diseases and the mechanisms of ciliogenesis in general. Cep104 has recently been identified as a key player in the regulation of cilia formation. Using a combination of sequence analysis, biophysics, and x-ray crystallography, we obtained new insights into the domain architecture and interaction network of the Cep104 protein. We solved the crystal structure of the tumor overexpressed gene (TOG) domain, identified Cep104 as a novel tubulin-binding protein, and biophysically characterized the interaction of Cep104 with CP110, Cep97, end-binding (EB) protein, and tubulin. Our results represent a solid platform for the further investigation of the microtubule-EB-Cep104-tubulin-CP110-Cep97 network of proteins. Ultimately, such studies should be of importance for understanding the process of cilia formation and the mechanisms underlying different ciliopathies.
Asunto(s)
Proteínas Asociadas a Microtúbulos/química , Centriolos/química , Centriolos/genética , Centriolos/metabolismo , Cilios/química , Cilios/metabolismo , Ciliopatías/genética , Ciliopatías/metabolismo , Cristalografía por Rayos X , Humanos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Dominios ProteicosRESUMEN
Centrioles are critical for the formation of centrosomes, cilia and flagella in eukaryotes. They are thought to assemble around a nine-fold symmetric cartwheel structure established by SAS-6 proteins. Here, we have engineered Chlamydomonas reinhardtii SAS-6-based oligomers with symmetries ranging from five- to ten-fold. Expression of a SAS-6 mutant that forms six-fold symmetric cartwheel structures in vitro resulted in cartwheels and centrioles with eight- or nine-fold symmetries in vivo. In combination with Bld10 mutants that weaken cartwheel-microtubule interactions, this SAS-6 mutant produced six- to eight-fold symmetric cartwheels. Concurrently, the microtubule wall maintained eight- and nine-fold symmetries. Expressing SAS-6 with analogous mutations in human cells resulted in nine-fold symmetric centrioles that exhibited impaired length and organization. Together, our data suggest that the self-assembly properties of SAS-6 instruct cartwheel symmetry, and lead us to propose a model in which the cartwheel and the microtubule wall assemble in an interdependent manner to establish the native architecture of centrioles.