ABSTRACT
ZYG-1 is a polo-like kinase essential for centriole assembly in Caenorhabditis elegans. The targeting of ZYG-1 to nascent centrioles is via its central cryptic polo-box (CPB) domain. To shed light on the molecular basis of ZYG-1 recruitment, it is necessary to obtain structural knowledge of the ZYG-1 CPB. Here, the expression, purification and preliminary crystallographic analysis of the ZYG-1 CPB are reported. The protein was overexpressed in Escherichia coli strain BL21 (DE3), purified by multi-step chromatography and crystallized using the vapour-diffusion method. Crystals of the wild-type protein exhibited an order-disorder pathology, which was solved by reductive lysine methylation. A complete anomalous data set was collected to 2.54â Å resolution at the Seâ K edge (λ = 0.9792â Å). The crystal belonged to space group P2, with unit-cell parameters a = 53.3, b = 60.09, c = 87.51â Å, ß = 93.31°. There were two molecules in the asymmetric unit.
Subject(s)
Caenorhabditis elegans Proteins/chemistry , Caenorhabditis elegans , Protein Kinases/chemistry , Amino Acid Sequence , Animals , Caenorhabditis elegans Proteins/biosynthesis , Caenorhabditis elegans Proteins/isolation & purification , Crystallization , Crystallography, X-Ray , Escherichia coli , Molecular Sequence Data , Protein Kinases/biosynthesis , Protein Kinases/isolation & purificationABSTRACT
Trypanosoma brucei BILBO1 (TbBILBO1) is an essential component of the flagellar pocket collar of trypanosomes. We recently reported the high resolution structure of the N-terminal domain of TbBILBO1. Here, we provide further structural dissections of its other three constituent domains: EF-hand, coiled coil, and leucine zipper. We found that the EF-hand changes its conformation upon calcium binding, the central coiled coil forms an antiparallel dimer, and the C-terminal leucine zipper appears to contain targeting information. Furthermore, interdimer interactions between adjacent leucine zippers allow TbBILBO1 to form extended filaments in vitro. These filaments were additionally found to condense into fibers through lateral interactions. Based on these experimental data, we propose a mechanism for TbBILBO1 assembly at the flagellar pocket collar.
Subject(s)
Cytoskeletal Proteins/metabolism , Protozoan Proteins/metabolism , Trypanosoma brucei brucei/metabolism , Amino Acid Sequence , Animals , Cell Line , Cytoskeletal Proteins/chemistry , Dimerization , Microscopy, Electron , Molecular Sequence Data , Protozoan Proteins/chemistry , Sequence Homology, Amino AcidABSTRACT
Plk4 family kinases control centriole assembly. Plk4s target mother centrioles through an interaction between their cryptic polo box (CPB) and acidic regions in the centriolar receptors SPD-2/Cep192 and/or Asterless/Cep152. Here, we report a crystal structure for the CPB of C. elegans ZYG-1, which forms a Z-shaped dimer containing an intermolecular ß sheet with an extended basic surface patch. Biochemical and in vivo analysis revealed that electrostatic interactions dock the ZYG-1 CPB basic patch onto the SPD-2-derived acidic region to promote ZYG-1 targeting and new centriole assembly. Analysis of a different crystal form of the Drosophila Plk4 (DmPlk4) CPB suggests that it also forms a Z-shaped dimer. Comparison of the ZYG-1 and DmPlk4 CPBs revealed structural changes in the ZYG-1 CPB that confer selectivity for binding SPD-2 over Asterless-derived acidic regions. Overall, our findings suggest a conserved mechanism for centriolar docking of Plk4 homologs that initiate daughter centriole assembly.
Subject(s)
Caenorhabditis elegans Proteins/chemistry , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/genetics , Centrioles/physiology , Models, Molecular , Protein Kinases/chemistry , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Amino Acid Sequence , Animals , Caenorhabditis elegans Proteins/genetics , Centrioles/metabolism , Dimerization , Molecular Sequence Data , Protein Binding , Protein Kinases/genetics , Protein Structure, Tertiary , Species Specificity , Structure-Activity RelationshipABSTRACT
The centriole is a conserved microtubule-based organelle essential for both centrosome formation and cilium biogenesis. It has a unique 9-fold symmetry and its assembly is governed by at least five component proteins (SPD-2, ZYG-1, SAS-5, SAS-6 and SAS-4), which are recruited in a hierarchical order. Recently published structural studies of the SAS-6 N-terminal domain have greatly advanced our understanding of the mechanisms of centriole assembly. However, it remains unclear how the weak interaction between the SAS-6 N-terminal head groups could drive the assembly of a closed ring-like structure, and what determines the stacking of multiple rings on top one another in centriole duplication. We recently reported that SAS-5 binds specifically to a very narrow region of the SAS-6 central coiled coil through its C-terminal domain (CTD, residues 391-404). Here, we further demonstrate by both static light scattering and small angle X-ray scattering that the SAS-5 N-terminal domain (NTD, residues 1-260) forms a tetramer. Specifically, we found that the tetramer is formed by SAS-5 residues 82-260, whereas residues 1-81 are intrinsically disordered. Taking these results together, we propose a working model for SAS-5-mediated assembly of the multi-layered central tube structure.
