title: Electron Microscopy Structural Insights into CPAP Oligomeric Behavior: A Plausible Assembly Process of a Supramolecular Scaffold of the Centrosome.
abstract: Centrosomal P4.1-associated protein (CPAP) is a cell cycle regulated protein fundamental for centrosome assembly and centriole elongation. In humans, the region between residues 897-1338 of CPAP mediates interactions with other proteins and includes a homodimerization domain. CPAP mutations cause primary autosomal recessive microcephaly and Seckel syndrome. Despite of the biological/clinical relevance of CPAP, its mechanistic behavior remains unclear and its C-terminus (the G-box/TCP domain) is the only part whose structure has been solved. This situation is perhaps due in part to the challenges that represent obtaining the protein in a soluble, homogeneous state for structural studies. Our work constitutes a systematic structural analysis on multiple oligomers ofHsCPAP897-1338, using single-particle electron microscopy (EM) of negatively stained (NS) samples. Based on image classification into clearly different regular 3D maps (putatively corresponding to dimers and tetramers) and direct observation of individual images representing other complexes ofHsCPAP897-1338(i.e., putative flexible monomers and higher-order multimers), we report a dynamic oligomeric behavior of this protein, where different homo-oligomers coexist in variable proportions. We propose that dimerization of the putative homodimer forms a putative tetramer which could be the structural unit for the scaffold that either tethers the pericentriolar material to centrioles or promotes procentriole elongation. A coarse fitting of atomic models into the NS 3D maps at resolutions around 20 Å is performed only to complement our experimental data, allowing us to hypothesize on the oligomeric composition of the different complexes. In this way, the current EM work represents an initial step toward the structural characterization of different oligomers of CPAP, suggesting further insights to understand how this protein works, contributing to the elucidation of control mechanisms for centriole biogenesis.
authors.affiliation: Biocomputing Unit, Macromolecular Structures, Centro Nacional de Biotecnología-CSICMadrid, Spain; Molecular and Computational Biology Program, Department of Biological Sciences, University of Southern CaliforniaLos Angeles, CA, USA.
authors.forename: Ana L
authors.initials: AL
authors.lastname: Alvarez-Cabrera
authors.affiliation: Structural Biology Unit, CIC bioGUNE Derio, Spain.
authors.forename: Sandra
authors.initials: S
authors.lastname: Delgado
authors.affiliation: Structural Biology Unit, CIC bioGUNE Derio, Spain.
authors.forename: David
authors.initials: D
authors.lastname: Gil-Carton
authors.affiliation: Protein Structure and Function Program, Faculty of Health and Medical Sciences, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen Copenhagen, Denmark.
authors.forename: Gulnahar B
authors.initials: GB
authors.lastname: Mortuza
authors.affiliation: Protein Structure and Function Program, Faculty of Health and Medical Sciences, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen Copenhagen, Denmark.
authors.forename: Guillermo
authors.initials: G
authors.lastname: Montoya
authors.affiliation: Biocomputing Unit, Macromolecular Structures, Centro Nacional de Biotecnología-CSIC Madrid, Spain.
authors.forename: Carlos O S
authors.initials: CO
authors.lastname: Sorzano
authors.affiliation: Institute of Biomedical Sciences, Academia Sinica Taipei, Taiwan.
authors.forename: Tang K
authors.initials: TK
authors.lastname: Tang
authors.affiliation: Biocomputing Unit, Macromolecular Structures, Centro Nacional de Biotecnología-CSIC Madrid, Spain.
authors.forename: Jose M
authors.initials: JM
authors.lastname: Carazo
keywords: centriole; centrosome; cilia; electron microscopy; negative staining; pericentiolar material (PCM)