Electron Microscopy Structural Insights into CPAP Oligomeric Behavior: A Plausible Assembly Process of a Supramolecular Scaffold of the Centrosome.

Ana L Alvarez-Cabrera,Sandra Delgado,Tang K Tang,Guillermo Montoya,Gulnahar B Mortuza,David Gil ,Carlos Óscar S Sorzano ,José Marı́a Carazo

doi:10.3389/fmolb.2017.00017

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 of HsCPAP897−1338, 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 of HsCPAP897−1338 (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.

Highlights

Centrosomes are found in most animal cells as the primary microtubule organizing centers (MTOC), being involved in important cellular processes, such as division, motility, and structural stabilization
In silico analysis of HsCPAP897−1338 secondary structure (Supplementary Figures 1A∗, 3B,C) predicts three different regions distributed as follows: 38% is mostly formed by α-helices and corresponds to the CC4/CC5 region, 19% constitutes the unstructured CCGb-linker, and 43% shows several short betastrands belonging to the G-box, which is in agreement with the solved structures of the equivalent domain of Centrosomal P4.1-associated protein (CPAP) in D. rerio and D. melanogaster (Cottee et al, 2013; Hatzopoulos et al, 2013; Zheng et al, 2014)
Taking together the analysis of our biochemical and biophysical data, the 2D images and the 3D volume obtained by the electron microscopy (EM) work, the solved crystallographic structure of the G-box domain and, the in silico structure modeling of the CC4/CC5 region and a gross estimation of the dimensions of the CCGb-linker of CPAP, we propose that this protein is highly flexible as a monomer, and that it presents a discrete but dynamic oligomeric behavior that includes the assembly of dimers, tetramers, and larger structures formed by stacks of tetramers

Summary

Introduction

Centrosomes are found in most animal cells as the primary microtubule organizing centers (MTOC), being involved in important cellular processes, such as division, motility, and structural stabilization. CPAP ( known as Centromere Protein J -CENPJ-, or as Sas-4 in D. melanogaster and SAS-4 in C. elegans) is a conserved and cell-cycle regulated protein (Tang et al, 2009) crucial for centrosome biogenesis. This versatile macromolecule plays roles in centriole elongation (by scaffolding cytoplasmic PCM complexes which posteriorly tether to centrioles; Zheng et al, 2014) and cilium disassembly (by providing a scaffold for the cilium disassembly complex, CDC; Gabriel et al, 2016); it contributes to maintain spindle pole integrity (Chou et al, 2016). CPAP can be localized in different structures within the centrosome (Hung et al, 2000; Kleylein-Sohn et al, 2007) and in the cytoplasm (Schmidt et al, 2009)

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Conclusion