Edited by: Kristina Djinovic-Carugo, University of Vienna, Austria
Reviewed by: Evangelia D. Chrysina, National Hellenic Research Foundation, Greece; Joost Schymkowitz, VIB-KU Leuven, Belgium
*Correspondence: Ana L.Alvarez-Cabrera
This article was submitted to Structural Biology, a section of the journal Frontiers in Molecular Biosciences
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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
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. This organelle is composed by two centrioles surrounded by a proteinaceous matrix of pericentriolar material (PCM), and is the base point for assembly o f cilia and flagella. Centrosomes serve as anchorage sites for a large number of regulatory processes (Doxsey et al.,
CPAP (also known as Centromere Protein J -CENPJ-, or as Sas-4 in
It has been suggested that the N-terminal domain of CPAP acts as a scaffold for other cytoplasmic proteins, while its C-terminal domain tethers the new ensemble PCM complexes to the centriole, thus allowing to continue the formation of normal and functional centrosomes (Gopalakrishnan et al.,
The C-terminus of CPAP includes a region known as the G-box or TCP domain, which is highly conserved through evolution, pointing to its primordial role in centriole/centrosome development. The determined crystallographic structures of the G-box/TCP domain revealed a solvent-exposed beta-sheet structure that remains stable by its own, despite lacking a hydrophobic core (Cottee et al.,
In human CPAP (
The full-length human cDNA of CPAP (1338 residues) was kindly provided by Professor Tang T. K., from the Institute of Biomedical Sciences, Academia Sinica. Taipei 115, Taiwan (Hung et al.,
After centrifugation (40,000 rpm/45 min) of the cell lysate, the soluble His-tagged protein in the clarified supernatant was purified by immobilized metal affinity chromatography (IMAC) with a Talon resin (Talon superflow. GE Healthcare) following the recommendations of the manufacturer. The collected fractions were analyzed by SDS-PAGE (Any KD Mini-PROTEAN TGX Gel. BIO-RAD), followed by staining with SimplyBlue SafeStain (Invitrogene); the CPAP identity of the observed protein bands was confirmed by Western Blot (against the His-tag) and Mass Spectrometry (MALDI TOF/TOF MS) analysis (data not shown).
Part of the protein samples eluted from IMAC (using a linear imidazole gradient) were concentrated and then loaded into either preparative or analytical Superdex 200 columns equilibrated with cold buffer H-300 (20 mM Hepes pH 7.4, 300 mM NaCl, 0.25 mM TCEP and 10% Glycerol) at a temperature of 4°C. The collected fractions were analyzed by SDS-PAGE (Any KD Mini-PROTEAN TGX Gel. BIO-RAD) stained with SimplyBlue SafeStain (Invitrogene). Some selected fractions from SEC were analyzed by NS-EM. Native-PAGE was used to determine the oligomeric state of flexible, non-globular particles.
The rest of the protein samples eluted from IMAC were pooled and dialyzed for 3 h at 4°C against buffer H-150 (20 mM Hepes pH 7.4, 150 mM NaCl, 0.25 mM TCEP and 10% Glycerol). During the dialysis process,
Predictions of order/globularity and “disorder” (highly flexible; Janin and Sternberg,
For negative staining (NS) of the samples, a drop of the protein solution was applied directly onto a glow-discharged EM grid (QUANTIFOIL. Formvar/Carbon. Cu 400 mesh grids), and allowed to be adsorbed on the grid surface (for few seconds or minutes, depending on protein concentration); then the drop was blotted with filter paper (Whatman grade No. 1) and the grid was washed by touching the surface with two consecutive drops of 0.75% (w/v) uranyl formate, blotting each time, and stained for 1 min with one more drop of the same staining agent. Finally, the grid was blotted again and allowed to air dry before observation.
NS-EM grids were examined in a JEOL JEM-1230 (accelerating voltage 100 kV) electron microscope, and images were recorded with a CCD camera ORIUS SC100 (4 × 2.7 k pixel) at 40,000x magnification. CTF corrected images were downsampled by a factor of 2 so the resulting image pixel size was 4.56 Å/pixel. All image preprocessing, particle selection and two-dimensional (2D) analysis steps were carried out following the general workflow of the image processing package Xmipp 3.1 (De la Rosa-Trevín et al.,
EM images of the
Atomic models for the different domains of
In our hands, the full-length
The sequence of
We faced problems obtaining reliable measurements of the protein concentration using both colorimetric (Bradford assay) and spectrophotometric (absorbance at 280 nm) methods. In the first case, very low and inconsistent measurements were obtained, possibly because of inefficient binding of the dye to the protein due to the low number of aromatic residues in
Variable SEC profiles obtained in a number of purifications of
The relatively broad elution profile obtained in some cases (Supplementary Figure
Visualization of NS-EM images of a number of
A putative assignment of the oligomeric states was performed based on elution profiles, native gels, EM images and maps, and a very coarse fitting of a number of copies of the atomic model of
A native PAGE of a SEC (Superdex 200 16/60) purified peak at elution volume of 64–70 ml (between apparent 120 and 170 kDa; Supplementary Figure
A 2D structural analysis of some of these flexible particles was made by measuring the general dimensions of the most representative images, which presented a cane-like shape with a “shaft” of ~15–17 nm long and a flexible “handle” of ~10 nm long. To give additional support to the assignment of a putative monomeric state to these flexible particles, a complete atomic model for a possible
EM analysis of several consecutive fractions eluted from a SEC (Superdex 200 10/300) purification of
During the averaging of the aligned images, the signal of highly flexible structures tends to be lost, although the signal of the base part uses to remain visible for being a most stable point. Indeed, in agreement with the aforementioned observations, the 2D class average classification of 36,699 single particle images (Figure
Combining 2D and 3D classification image processing methods allowed us to obtain a final 20 Å resolution map (using the FSC = 0.143 criterion) of an asymmetrical toroid-like structure (Figure
When the 3D map is displayed at a lower contour level (Figure
A peak fraction from a gel filtration (Superdex 200 10/300) purification step of
A 23 Å resolution 3D map (using the FSC = 0.143 criterion) of the putative
Highly flexible regions, as the CCGb-linker of CPAP is predicted to be (Supplementary Figure
It is noteworthy that the 3D map of the putative
Elongated rope-like structures were formed during the dialysis process of lowering the buffer salt concentration from 300 mM NaCl (buffer H-300) to 150 mM NaCl (buffer H-150; see Section “Ion Exchange Chromatography” from Section Materials and Methods). The dialyzed sample was further subjected to an anion exchange purification step. NS-EM of protein fractions eluted between 350 and 450 mM NaCl from the anion exchange column, showed modular rope-like structures of different lengths. At first glance, these structures seem to be formed by linear stacks of a variable number of rectangular modules with dimensions varying between ~7–8.5 nm (
It has been proposed that higher order assemblies of CPAP could act in a synergistic way as a platform to provide an interface mediating the tethering of PCM to the centriole (Gopalakrishnan et al.,
In this study we report that
Taking into account the multiple binding partners of CPAP, it is not surprising that a considerable part of this protein is predicted to be largely unstructured, being this a characteristic that can confer the structural flexibility necessary to interact with different proteins/complexes (Dunker et al.,
It is well-known that concentration can be a driving factor affecting the oligomerization status of many proteins, which in turn, modifies its basic structure and, subsequently, its function (Giese and Vierling,
CPAP/SAS-4 has been localized in the proximity of the lumen centriole surface of microtubules (Kleylein-Sohn et al.,
It has been reported that the whole structure and protein composition of centrosomes is poorly affected by NaCl and KCl at concentrations as high as 2M (Klotz et al.,
Taking together all the data presented in this work, we propose a putative model for the formation of higher order modular filaments of CPAP (Figure
The suggested tentative organization of the G-box and CC4/CC5 domains on opposite sides of the putative tetramer structure of
The presented work reinforces the idea that CPAP forms organized higher order structures allowing it to act as a scaffold that connects PCM proteins and complexes with the nascent centriole (Gopalakrishnan et al.,
Our results showing that
The EM maps of the putative dimer and the putative tetramer of
AA and JC conceived the project and designed the work. AA carried out and designed most experiments and sample preparation. GBM assisted the protein purification experiments. AA, SD, and DG carried out the EM data collection. AA, CS, and JC performed the image analysis and 3D reconstruction of HsCPAP897−1338 complexes. AA and JC wrote the paper with significant inputs from GM, CS and TT. All authors reviewed the results, provided critical comments and input, proof- read, and approved the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The content of this manuscript is based on a Ph.D. thesis (Alvarez-Cabrera,
The Supplementary Material for this article can be found online at: