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Structural analysis of the G-box domain of the microcephaly protein CPAP suggests a role in centriole architecture.

Hatzopoulos GN, Erat MC, Cutts E, Rogala KB, Slater LM, Stansfeld PJ, Vakonakis I - Structure (2013)

Bottom Line: Centrioles are evolutionarily conserved eukaryotic organelles composed of a protein scaffold surrounded by sets of microtubules organized with a 9-fold radial symmetry.The G-box comprises a single elongated β sheet capable of forming supramolecular assemblies.Structural and biophysical studies highlight the conserved nature of the CPAP-STIL complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.

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Molecular Architecture of HsCPAP(A) Schematic representation of CPAP/SAS-4 from human, D. rerio, D. melanogaster, and C. elegans. Orange, yellow, and red boxes correspond to the protein tubulin binding (PN2-3), microtubule binding (A5N), and G-box domains, respectively. The predicted coiled-coil domains are indicated by green helices; the long coiled coil adjacent to the G-box features a short break in some species. Residue boundaries and the extent of our protein constructs are indicated. Asterisks in HsCPAP denote the location of MCPH causing mutations; M1 and M2 correspond to premature stop codons.(B and C) CD spectrum of HsCPAPCC (B) and change in molar elipticity (C) at 222 nm showing a cooperative transition upon thermal unfolding of the same construct are shown.(D) SEC-MALS analysis of HsCPAPCC. The UV absorbance profile from SEC (black line) is overlain with the MALS estimation of molecular mass (red line), which agrees well with that of a dimer (39.5 kDa).(E) SDS-PAGE of HsCPAPCC under reducing (+βMe) and nonreducing (−βMe) conditions. Arrowheads point to protein bands corresponding to monomeric (M) and disulfide-linked dimeric (D) forms.(F and G) CD spectra (F) and thermal unfolding profile (G) at 218 nm of DrCPAPG-box are shown.(H) SEC-MALS analysis of DrCPAPG-box as in (D). The estimated molecular mass agrees well with a momoner (20.8 kDa).(I) Molecular model of CPAP with a parallel coiled coil and a C-terminal domain connected by a flexible linker is shown.See also Figure S1.
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fig1: Molecular Architecture of HsCPAP(A) Schematic representation of CPAP/SAS-4 from human, D. rerio, D. melanogaster, and C. elegans. Orange, yellow, and red boxes correspond to the protein tubulin binding (PN2-3), microtubule binding (A5N), and G-box domains, respectively. The predicted coiled-coil domains are indicated by green helices; the long coiled coil adjacent to the G-box features a short break in some species. Residue boundaries and the extent of our protein constructs are indicated. Asterisks in HsCPAP denote the location of MCPH causing mutations; M1 and M2 correspond to premature stop codons.(B and C) CD spectrum of HsCPAPCC (B) and change in molar elipticity (C) at 222 nm showing a cooperative transition upon thermal unfolding of the same construct are shown.(D) SEC-MALS analysis of HsCPAPCC. The UV absorbance profile from SEC (black line) is overlain with the MALS estimation of molecular mass (red line), which agrees well with that of a dimer (39.5 kDa).(E) SDS-PAGE of HsCPAPCC under reducing (+βMe) and nonreducing (−βMe) conditions. Arrowheads point to protein bands corresponding to monomeric (M) and disulfide-linked dimeric (D) forms.(F and G) CD spectra (F) and thermal unfolding profile (G) at 218 nm of DrCPAPG-box are shown.(H) SEC-MALS analysis of DrCPAPG-box as in (D). The estimated molecular mass agrees well with a momoner (20.8 kDa).(I) Molecular model of CPAP with a parallel coiled coil and a C-terminal domain connected by a flexible linker is shown.See also Figure S1.

Mentions: Human CPAP (HsCPAP) is a 1338 amino acids protein with a glycine-rich C-terminal domain (G-box) (Figure 1A). Previous work showed that separate HsCPAP motifs bind tubulin heterodimers and destabilize microtubules (PN2-3) (Cormier et al., 2009; Hung et al., 2004) or bind and stabilize microtubules (A5N) (Hsu et al., 2008). PN2-3, A5N, and the G-box are the most conserved regions between HsCPAP and its functional orthologs in D. rerio, Drosophila melanogaster, and Caenorhabditis elegans (Gopalakrishnan et al., 2011; Kitagawa et al., 2011a; Tang et al., 2011). All four orthologs also feature a predicted ∼150–200 residue coiled coil adjacent to the G-box; in contrast, the three N-terminal coiled coils of HsCPAP are not universally predicted, and they are presumed to be of low stability or unstructured as judged by their short length and biophysical properties (Cormier et al., 2009).


Structural analysis of the G-box domain of the microcephaly protein CPAP suggests a role in centriole architecture.

Hatzopoulos GN, Erat MC, Cutts E, Rogala KB, Slater LM, Stansfeld PJ, Vakonakis I - Structure (2013)

Molecular Architecture of HsCPAP(A) Schematic representation of CPAP/SAS-4 from human, D. rerio, D. melanogaster, and C. elegans. Orange, yellow, and red boxes correspond to the protein tubulin binding (PN2-3), microtubule binding (A5N), and G-box domains, respectively. The predicted coiled-coil domains are indicated by green helices; the long coiled coil adjacent to the G-box features a short break in some species. Residue boundaries and the extent of our protein constructs are indicated. Asterisks in HsCPAP denote the location of MCPH causing mutations; M1 and M2 correspond to premature stop codons.(B and C) CD spectrum of HsCPAPCC (B) and change in molar elipticity (C) at 222 nm showing a cooperative transition upon thermal unfolding of the same construct are shown.(D) SEC-MALS analysis of HsCPAPCC. The UV absorbance profile from SEC (black line) is overlain with the MALS estimation of molecular mass (red line), which agrees well with that of a dimer (39.5 kDa).(E) SDS-PAGE of HsCPAPCC under reducing (+βMe) and nonreducing (−βMe) conditions. Arrowheads point to protein bands corresponding to monomeric (M) and disulfide-linked dimeric (D) forms.(F and G) CD spectra (F) and thermal unfolding profile (G) at 218 nm of DrCPAPG-box are shown.(H) SEC-MALS analysis of DrCPAPG-box as in (D). The estimated molecular mass agrees well with a momoner (20.8 kDa).(I) Molecular model of CPAP with a parallel coiled coil and a C-terminal domain connected by a flexible linker is shown.See also Figure S1.
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fig1: Molecular Architecture of HsCPAP(A) Schematic representation of CPAP/SAS-4 from human, D. rerio, D. melanogaster, and C. elegans. Orange, yellow, and red boxes correspond to the protein tubulin binding (PN2-3), microtubule binding (A5N), and G-box domains, respectively. The predicted coiled-coil domains are indicated by green helices; the long coiled coil adjacent to the G-box features a short break in some species. Residue boundaries and the extent of our protein constructs are indicated. Asterisks in HsCPAP denote the location of MCPH causing mutations; M1 and M2 correspond to premature stop codons.(B and C) CD spectrum of HsCPAPCC (B) and change in molar elipticity (C) at 222 nm showing a cooperative transition upon thermal unfolding of the same construct are shown.(D) SEC-MALS analysis of HsCPAPCC. The UV absorbance profile from SEC (black line) is overlain with the MALS estimation of molecular mass (red line), which agrees well with that of a dimer (39.5 kDa).(E) SDS-PAGE of HsCPAPCC under reducing (+βMe) and nonreducing (−βMe) conditions. Arrowheads point to protein bands corresponding to monomeric (M) and disulfide-linked dimeric (D) forms.(F and G) CD spectra (F) and thermal unfolding profile (G) at 218 nm of DrCPAPG-box are shown.(H) SEC-MALS analysis of DrCPAPG-box as in (D). The estimated molecular mass agrees well with a momoner (20.8 kDa).(I) Molecular model of CPAP with a parallel coiled coil and a C-terminal domain connected by a flexible linker is shown.See also Figure S1.
Mentions: Human CPAP (HsCPAP) is a 1338 amino acids protein with a glycine-rich C-terminal domain (G-box) (Figure 1A). Previous work showed that separate HsCPAP motifs bind tubulin heterodimers and destabilize microtubules (PN2-3) (Cormier et al., 2009; Hung et al., 2004) or bind and stabilize microtubules (A5N) (Hsu et al., 2008). PN2-3, A5N, and the G-box are the most conserved regions between HsCPAP and its functional orthologs in D. rerio, Drosophila melanogaster, and Caenorhabditis elegans (Gopalakrishnan et al., 2011; Kitagawa et al., 2011a; Tang et al., 2011). All four orthologs also feature a predicted ∼150–200 residue coiled coil adjacent to the G-box; in contrast, the three N-terminal coiled coils of HsCPAP are not universally predicted, and they are presumed to be of low stability or unstructured as judged by their short length and biophysical properties (Cormier et al., 2009).

Bottom Line: Centrioles are evolutionarily conserved eukaryotic organelles composed of a protein scaffold surrounded by sets of microtubules organized with a 9-fold radial symmetry.The G-box comprises a single elongated β sheet capable of forming supramolecular assemblies.Structural and biophysical studies highlight the conserved nature of the CPAP-STIL complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.

Show MeSH
Related in: MedlinePlus