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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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Structural Analysis of DrCPAPG-box(A and B) Shown is schematic representation of DrCPAPG-box in two perpendicular views. The secondary structure elements, protein dimensions, and the β sheet twist are shown.(C and D) Electrostatic (C) and hydrophobic (D) interactions on the surface of the DrCPAPG-box β sheet are shown. The residues involved are shown in CPK and space-filling representation, respectively.(E) Schematic representation of the DrCPAPG-box fibril. Three successive G-box domains are colored pink, blue, and green, with the β1 and β17 strands and the fibril periodicity shown.(F) Distributions of hydrodynamic radii (Rh) of DrCPAPG-box assemblies in solution at three different concentrations as measured by DLS. The distributions are broad, with large assemblies populated even at the lowest concentration (blue line); their prevalence increases as a function of protein concentration.(G) Mean Rh of DrCPAPG-box assemblies by DLS as a function of protein concentration. Rh at the infinite dilution point (pink square) was calculated from the DrCPAPG-box structure. Error bars correspond to one SD from triplicate measurements.See also Figure S2 and Movie S1.
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fig2: Structural Analysis of DrCPAPG-box(A and B) Shown is schematic representation of DrCPAPG-box in two perpendicular views. The secondary structure elements, protein dimensions, and the β sheet twist are shown.(C and D) Electrostatic (C) and hydrophobic (D) interactions on the surface of the DrCPAPG-box β sheet are shown. The residues involved are shown in CPK and space-filling representation, respectively.(E) Schematic representation of the DrCPAPG-box fibril. Three successive G-box domains are colored pink, blue, and green, with the β1 and β17 strands and the fibril periodicity shown.(F) Distributions of hydrodynamic radii (Rh) of DrCPAPG-box assemblies in solution at three different concentrations as measured by DLS. The distributions are broad, with large assemblies populated even at the lowest concentration (blue line); their prevalence increases as a function of protein concentration.(G) Mean Rh of DrCPAPG-box assemblies by DLS as a function of protein concentration. Rh at the infinite dilution point (pink square) was calculated from the DrCPAPG-box structure. Error bars correspond to one SD from triplicate measurements.See also Figure S2 and Movie S1.

Mentions: DrCPAPG-box consists of 17 antiparallel β strands that form a ∼75 Å long and ∼23 Å wide β sheet, with an ∼168° of twist across its length (Figures 2A and 2B). Remarkably, all residues of this structure are solvent exposed; thus, it lacks a well-defined hydrophobic core. A search for similar structures in the Protein Data Bank shows that only OspA, which features a large exposed β sheet (Makabe et al., 2006), is structurally related. However, DrCPAPG-box does not include globular domains capping the β sheet, in contrast to OspA, yet it remains highly soluble and cooperatively folded (Figure 1G). Analysis of the model suggests that DrCPAPG-box is stabilized through a number of interstrand ionic interactions (Figure 2C), as well as through clusters of hydrophobic residues that span multiple β strands (Figure 2D). In addition, significant burial of hydrophobic surface area occurs between nonpolar side chains and the aliphatic part of long polar groups, such as arginine and lysine residues. To better understand DrCPAPG-box stability, we performed a 100 ns atomistic molecular dynamics simulation starting from the crystallographic structure. As shown in Figure S2, DrCPAPG-box remains folded during the simulation with no signs of β sheet unraveling. The domain length (Figure S2A), the number of main chain hydrogen bonds formed (Figure S2B), and the hydrophobic area buried (Figure S2C) remain essentially unaltered throughout the simulation trajectory (Figure S2D; Movie S1).


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)

Structural Analysis of DrCPAPG-box(A and B) Shown is schematic representation of DrCPAPG-box in two perpendicular views. The secondary structure elements, protein dimensions, and the β sheet twist are shown.(C and D) Electrostatic (C) and hydrophobic (D) interactions on the surface of the DrCPAPG-box β sheet are shown. The residues involved are shown in CPK and space-filling representation, respectively.(E) Schematic representation of the DrCPAPG-box fibril. Three successive G-box domains are colored pink, blue, and green, with the β1 and β17 strands and the fibril periodicity shown.(F) Distributions of hydrodynamic radii (Rh) of DrCPAPG-box assemblies in solution at three different concentrations as measured by DLS. The distributions are broad, with large assemblies populated even at the lowest concentration (blue line); their prevalence increases as a function of protein concentration.(G) Mean Rh of DrCPAPG-box assemblies by DLS as a function of protein concentration. Rh at the infinite dilution point (pink square) was calculated from the DrCPAPG-box structure. Error bars correspond to one SD from triplicate measurements.See also Figure S2 and Movie S1.
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fig2: Structural Analysis of DrCPAPG-box(A and B) Shown is schematic representation of DrCPAPG-box in two perpendicular views. The secondary structure elements, protein dimensions, and the β sheet twist are shown.(C and D) Electrostatic (C) and hydrophobic (D) interactions on the surface of the DrCPAPG-box β sheet are shown. The residues involved are shown in CPK and space-filling representation, respectively.(E) Schematic representation of the DrCPAPG-box fibril. Three successive G-box domains are colored pink, blue, and green, with the β1 and β17 strands and the fibril periodicity shown.(F) Distributions of hydrodynamic radii (Rh) of DrCPAPG-box assemblies in solution at three different concentrations as measured by DLS. The distributions are broad, with large assemblies populated even at the lowest concentration (blue line); their prevalence increases as a function of protein concentration.(G) Mean Rh of DrCPAPG-box assemblies by DLS as a function of protein concentration. Rh at the infinite dilution point (pink square) was calculated from the DrCPAPG-box structure. Error bars correspond to one SD from triplicate measurements.See also Figure S2 and Movie S1.
Mentions: DrCPAPG-box consists of 17 antiparallel β strands that form a ∼75 Å long and ∼23 Å wide β sheet, with an ∼168° of twist across its length (Figures 2A and 2B). Remarkably, all residues of this structure are solvent exposed; thus, it lacks a well-defined hydrophobic core. A search for similar structures in the Protein Data Bank shows that only OspA, which features a large exposed β sheet (Makabe et al., 2006), is structurally related. However, DrCPAPG-box does not include globular domains capping the β sheet, in contrast to OspA, yet it remains highly soluble and cooperatively folded (Figure 1G). Analysis of the model suggests that DrCPAPG-box is stabilized through a number of interstrand ionic interactions (Figure 2C), as well as through clusters of hydrophobic residues that span multiple β strands (Figure 2D). In addition, significant burial of hydrophobic surface area occurs between nonpolar side chains and the aliphatic part of long polar groups, such as arginine and lysine residues. To better understand DrCPAPG-box stability, we performed a 100 ns atomistic molecular dynamics simulation starting from the crystallographic structure. As shown in Figure S2, DrCPAPG-box remains folded during the simulation with no signs of β sheet unraveling. The domain length (Figure S2A), the number of main chain hydrogen bonds formed (Figure S2B), and the hydrophobic area buried (Figure S2C) remain essentially unaltered throughout the simulation trajectory (Figure S2D; Movie S1).

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