Figure 1. Biochemical and structural characterisation of the CPAP TCP domain and its interaction with STIL.

(A) Schematic representation of D. rerio CPAP and STIL. CPAP is a 1124 amino acid (aa) protein with three predicted coiled coil (cc) domains and a C-terminal TCP domain. STIL is a 1263 aa protein with one predicted cc domain and several conserved regions (CR). The proline-rich CR2 domain is enlarged and coloured according to Consurf conservation scores (Glaser et al., 2003) from cyan (variable) to burgundy (conserved). The constructs used in this study are indicated by bars. (B) Two views of the TCP domain structure (green) in complex with the STIL peptide (orange), rotated by 180°. Images on the left of each view show a ribbon representation and images on the right show the TCP domain as a molecular surface coloured according to Consurf conservation scores. Note the presence of a conserved patch (dashed circle) along the edge of the TCP domain where the STIL peptide is bound. This patch contains aromatic residues (black sticks) that would be well placed to interact with conserved prolines in the C-terminal part of the STIL CR2 region that we had to omit for crystallisation. ITC experiments (Figure 1D) suggest that these putative additional contacts would only contribute weakly to overall binding. (C) Detailed view of the D. rerio CPAP–STIL interaction interface coloured according to Consurf conservation scores. Interface residues are shown in sticks, and the TCP domain is shown as a semi-transparent molecular surface. Contact residues are labelled in green (CPAP) and orange (STIL). Dotted yellow lines indicate hydrogen-bonds. The dark orange sphere represents a bound water molecule. (D) ITC analysis using the STIL constructs shown in Figure 1A. The excess heat measured on titrating STIL into CPAP at 25°C was fitted to a single set of binding sites model. Fitted K_D values are indicated together with their standard deviations. (E and F) Ribbon models of the apo-structures of the D. rerio CPAP TCP domain: (E) WT apo-structure; (F) E1021V (MCPH mutation) apo-structure (V1021 represented as red spheres).

DOI: http://dx.doi.org/10.7554/eLife.01071.003

Figure 1—figure supplement 1. Multiple sequence alignment of the conserved proline-rich region of STIL (CR2).

The numbering refers to D. rerio STIL. The alignment is coloured by conservation according to the Consurf conservation score from cyan (variable) to burgundy (conserved).

DOI: http://dx.doi.org/10.7554/eLife.01071.004

Figure 1—figure supplement 2. The TCP domain sequence repeats.

(A) Alignment of the sequence repeats of D. rerio CPAP^937–1124. Residues are coloured according to the Clustalx colour scheme. R, Repeat. Repeat 1 was not visible in the electron density map of D. rerio apo-CPAP^937–1124 but could be seen partially in the structure of the complex between D. rerio CPAP^937–1124 and STIL^408–428. (B) Sequence logo of the CPAP^937–1124 repeat with the relative residue frequencies at each position. Prominent features of this repeat are two PDG motifs and the high frequency of aromatic residues adjacent to the first PDG motif in position 6 of the repeat. (C) Left: ribbon presentation of the D. rerio apo CPAP^937–1124 structure with its sequence repeats rainbow-coloured from N- to C-terminus. R, Repeat. An individual structural repeat consists of a β-hairpin. The aromatic residues found in position 6 of the repeat are shown in black sticks. These aromatic residues run along the edge of one side of the β-sheet, where the proline-rich STIL peptide binds. The PDG motifs frequently constitute the β-turns of the TCP repeats. Boxed are three of these turns that are presented on the right as a close-up. In this close-up, residues of the PDG motif are labelled and shown in sticks. The Asp residue in this motif hydrogen-bonds (dotted black lines) to the main-chain of the (n) + 1 neighbouring residue.

DOI: http://dx.doi.org/10.7554/eLife.01071.005

Figure 1—figure supplement 3. The TCP domain of CPAP is predominantly monomeric in solution.

(A) Panel showing size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) chromatograms. CPAP^937–1124 was injected at concentrations of approximately 70 μM (light grey), 460 μM (dark grey) and 2.4 mM (black). The corresponding chromatogram traces (thin lines) show the refractive index signal. The concentrations of the CPAP^937–1124 monomer measured at the peaks are indicated, and heavy solid lines across the peaks show the calculated molar masses. When averaged across the central 50% of the peaks, these molar masses were 22 kDa, 23 kDa, and 29 kDa, respectively. The theoretical molecular weight of a CPAP^937–1124 monomer is 22 kDa. The Rh of CPAP^937–1124 determined at the intermediate concentration was 2.9 ± 0.15 nm, which is significantly larger than expected for a globular protein of this mass and thus is consistent with the extended crystallographic structure. SEC-MALS measurements were performed using a Wyatt Heleos II 18 angle light scattering instrument coupled to a Wyatt Optilab rEX online refractive index detector. Detector 12 in the Heleos instrument was replaced with Wyatt's QELS detector for dynamic light scattering measurement. Samples (100 μl) were resolved on a Superdex S-200 10/300 analytical gel filtration column (GE Healthcare, Little Chalfont, UK) running at 0.5 ml/min in 25 mM bis Tris pH 7.2, 100 mM NaCl buffer before passing through the light scattering and refractive index detectors in a standard SEC-MALS format. Protein concentration was determined from the excess differential refractive index based on 0.186 RI increment for 1 g/ml protein solution. The concentration and the observed scattered intensity at each point in the chromatograms were used to calculate the absolute molecular mass from the intercept of the Debye plot using Zimm's model as implemented in Wyatt's ASTRA software. Autocorrelation analysis of data from the dynamic light scattering detector was also performed using Wyatt's ASTRA software, and the translational diffusion coefficients determined were used to calculate the hydrodynamic radius using the Stokes-Einstein equation and the measured solvent viscosity of 9.3 e-3 Poise. (B) Small-angle X-ray scattering (SAXS) experiment with approximately 20 μM D. rerio CPAP^937–1124 in 25 mM bis Tris pH 7.2, 100 mM NaCl, 2 mM DTT. Shown in blue is the experimentally measured SAXS curve of CPAP^937–1124 with the experimental error indicated by black bars. The orange line shows the fitted theoretical SAXS curve of CPAP^937–1124 derived from its crystal structure. Fitting was done using CRYSOL (Svergun et al., 1995) and resulted in a χ-value of 1.416. A.U., arbitrary units. At higher CPAP^937–1124 concentrations the fit became less good due to the tendency of CPAP^937–1124 to self-associate at these concentrations as revealed by a gradual increase of the derived Rg values. SAXS data were collected at the European Synchrotron Radiation Facility (ESRF), Grenoble, France, at beamline ID14–3. Measurements were done at 10°C at a wavelength of 0.931 Å with the standard beamline settings using a PILATUS 1M detector (Dectris, Baden, Switzerland). To minimise radiation damage, a flow cell was used for the measurements. Collected data was buffer subtracted using PRIMUS (Konarev et al., 2003) and the beamstop shadow removed by cutting the data at a q-value of 0.055 nm⁻¹. Above a q-value of 3.8 nm⁻¹ the data became too noisy to be interpretable and the data were therefore cut at this value.

DOI: http://dx.doi.org/10.7554/eLife.01071.006

Figure 1—figure supplement 4. The TCP domain resembles engineered peptide-assembly mimics used to study β-rich self-assemblies.

Side-by-side comparison of the apo-structure of the D. rerio TCP-domain of CPAP (left) with the structure of an engineered peptide-assembly mimic based on Borrelia OspA (right) that is used to study β-rich self-assemblies (PDB code 2FKJ, chain A). Structures are shown as ribbon presentations and are rainbow-coloured from N- to C-terminus. Note that the conformation of the peptide-self-assembly mimic is maintained by two globular domains capping both ends of its β-sheet. In contrast, the TCP domain entirely lacks a hydrophobic core.

DOI: http://dx.doi.org/10.7554/eLife.01071.007

Figure 1—figure supplement 5. Multiple sequence alignment of the TCP domain of CPAP.

The numbering refers to D. rerio CPAP. The alignment is coloured by conservation according to the Consurf conservation score from cyan (variable) to burgundy (conserved).

DOI: http://dx.doi.org/10.7554/eLife.01071.008