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. 2013 Sep 17;2:e01071. doi: 10.7554/eLife.01071

Figure 2. Mutational analysis of the CPAP:STIL interaction in vitro and conservation of the interaction across species.

(A) Graphs showing the binding constants (KD) determined by ITC for the interaction between WT and mutant constructs of CPAP937–1124 and STIL404–448. Left panel, WT and various mutant forms of CPAP937–1124 binding to WT STIL404–448 (T986 is a non-interacting residue included as a negative control). Error bars, standard deviation. Right panel, WT and various mutant STIL404–448 constructs binding to WT CPAP937–1124 (N422 is a non-interacting residue included as a negative control). Error bars, standard deviation. The wild-type measurements are the same as shown in Figure 1D and are shown again for comparison to the mutants. (B and C) Close-up view of the CPAP (green):STIL (orange) interaction interface from D. rerio (B) and Drosophila (C). Interface residues are shown as sticks, in yellow is the Glutamate residue in Drosophila and D. rerio CPAP that is equivalent to E1235 in human CPAP (mutated in MCPH). Residues of the D. rerio protein mutated for ITC experiments are ringed in green (CPAP) or red (STIL). Dotted black lines indicate hydrogen-bonds. The conserved bound water molecule is shown as a red sphere.

DOI: http://dx.doi.org/10.7554/eLife.01071.010

Figure 2.

Figure 2—figure supplement 1. Characterisation of the D. rerio TCP domain mutants and STIL peptide mutants used for thermodynamic analysis.

Figure 2—figure supplement 1.

(A) Coomassie stained SDS-PAGE gel of purified, recombinant CPAP937–1124 and its mutants (as labelled in colours above the gel). (B) Buffer-subtracted circular dichroism (CD) spectra of D. rerio CPAP937–1124 and its mutants in 10 mM Na-Phosphate pH 7.3 at approximately 200 μg/ml. Spectra were recorded on a JASCO J-810 from 260 to 190 nm in 0.2 nm steps at 20°C and are colour-coded as in (A). The data were cut at 200 nm as the detector was saturated below this wavelength for some constructs as indicated by a high HT voltage. (C) Coomassie stained SDS-PAGE gel showing purified, recombinant D. rerio STIL404–448 and its mutants.
DOI: http://dx.doi.org/10.7554/eLife.01071.011