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Uncoupling the Folding and Binding of an Intrinsically Disordered Protein - PubMed

  • ️Mon Jan 01 2018

Uncoupling the Folding and Binding of an Intrinsically Disordered Protein

Anusha Poosapati et al. J Mol Biol. 2018.

Abstract

The relationship between helical stability and binding affinity was examined for the intrinsically disordered transactivation domain of the myeloblastosis oncoprotein, c-Myb, and its ordered binding partner, KIX. A series of c-Myb mutants was designed to either increase or decrease helical stability without changing the binding interface with KIX. This included a complimentary series of A, G, P, and V mutants at three non-interacting sites. We were able to use the glycine mutants as a reference state and show a strong correlation between binding affinity and helical stability. The intrinsic helicity of c-Myb is 21%, and helicity values of the mutants ranged from 8% to 28%. The c-Myb helix is divided into two conformationally distinct segments. The N-terminal segment, from K291-L301, has an average helicity greater than 60% and the C-terminal segment, from S304-L315, has an average helicity less than 10%. We observed different effects on binding when these two segments were mutated. Mutants in the N-terminal segment that increased helicity had no effect on the binding affinity to KIX, while helix destabilizing glycine and proline mutants reduced binding affinity by more than 1 kcal/mol. Mutants that either increased or decreased helical stability in the C-terminal segment had almost no effect on binding. However, several of the mutants reveal the presence of multiple conformations accessible in the bound state based on changes in enthalpy and linkage analysis of binding free energies. These results may explain the high level of sequence identity (>90%), even at non-interacting sites, for c-Myb homologues.

Keywords: binding affinity; c-Myb transactivation domain; coupled folding and binding; fractional helicity; intrinsically disordered protein.

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Figures

Figure 1
Figure 1. CD spectra of WT c-Myb TAD, two proline mutants, and G311A

Molar residue ellipticity is plotted against wavelength. The black curve shows the molar residue ellipticity of WT c-Myb TAD. Grey curves show the molar residue ellipticity for G311A, P289A, and P289A/P316A. Molar residue ellipticity values in the figure are the mean of two CD measurements collected using the same sample.

Figure 2
Figure 2. Measuring c-Myb TAD binding to KIX

Black circles show enthalpy per mole of injectant, measured using isothermal titration calorimetry, plotted as a function of [c-Myb TAD]/[KIX]. Black lines show the fit to the data obtained using a single site binding model. Shown here is the binding of A) WT c-Myb TAD to KIX B) c-Myb P289A to KIX and C) c-Myb P289A P316A to KIX.

Figure 3
Figure 3. Residue specific helical populations of WT c-Myb TAD and proline mutants

Plots showing alpha carbon secondary chemical shifts (ΔδCα, black bars) and the percent helicity values calculated using δ2d (red lines) for WT and the proline mutants of c-Myb TAD. Blue lines in B and C show overlay of WT δ2d values. X-axis shows the amino acid sequence with mutation sites in red font. A) ΔδCα and δ2d plots for WT c-Myb TAD, B) ΔδCα and δ2d plots for P289A, and C) ΔδCα and δ2d plots for P289A/P316A.

Figure 4
Figure 4. Interactions between c-Myb TAD and KIX

A) c-Myb TAD residues shown in blue form an alpha helix and are in direct contact with the KIX residues shown in green. Other c-Myb TAD residues shown form an alpha helix and do not make direct contact with KIX. Residues shown in red selected for mutagenesis. B) Structure of the c-Myb TAD-KIX complex (PDB ID 1SB0). A surface model of KIX is shown in green and c-Myb TAD is shown as a ribbon structure in red. Side chains are shown for solvent-exposed amino acid residues that do not directly contact KIX.

Figure 5
Figure 5. CD spectra of L300, S304, N307 and charge mutants

Molar residue ellipticity is plotted against wavelength. In each panel the black curve shows the molar residue ellipticity of WT c-Myb TAD and grey curves show the mutants. A) CD spectra for L300 mutants. B) CD spectra for S304 mutants. C) CD spectra for N307 mutants. D) CD spectra for charge mutants. Molar residue ellipticity values in the figure are the mean of two CD measurements collected using the same sample.

Figure 6
Figure 6. Measuring L300 mutants binding to KIX

Black circles show enthalpy per mole of injectant, measured using isothermal titration calorimetry, plotted as a function of [c-Myb TAD]/[KIX]. Black lines show the fit to the data obtained using a single site binding model. Shown here is the binding of a) L300V to KIX b) L300G to KIX and C) L300P to KIX.

Figure 7
Figure 7. Residue specific helical populations of L300G and L300P

Plots showing alpha carbon secondary chemical shifts (ΔδCα, black bars) and the percent helicity values calculated using δ2d (red lines) for L300 mutants. Blue lines in A and B show overlay of WT δ2d values. X-axis shows the amino acid sequence with mutation sites in red font. A) ΔδCα and δ2d plots for L300G, B) ΔδCα and δ2d plots for L300P.

Figure 8
Figure 8. Chemical shift mapping of KIX binding to c-Myb TAD

Calculation of average amide nitrogen and proton chemical shift changes for KIX is described in the materials and methods. The plots show residue specific chemical shift changes of A) KIX binding to WT cMyb TAD and B) KIX binding to L300G and C) KIX binding to L300P. Asterisks label KIX residues that directly contact c-Myb TAD in the solution structure. The average combined resolution for the 1H and 15N dimensions in the HSQC was 0.03 ppm. Chemical shift changes greater than this value are considered.

Figure 9
Figure 9. Correlation analysis of chemical shift mapping

A) Correlation plot of the average chemical shift changes for KIX when bound to either L300G or WT c-Myb TAD. B) Correlation plot of the average chemical shift changes for KIX when bound to either L300P or WT c-Myb TAD. C) Overlay of 1H-15N HSQC resonances for Y640. D) Overlay of 1H-15N HSQC resonances for K633. In B) and C) blue peaks are from free KIX, red peaks are from KIX bound to WT c-Myb TAD, green peaks are from KIX bound to L300G, and magenta peaks are from KIX bound to L300P

Figure 10
Figure 10. Linking binding free energy (ΔG) and helical stability of WT and mutant c-Myb peptides

A) Plot of binding free energy (ΔG) from ITC is versus percent helicity values for residues 289–316. B) Expanded version of A with labels for each data point. Blue points show mutants that may change the structure of the c-Myb/KIX complex. C) Plot of helical stability versus ΔΔG of binding. The ΔG of binding for the glycine mutants was subtracted from the values of WT and the A, V, and P mutants at each site. Helical stability is plotted a percent helicity of a glycine mutant at a given position divided by the percent helicity of the WT and the A, V, and P mutants at the same site. The S304V mutant was excluded from the plot.

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