Conformational Dynamics of the Partially Disordered Yeast Transcription Factor GCN4

Abstract

Molecular dynamics (MD) simulations have been employed to study the conformational dynamics of the partially disordered DNA binding basic leucine zipper domain of the yeast transcription factor GCN4. We demonstrate that back-calculated NMR chemical shifts and spin-relaxation data provide complementary probes of the structure and dynamics of disordered protein states and enable comparisons of the accuracy of multiple MD trajectories. In particular, back-calculated chemical shifts provide a sensitive probe of the populations of residual secondary structure elements and helix capping interactions, while spin-relaxation calculations are sensitive to a combination of dynamic and structural factors. Back calculated chemical shift and spin-relaxation data can be used to evaluate the populations of specific interactions in disordered states and identify regions of the phase space that are inconsistent with experimental measurements. The structural interactions that favor and disfavor helical conformations in the disordered basic region of the GCN4 bZip domain were analyzed in order to assess the implications of the structure and dynamics of the apo form for the DNA binding mechanism. The structural couplings observed in these experimentally validated simulations are consistent with a mechanism where the binding of a preformed helical interface would induce folding in the remainder of the protein, supporting a hybrid conformational selection / induced folding binding mechanism.

Figure 1

Fractional helicity for each residue of GCN4 bZip domain observed in four 100 ns MD trajectories initiated from starting structures with varying degrees of helicity. Helical conformations were assigned for each MD snapshot using the program STRIDE. Helix populations predicted from the experimental chemical shifts using the δ2D program are shown as a dotted line. The sequence of the GCN4 bZip domain is displayed above the graph with residues in the basic region colored blue and residues in the leucine zipper colored red.

Figure 2

Deviations between the experimentally measured Cα, C', and H_N chemical shifts (δ_Exp) and the average Sparta+ chemical shift predictions (δ_MD) for the basic region residues of the GCN4 bZip domain from four 100 ns MD simulations initiated from starting structures with varying degrees of helicity.

Figure 3

Populations of H1 helix stabilizing C-cap hydrogen bonds from chemical shifts. A) An illustrative conformation displaying hydrogen bond pairs formed by C-terminal residues of the partially populated H1 helix in MD simulations. B) Normalized distribution of Sparta+ predicted chemical shifts for the C' and H_N atoms involved in the hydrogen bonds observed in the MD trajectories. The average value of the shift predictions for trajectories 1, 2, 3, and 4 are displayed as a red circle, a green square, a blue diamond, and a brown triangle respectively. The experimentally measured value is shown as a black diamond.

Figure 4

Comparison of experimental and simulated generalized order parameters (S²) from four 100 ns MD simulations initiated from starting structures with varying degrees of helicity. Simulated S² values were calculated after superposition of backbone atoms of the leucine zipper (residues 26-54). Error bars of the simulated S² values reflect the standard errors of the mean of each residue for five 18.9 ns analysis blocks. The sequence of the GCN4 bZip domain is displayed above the graph with residues in the basic region colored blue and residues in the leucine zipper colored red.

Figure 5

The fractional helicity of each residue observed in trajectory 2, the trajectory in the best agreement with experimental NMR data, is mapped onto the crystal structure of the GCN4 bZip domain bound to DNA (PDB code 1YSA) according to the color bar shown. Side chains are displayed for residues which make contacts with the DNA.