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. 2020 Jul 17;5(29):18331-18341.
doi: 10.1021/acsomega.0c02051. eCollection 2020 Jul 28.

Identification of an α-MoRF in the Intrinsically Disordered Region of the Escargot Transcription Factor

Teresa Hernández-Segura  1   2 , Nina Pastor  1
Affiliations

Identification of an α-MoRF in the Intrinsically Disordered Region of the Escargot Transcription Factor

Teresa Hernández-Segura et al. ACS Omega. .

Abstract

Molecular recognition features (MoRFs) are common in intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs). MoRFs are in constant order-disorder structural transitions and adopt well-defined structures once they are bound to their targets. Here, we study Escargot (Esg), a transcription factor in Drosophila melanogaster that regulates multiple cellular functions, and consists of a disordered N-terminal domain and a group of zinc fingers at its C-terminal domain. We analyzed the N-terminal domain of Esg with disorder predictors and identified a region of 45 amino acids with high probability to form ordered structures, which we named S2. Through 54 μs of molecular dynamics (MD) simulations using CHARMM36 and implicit solvent (generalized Born/surface area (GBSA)), we characterized the conformational landscape of S2 and found an α-MoRF of ∼16 amino acids stabilized by key contacts within the helix. To test the importance of these contacts in the stability of the α-MoRF, we evaluated the effect of point mutations that would impair these interactions, running 24 μs of MD for each mutation. The mutations had mild effects on the MoRF, and in some cases, led to gain of residual structure through long-range contacts of the α-MoRF and the rest of the S2 region. As this could be an effect of the force field and solvent model we used, we benchmarked our simulation protocol by carrying out 32 μs of MD for the (AAQAA)3 peptide. The results of the benchmark indicate that the global amount of helix in shorter peptides like (AAQAA)3 is reasonably predicted. Careful analysis of the runs of S2 and its mutants suggests that the mutation to hydrophobic residues may have nucleated long-range hydrophobic and aromatic interactions that stabilize the MoRF. Finally, we have identified a set of residues that stabilize an α-MoRF in a region still without functional annotations in Esg.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Disorder probability analysis of the Esg protein. The plot shows the average probability of disorder by residue. The light and dark green color lines show the disordered and ordered regions, respectively.
Figure 2
Figure 2
Initial ensemble of the S2 region in the NTD of Esg, obtained by HHpred (HHP), I-Tasser (IT), Phyre2 (PI), QUARK (Q), and SPARK-X (SP) predictors. Each model shows secondary structural elements and is color-coded from red to blue, progressing from the N- to the C-terminus.
Figure 3
Figure 3
Structural diversity and degree of compaction of the S2 region of Esg. (A) Energy landscape built with Cα-atom RMSD (Å) and Rg (Å). (B) Histogram of the Cα-atom RMSD (Å) distribution in the ensemble during 54 μs of simulation. (C) Histogram of the Rg (Å) distribution in the ensemble during 54 μs of simulation. (D) Average inter-residue distances (Å) as a function of sequence distance during 54 μs of simulation.
Figure 4
Figure 4
Heat maps representing pairwise RMSD (Å) calculated for the Cα-atoms of the (A) SP8 (frames 1–2000) and SP9 (frames 2001–4000) runs and (B) IT4 (frames 1–2000) and SP7 (frames 2001–4000) runs. Each plot shows the location of the minimum and maximum pairwise RMSD. (C) Initial structures of the SP8 and SP9 models, and overlapping of the minimum of pairwise RMSD. (D) Initial structures of the IT4 and SP7 models, and overlapping of the minimum of pairwise RMSD.
Figure 5
Figure 5
Secondary structure and tertiary contacts of the S2 region. (A) Percentage of the time found as a helix for each residue. (B) Heat map representing the contacts between residue pairs; the dashed line square marks the initial position of helices. (C) Heat map representing the hydrogen bonds involving side chain atoms between residue pairs; the dashed line square marks the C-terminal helix. (D) α-Helix conformation depicting the interactions between E141 with R144 and T145. The main chain is shown as a purple ribbon, and the amino acids are shown as sticks in CPK colors.
Figure 6
Figure 6
Percentage of the helicity per residue of the α-MoRF (from residues 134 to 152) of each mutant, with respect to the wild-type (black line). The value for each residue is marked by a dot.
Figure 7
Figure 7
Ensemble of (AAQAA)3 during 32 μs of MD simulation. (A) Fraction helix of (AAQAA)3 computed over 10 ns blocks; the first 16 μs correspond to the simulation starting from the extended conformation and the last 16 μs correspond to the simulation starting from a perfect helix. (B) Percentage of the helicity per residue, averaged over the 32 μs (red line) compared to the initial ensemble (blue line). (C) (AAQAA)3 conformations representing coil–helix transitions at different times during the simulations.
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