The molecular basis of conformational instability of the ecdysone receptor DNA binding domain studied by in silico and in vitro experiments

The heterodimer of the ecdysone receptor (EcR) and ultraspiracle (Usp), members of the nuclear receptors superfamily, regulates gene expression associated with molting and metamorphosis in insects. The DNA binding domains (DBDs) of the Usp and EcR play an important role in their DNA-dependent heterodimerization. Analysis of the crystal structure of the UspDBD/EcRDBD heterocomplex from Drosophila melanogaster on the hsp27 gene response element, suggested an appreciable similarity between both DBDs. However, the chemical denaturation experiments showed a categorically lower stability for the EcRDBD in contrast to the UspDBD. The aim of our study was an elucidation of the molecular basis of this intriguing instability. Toward this end, we mapped the EcRDBD amino acid sequence positions which have an impact on the stability of the EcRDBD. The computational protein design and in vitro analyses of the EcRDBD mutants indicate that non-conserved residues within the α-helix 2, forming the EcRDBD hydrophobic core, represent a specific structural element that contributes to instability. In particular, the L58 appears to be a key residue which differentiates the hydrophobic cores of UspDBD and EcRDBD and is the main reason for the low stability of the EcRDBD. Our results might serve as a benchmark for further studies of the intricate nature of the EcR molecule

The analysis of the EcRDBD mutants' binding to the <i>hsp27_pal</i>.

<p>The electrophoretic mobility-shift assays (EMSA) were conducted with the indicated EcRDBD (E) or the UspDBD (U), separately (panel A, lanes 1, 3–10 and 20–21; panel C, lanes 2–7 and 14–15) or with an equimolar mixture of both the indicated EcRDBD and UspDBD (panel A, lanes 11, 13–19; panel C, lanes 8–13). The protein (CI – monomer, CII – dimer) complexes formed with the <i>hsp27_pal</i> were denoted as: U, E or UE for the UspDBD, indicated EcRDBD and both DBDs heterodimer, respectively. F, free DNA probe, WT – EcRDBD_WT. The WT, U, UE and F lanes were included as controls. The positions of the corresponding complexes are marked on the left. The total protein concentrations were: 200 nM (panel A, lanes 1, 3–8 and 13–19) and 50 nM (panel C, lanes 2–6 and 9–13), using half of the amounts of each component that were used with a single DBD. The EMSA results were quantitatively analyzed as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086052#pone.0086052-Orowski1" target="_blank">[12]</a>, which is shown in panel B (data from panel A, lanes 1, 3–8 and 13–19, respectively) and panel D (data from panel C, lanes 2–6 and 9–13, respectively).</p

Graphical presentation of guanidine hydrochloride (GdmCl) half concentration (C_1/2), melting temperature (T_m) and the RosettaDesign scoring obtained for the EcRDBD mutants in comparison with the EcRDBD_WT and UspDBD.

<p>Aligned histograms were shown for convenience of the data comparative analysis.</p

The GdmCl denaturation profiles of the EcRDBD mutants.

<p>The chemical denaturation experiments were conducted at 20°C, at protein concentrations of 2.5 µM, and respective denaturation curves were recorded using fluorescence measurement. Further details are given under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086052#s4" target="_blank"><i>Materials and Methods</i></a>. One of the three representative profiles for this experiment is shown. Panel A: filled circles – EcRDBD_WT (WT), asterisks – L16R, filled triangles – M49W, open diamonds – L58F, open triangles – C61A, open circles – V64E, x symbols – V64M, filled squares – UspDBD. Panel B: filled circles – EcRDBD_WT (WT), open diamonds – L16R/M49W/L58F, open circles – L16R/L58F, filled triangles – M49W/L58F, asterisks – L16R/M49W, filled squares – UspDBD.</p

Quantitative estimation of the secondary structure contents for the EcRDBD_WT, EcRDBD mutants and UspDBD.

<p>Calculation were carried out for the 119-amino acid EcRDBD polypeptide and for the 106-amino acid UspDBD polypeptide spectra at 20°C. The SELCON3, CDSSTR and CONTIN/LL programs were used and results were averaged. RMSD is a CONTIN/LL fit parameter, with low values indicative of close correspondence between calculated secondary structure and experimental data <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086052#pone.0086052-Dantas2" target="_blank">[23]</a>.</p

Root-mean-square deviation (RMSD) profiles with respect to the EcRDBD mutant structures during MD simulations.

<p>The trajectories of the backbone root-mean-square deviation (RMSD) of the EcRDBD point mutant structures (black lines) in comparison with the EcRDBD_WT (gray lines, WT) and the UspDBD (gray, thin lines). The RMSDs were calculated for 10 ns MD simulations at 300 K with respect to energy-minimized structures. The thermalisation time up to 300 K is not shown.</p

Guanidine hydrochloride (GdmCl) half concentration (C_1/2) and melting temperature (T_m) obtained for the EcRDBD_WT, its mutants and the UspDBD in chemical and thermal denaturation experiments in comparison with the RosettaDesign scoring.

<p>The GdmCl concentration was taken for 50% of the protein fraction unfolded each and melting temperature values were assigned to the thermal denaturation curve derivative maxima (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086052#pone-0086052-g006" target="_blank">Figure 6</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086052#pone-0086052-g007" target="_blank">Figure 7</a>, insets and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086052#pone-0086052-g008" target="_blank">Figure 8</a>). An asterisk sign for the EcRDBD_WT score indicates value calculated for a side chain rotamers redesign structure (without any substitutions). The lower RosettaDesign score value the better structure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086052#pone.0086052-Rastinejad2" target="_blank">[17]</a>.</p

Root-mean-square fluctuation (RMSF) profiles with respect to EcRDBD mutant structures during MD simulations.

<p>The side chain root-mean-square fluctuations (RMSF) of the EcRDBD point mutants and the UspDBD (solid lines) in comparison with the EcRDBD_WT (dashed lines) calculated for the last 2 ns time period of MD simulations (from 8^th to 10^th nanosecond), panels A–G. Panel H represents the comparison between the RMSF profiles of the L58F point mutant (black line) and the UspDBD (gray line). The substituted EcRDBD positions are displayed by arrows. The secondary structure elements (α-helices 1 and 2) and C-terminal extension (CTE) sequence <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086052#pone.0086052-NiedzielaMajka1" target="_blank">[5]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086052#pone.0086052-Grad1" target="_blank">[6]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086052#pone.0086052-Orowski1" target="_blank">[12]</a> are indicated by gray areas and labeled by: H1, H2 and CTE labels.</p

Analysis of amino acid sequences and 3D structures of the EcRDBD and UspDBD.

<p>(A) Alignment of EcRDBD_WT and UspDBD sequences. The alignment was done using the <i>needle</i> program (<a href="http://www.ebi.ac.uk/Tools/emboss/align/index.html" target="_blank">http://www.ebi.ac.uk/Tools/emboss/align/index.html</a>) and revealed 46.0% of the sequences similarity and 38.1% of identity (<i>needle</i> score: 253.5). The residue numbering is relative to the first C residue coordinating the zinc ion of the DBD zinc module. The EcRDBD_WT sequence positions substituted by the RosettaDesign program <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086052#pone.0086052-Dantas1" target="_blank">[20]</a>, the conserved C residues coordinating the zinc ions and the terminal residues not visible in the crystal data <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086052#pone.0086052-Jakb1" target="_blank">[11]</a> were highlighted in red, yellow and gray, respectively. Blue dots indicate residues that form the hydrophobic core that stabilizes the domain. The α-helix structures, T-box and A-box were marked outside of each sequence by black, green and red lines, respectively. (B) The crystal structures of the EcRDBD_WT (red) and UspDBD (black) are superimposed together by their Cα. The RMSD value for the superimposition of the DBD fragments (residues from C1 to M66 in both cases) is equal to 0.747 Å. The main α-helices 1 and 2 of the DBD core, N- and C-termini of the domains and the C-terminal extension were labeled by: H1 and H2, N, C and CTE labels, respectively. The domain structures were taken from the UspDBD/EcRDBD heterocomplex on a natural response element <i>hsp27_pal</i> (PDB: 2HAN) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086052#pone.0086052-Jakb1" target="_blank">[11]</a>. (C) and (D) EcRDBD_WT and UspDBD energy-minimized structures (white) superimposed upon their structures obtained after a 10 ns time period of each MD simulation (green). (E–J, left) Side chain conformations of the chosen EcRDBD substituted residues together with their adjacent residues after 10 ns MD simulations. The shortest distances between the residues and salt bridges (in Å) are shown as dashed lines. (E–J, right) The whole EcRDBD point mutant energy-minimized structures (white) superimposed upon their structures obtained after a 10 ns time period of each MD simulation (green). The substituted residues were shown as sticks (before and after the MD simulations as black and red, respectively).</p