Interhelical loops within the bHLH domain are determinant in maintaining TWIST1-DNA complexes
Charlotte Bouard, Raphael Terreux, Jennifer Hope, Julie Anne Chemelle, Alain Puisieux, Stéphane Ansieau, Lea Payen-Gay [1]
Molecular Tour
We have established homology models of the human TWIST1 homodimer and heterodimers (formed with the E12 partner) bound to their target DNA sequences. The (PDB entry 2ql2) meets all the criteria for constituting an appropriate template. In their bHLH domains, TWIST1 is 48% and 29% identical to the murine NEUROD1 and E47 proteins. Moreover, the 5’-CATCTG-3’ E-box sequence included in this X-ray structure is a TWIST-responsive element (Connerney, 2006; De Masi, 2011). Although the C-terminal sequences adjacent to the bHLH domain, constitute a protein interaction site essential for TWIST function (Spicer, 1996; Bialek, 2004), we reasoned that a structural model restricted to the bHLH domains should provide additional insights into the TWIST-DNA interface and highlight functional differences between TWIST1 complexes. Using a homology strategy, in silico models based on the murine NEUROD1/E47 X-ray structure were established and also subjected to dynamics simulations.
The established homology models (including DNA sequences) were visualized with the VMD 1.9.1 software. The resulting model was inserted into a parapipedic TIP3P solvent box by means of the add solvation box module of the VMD 1.9.1 software. Conditions were computed to reach neutral charges before adding sodium and chloride to concentrations corresponding to physiological conditions. It was computed on a 144 xeon core CPU cluster supercomputer (SGI Altix). Simulations were carried out at constant temperature (300 K) and pressure (1 atm) and by implementing the widely used CHARMM 27 force fields. The time step was set at 1 fs and Langevin dynamics was performed with a target piston pressure of 1.01325 bar and a damping coefficient of 5 ps-1. There is no coupling of the Langevin temperature with hydrogen. The PME algorithms were applied with a grid extended by 10 Å from the PBC size. The electrostatic cut-off was set at 14 Å. A conformation was sampled every 10 ps. The equilibrium state was reached around 30 ps for all studied models. In the attached video 1, the TWIST1/TWIST1 in silico models are represented as yellow ribbons. Lateral chains of residues within the T-loop and E-loop of bHLH domains are visualized in orange, while DNA is represented in dark grey.
According to the literature, a deficiency of TWIST1 expression leads to Saethre-Chotzen syndrome, which is characterized by premature fusion of the cranial sutures and specific minor limb abnormalities (Bourgeois, 1998;El Ghouzzi, 1999; El Ghouzzi, 1997 ;El Ghouzzi, 2000;El Ghouzzi, 2001; Firulli, 2005;Yousfi, 2002). Consequently, to gain further insights into the role of the interhelical loops in TWIST1 dimer function and specificity, we next focused on additional Saerthe-Chotzen associated TWIST1 variants with aberrant insertion of 7 amino acids into the interhelical loop at position 135 or 139 (Ins-135 or Ins-139) due to the presence of a 21-bp tandem repeat in the TWIST1 gene (El Ghouzzi, 1997).
We superposed the homodimeric complex structures generated for (blue), its Ins-135 (red), and Ins-139 (grey) variants (Figure 1). We clearly notice that these insertions are wider compared to the wild-type TWIST1 and not equivalent in the conformations of the interhelical loops of the complexes, as the Ins-135 insertion disrupts much fewer contacts between interhelical loops and DNA. Furthermore, the structural analysis highlights, between the two amphipathic α-helices, an interhelical loop whose conformation differs between the monomers forming a given TWIST1-containing dimer and according to the partner of TWIST1 in the dimer. We next aimed to identify residues essential for interhelical loop conformation. Although these loops do not have a structural organization strictly speaking, H-bond formation can stiffen their structure. By limiting our study to H-bonds with an occupancy exceeding 5% of the total simulation and by excluding H-bonds between residues of different protein partners or involved in α-helix structures, we were led to focus on four TWIST1 residues: Lys145, Ser144, Arg132 and Arg118. We highlighted important H-bonds (in purple) established between residues (Lys145-HZ2/Asn125-OD1, Ser144-O/Thr148-HG) or DNA (Ser144-HG/Cyt(-1*)-O1P (H-bond) and Arg118-HH12/Ade(+4*)-O2P (H-bond) on the E-loop, during dynamics simulation of the dimer TWIST1/TWIST1 (Figures 2 and 3, the T- and E-loops are respectively represented in yellow and grey ribbons).
In conclusion, our data support the view that interhelical loops within the bHLH play a determining role in maintaining TWIST1-DNA complex structures and provide a structural explanation for the loss of function associated with several TWIST1 mutations/insertions observed in SC patients.
