Computational protocol: Interhelical loops within the bHLH domain are determinant in maintaining TWIST1–DNA complexes

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[…] The primary sequences of human TWIST1 (Q15672), TWIST2 (Q8WVJ9), E12 (P15923-1), HAND1 (O906004) HAND2 (P61296), NEUROD1 (Q13562), E47 (P15923-2), and MYOD1 (P15172) were downloaded from the UniProtKB/Swiss-Prot website. The mouse NEUROD1 and E47 sequences were obtained from the 2ql2 PDB files. Primary sequences, restricted to the bHLH domains, were aligned with the ClustalW 2.0 software ().The X-ray structures of the human MYC/MAX dimer (PDB code lnkp) (), mouse MYOD1/MYOD1 complex (PDB code lmdy) (), and mouse NEUROD1/E47 complex (PDB code 2ql2) () were downloaded from the RCSB protein data bank. Two X-ray structures of mouse NEUROD1/E47 have been reported (2ql2A/2ql2B; 2ql2C/2ql2D), which slightly diverge (root-mean-square deviation, RMSD). The calculated RMSD (based on align seed residue and list distance parameters) on C-chains is weak and evaluated at 0.618 Å, suggesting that the two models can be considered similar.Sequence alignments were submitted to the SWISS-MODEL Workspace () to generate models for homodimeric and heterodimeric TWIST1 complexes by homology with the NEURODl(2ql2D)/E47(2ql2C) or NEUROD1(2ql2B)/E47(2ql2A) template, respectively, as described in (). The RMSDs between the models generated by Swiss Model Workspace and the folds are, respectively, 1.065/0.069 Å and 1.60/0.064 Å for TWIST1/E12 (modeled reference 2ql2B/A) and TWIST1/TWIST1 (modeled with the reference 2ql2D/C). The choice between the two homology models was finally made by calculating the RMSD of each dimer with respect to its reference. Homology modeling was performed with the same DNA sequence as used in the reported NEUROD1/E47 X-ray structure. The dynamics simulations represent series of possible conformational states of the DNA-bound complexes.To gain further insight into TWIST1 dimer function and specificity, we generated additional models with two functionally active TWIST1 variants expressed in SC patients. These variants result from a 21-bp tandem repeat insertion into the TWIST1 gene, leading to the aberrant presence of seven extra amino acids in the interhelical loop at position 135 or 139 (Ins-135 or Ins-139, Figure S5) (). We, thus, built homology models of the following complexes: TWIST1/TWIST1, TWIST1 Ins-135/TWIST1 Ins-135, TWIST1 Ins-139/TWIST1 Ins-139, TWIST1/E12, TWIST1 Ins-135/E12, and TWIST1 Ins-139/E12.Minimizations (>10,000 steps with the conjugated gradient algorithm) were carried out with the Sybyl-X 1.1 software package, elaborated by the Tripos company. We applied the Tripos force field with the Gasteiger–Marsilli partial charges and a dielectric constant of 80 to simulate an implicit water phase (the dielectric constant of water is 20.10 at 20 °C). No restrains was applied to our models. This step principally refines and corrects the positions of residue side chains. The effects of minimization on the homology models were estimated approximately by calculating their RMSDs before and after the minimization step. In the heterodimer, the RMSDs of the E12 and TWIST1 peptides were 1.58 and 1.97 Å, respectively. In the homodimer, the RMSDs of the two TWIST1 peptides were 3.21 and 1.69 Å. The E-loop, thus, seems more disordered that the T-loop in the homodimeric complex, in agreement with the NeuroDl/E47 X-ray structure (). Nonetheless, the RMSD calculation during the minimization step showed that the E-loop structure converges to a stable conformation, which could be used in dynamic simulations. [...] The established homology models (including DNA sequences) were visualized with the VMD1.9.1 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. A distance of 15 Å was set between the surfaces of the protein and the limit of the solvent box.Conditions were computed to reach neutral charges before adding sodium and chloride to concentrations corresponding to physiological conditions. The model was minimized with the NaMD 2.8 bl software for 1000 steps before the molecular dynamics simulations (). 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 5ps−−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 periodic boundary condition size (). The electrostatic cut-off was set at 14 Å. A conformation was sampled every 10 ps. As the solvent was described, the dielectric constant was set at 1. To identify steady conformations, 2D-RMSD calculations were carried out on 100 conformations selected, with a stride of 10, from the 1000 conformations produced during the 10-ns simulation. The equilibrium state was reached around 30-ps for all studied models. […]

Pipeline specifications

Software tools Clustal W, SWISS-MODEL, Sybyl-X, VMD, NAMD, CHARMM, STRIDE
Databases UniProt UniProtKB
Application Protein structure analysis
Organisms Mus musculus, Homo sapiens
Diseases Acrocephalosyndactylia