BACKGROUND
twist <dig> is a transcription factor belonging to the basic helix-loop-helix  superfamily.
during embryogenesis, it functions as an inducer of the epithelial-to-mesenchymal transition , a transdifferentiation process promoting the transient conversion of epithelial cells into mesenchymal cells  <cit> .
the twist <dig> protein is highly conserved from drosophila to humans, where it plays a key role during embryonic morphogenesis, mesoderm patterning and development.
it is involved in cell-type determination and differentiation during myogenesis, cardiogenesis, neurogenesis, hematopoiesis and osteogenesis  <cit> .
the aberrant expression of twist <dig> has recently been implicated in cancer development, by fostering tumorigenesis and promoting the invasion-metastasis cascade  <cit> .
one of the major functions of proteins belonging to the bhlh superfamily, which includes twist <dig>  twist <dig>  e <dig>  e <dig>  hand <dig> and hand <dig>  <cit> , is their interaction, once dimerized, with e-box sequences .
twist <dig> proteins can form either homodimeric  or heterodimeric complexes, mainly in association with e2a proteins.
this dimerization is a prerequisite for the recognition of e-box sequences, and involves basic domains and key residues of the bhlh domains  <cit> .
these complexes can bind to numerous variable canonical core recognition e-box sequences with different levels of affinity, and display distinct and sometimes even antagonistic functions during embryonic development  <cit> .
the differences in the composition of the e-box sequences, in effect, largely modulate the in vivo function of cellular transactivation by closely related transcription factors  <cit> .
the saethre-chotzen syndrome  is an autosomal dominant craniosynostosis characterized by facial and limb deformities caused by loss-of-function mutations of the twist <dig> gene on chromosome 7p <dig>  <cit> .
over  <dig> twist mutations have been described in scs patients, a majority of which are present in the bhlh domains, mediating protein dimerization, and involving a single base pair substitution .
these substitutions either create premature termination codons, which lead to truncated proteins, or substitute highly conserved residues in the bhlh region.
these mutations affect the dimerizing ability and the dna-binding properties of twist <dig>  deletions  and insertions  of nucleotides have also been reported in scs patients.
one such mutation, termed r154g, is the naturally-occurring, rare substitution of the arginine  <dig> amino acid with a glycine residue on the twist <dig> protein  <cit> .
arginine binds phosphate anions with a high affinity, and is often present in the active sites of proteins that bind phosphorylated substrates.
as such, arginine residues play crucial roles in maintaining the overall balance of the charges within a protein.
the arginine residue plays a role in maintaining the overall charge balance of a protein.
in addition, the nh <dig> groups of arginine are positioned so as to carry out h-bonds with partners or close residues of the protein, and strengthen the conformational structure of the dimer, while the biochemical properties of proline impede such an interaction.
this arginine  <dig> residue is not conserved in other bhlh proteins , which contain a glutamine, serine or asparagine residue at this position, residues that provide only limited physicochemical changes compared to an arginine residue.
however, the r <dig> residue is instrumental in maintaining proper dimerizing functions of the twist protein, as demonstrated by spicer et al.
<cit> , who performed a proline point-mutation  to ensure stronger physicochemical changes, since the lateral cycle usually disrupts the alpha helix of the hlh , and reported an impaired dimerization.
this was later confirmed by our team in vitro in mammary epithelial cells by immunoprecipitation experiments  <cit> .fig.
<dig> comparison of the persistence of the wild-type twist1/e <dig>  dimer and mutated r154p te dimer with and without dna.
a primary sequence alignment of the bhlh domains of the neurod <dig>  twist <dig>  and e2a proteins.
residues within the interhelical loops are underlined, h and m stand for human and murine, respectively.
the arrows indicate the localization of the r <dig> residue and its equivalent on the other proteins.
b-c 3d representation of the conserved twist <dig> /e <dig>  complex in the te and te r154p dimers in frontal  and lateral  views.
the localization of the  arginine and the  proline  <dig> residues in the twist1/e <dig> and twist1/e <dig> r154p complexes on the twist <dig> ribbon is highlighted in cpk.
d-e the root mean square fluctuation  of twist <dig> and e <dig> amino acids were estimated during 10 ns in silico molecular dynamics  simulations using the vmd  <dig> .
<dig> software.
the graphical representation showed the calculated rmsf in angstroms  for each  twist <dig> or  e <dig> residue in the mutated r154p te model with  or without  dna anchor, and the te model with  or without a dna anchor .
f-g box plots representing the root mean square deviation  by considering all of the atoms except hydrogen  during 10 ns in silico molecular dynamics  simulations.
these distances were estimated during the md simulations using the vmd  <dig> .
<dig> software .
the noh 1d-rmsd is shown in the  mutated r154p te model with  or without  dna anchor, and  the te model with  or without a dna anchor
our present aim was thus to investigate whether our previously published in silico approach  <cit>  was able to predict the decrease in twist1/e <dig> dimerization observed in vitro and to decipher the molecular mechanisms involved, by comparing the dimerizing ability of the wild-type twist1/e <dig>  complex with that of the mutated r154p te  complex  <cit> .
furthermore, we examined whether these complexes bound to functional e-box sequences of targeted gene promoters using md simulations, as previously described  <cit> .
this study was based both on the use of a recently published 3d dynamic structural model of the te dimer  <cit> , initially generated using the murine neurod1/e <dig> crystal structure  <cit> , and on functional in vitro assays conducted on cancer cells to validate these computer-based models.
methods
in silico analysis
selection of x-ray structure and in silico model building
when initiating our study, we first selected the most appropriate 3d comparative model of the twist1/e <dig> complex from the structures available in the protein data bank .
this choice mainly resided in the level of homology of these structures with twist <dig> and e <dig> and in the composition of the dna sequence in the x-ray structure.
indeed, at the level of protein identity two candidates were identified, namely the human scl/e <dig> complex  and murine neurod1/e <dig> complex , displaying 58% and 48% homology  with twist <dig>  respectively, and 100% and 86% homology  with e <dig>  respectively .
we next focused on the dna sequence used in the generation of the model.
indeed, all four models were generated using a dna sequence to stabilize the structures and facilitate the crystallization process.
having previously shown that the composition of dna bases of the e-box plays a crucial role for dna binding by bhlh dimers and for their transactivation activity  <cit> , and that the e-box sequence  used to generate the neurod1/e <dig> structure is a highly specific e-box sequence of the te complex  <cit> , we finally selected this model for our study.table  <dig> 3d bhlh structures available in the protein data bank
table containing four protein complexes structurally close to twist1/e <dig> .
the information included in this table enabled us to select the most appropriate crystal structure, based primarily on the percentage of identity , the dna sequence in x-ray structure and the resolution of structure.
the closest match  was neurod1/e47
in effect, although the “cagatg” e-box sequence contained in the scl/e <dig> structure could be a potential dna binding site of twist <dig>  as predicted by de masi et al.
<cit> , and confirmed by emsa experimentation in particular for the dna binding of twist1/e <dig> dimer  <cit> , the specificity of this box for twist <dig> homo- or heterodimers has not yet been determined.
lastly, the crystallization resolution of the neurod1/e <dig> structure was the highest between these two models at  <dig>  Å.
the mouse neurod <dig> and e <dig> 3d structure was thus obtained from pdb files, and the dna sequence used to generate the model was kept in place, so as to preserve the proper position of the dimer in the dna groove, and avoid altering residue-base interactions.
sequences, restricted to the bhlh domains, were aligned using the clustalw  <dig>  software .
the alignments were then manually refined, taking residue properties  into account.
two x-ray structures of the mouse neurod1/e <dig> complex have previously been reported , which differ slightly .
this divergence being fully corrected by a series of minimization steps  followed by 10 ns molecular dynamics  simulations, the two models were considered equivalent, generating comparable homology models.
sequence alignments were submitted to the swiss-model workspace to generate models of heterodimeric twist <dig> complexes by homology with the neurod <dig> template /e <dig> .
minimizations  were carried out with the sybyl-x  <dig>  software package, using the tripos method with the amber force field and a dielectric constant of  <dig> in order to refine and correct the positions of residue side chains.
as mentioned above, the conserved twist1/e <dig>  dimer model used throughout this study included a dna sequence named the e-box   <cit> .
the r154p mutated te complex was generated by substituting the arginine  <dig> residue by a proline residue, using the mutation function of the sybyl-x  <dig>  software package.
the resulting in silico models studied herein were, therefore, the te complex  bound or not to the e-box sequence, as well as the r154p mutated  te complex bound or not to the e-box sequence.
all of the models were generated following the same algorithm and using the same parameters.
molecular dynamics simulations
the established homology models were visualized with the vmd  <dig> .
<dig> software.
the resulting model was inserted into a parapipedic tip3p solvent box by means of the add solvation box module of the vmd  <dig> .
<dig> software.
a distance of 15 Å was set between the surface 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  <dig>  b <dig> software for  <dig> steps and molecular dynamics simulations, and was computed on a  <dig> xeon core cpu cluster supercomputer .
simulations were carried out at constant temperature  and pressure  and by implementing the widely used charmm  <dig> force fields.
the time frame was set at 1-fs and the langevin and pme algorithms were applied.
a conformation was sampled every 10-ps.
in the first instance, the effect of the r154p mutation of twist <dig> on the molecular interactions of the te dimer with the e-box sequence was investigated in comparison with the effect of the wt twist <dig> protein, over a 10 ns experimental time-course.
in the second instance, the impact of the anchor provided by the dna sequence  on the binding efficacy of the wt or r154p mutated te dimer was studied by conducting md simulations over a time course of 10 ns.
definition of the domains studied
several domains were arbitrarily assigned to regions of the te dimer in figs.
<dig> and  <dig>  indeed, in fig.
2a and b, we studied the position of the te dimer in the dna groove by studying the distance between basic domains of twist <dig> and e <dig> in the te and te r154p md simulations.
we considered that the basic domain of twist <dig> is composed of  <dig> amino acids: t <dig>  q <dig>  r <dig>  v <dig>  m <dig>  a <dig>  n <dig>  v <dig>  r <dig>  e <dig>  r <dig>  q <dig>  r <dig>  t <dig>  q <dig>  s <dig>  l <dig>  n <dig> and e126; whereas that of e <dig> is composed of the following  <dig> amino acids: e <dig>  r <dig>  r <dig>  v <dig>  a <dig>  n <dig>  n <dig>  a <dig>  r <dig>  e <dig>  r <dig>  l <dig>  r <dig>  v <dig>  r <dig>  d <dig>  i <dig> and n <dig>  in the fig.
2c, d, e and f, we defined three domains as “bottom”, “middle” and “top”.
the former encompasses methionine  <dig>  of twist <dig> and valine  <dig>  of e <dig>  localized at the bottom of te dimer and near the dna sequence, while “middle” designates residues leucine  <dig>  of twist <dig> and aspartic acid  <dig>  of e <dig>  localized in the middle of te dimer, and finally, the “top” is constituted of isoleucine  <dig>  of twist <dig> and leucine  <dig> of e <dig> localized at the top of te dimer.fig.
<dig> comparison of the persistence of the wild-type twist1/e <dig>  dimer and mutated r154p te dimer.
a 3d representation of the conserved twist <dig> /e <dig>  complex.
description of the position of the basic domain residues is represented as a cartoon and cpk in grey for twist <dig>  and in green for e <dig> .
b the ca-ca distances between pairs of basic domain residues on twist <dig> and e <dig> were estimated during 10 ns in silico molecular dynamics  simulations using the vmd  <dig> .
<dig> software.
the graphical representation shows the calculated mean distances between pairs of twist1/e <dig> residues in angstroms  in the mutated r154p te model with  or without  dna anchor, and the te model with  or without a dna anchor .
c 3d representation of the conserved twist <dig> /e <dig>  complex bound to dna along with the e-box sequence , carried out using the vmd  <dig> .
<dig> software.
the residues are numbered in each strand of the 5′-catctg-3′ e-box core.
the three pairs of residues are shown, namely m112-v <dig>  l124-d <dig>  and i134-l <dig>  which were located at the bottom, in the middle and at the top of the dimer, respectively.
d-f box plots representing the distances  between pairs of residues of the dimer as a function of time , during the te  and te r154p  10 ns in silico molecular dynamics  simulations.
these distances were estimated during the md simulations using the vmd  <dig> .
<dig> software .
the lengths of h-bond interactions were represented via graphpad prism 5: “smooth, differentiate or integrate curve, with  <dig> neighbors”.
interactions between the  m <dig> of twist <dig> and v <dig> of e <dig> residue pair, close to the bottom of the dimer,  l <dig> of twist <dig> and d <dig> of e <dig> residue pair, close to the middle of the dimer, and  i <dig> of twist <dig> and l <dig> of e <dig> residue pair, close to the top of the dimer
fig.
<dig> consequences of impaired twist1/e <dig>  dimerization on dna binding.
3d representation of the conserved twist <dig> /e <dig>  complex.
description of the position of the two series of residues  boxes a and d, and  b and c boxes, in the te dimer are represented as a cartoon and cpk.
the first dimerization blocs composed of boxes a and d , b and c  are represented by cylindrical grey and green solid surfaces, on the twist <dig> and e <dig> proteins, respectively.
the localization of r <dig> on the twist <dig> ribbon is highlighted in cpk.
c evaluation of the impact of the r154p mutation on the number of h-bonds, in each individual box a to d by studing the percentage of variation of h-bond interactions between residues during te and e r154p md simulations, established between residue-residue and residue-dna  or exclusively between residues .
d bar chart representing the variation of h-bond interactions between residues of the te versus r154p te complex.
the sum of the interactions occurring between residues within the box a  and box b  of the wild type or mutated twist <dig> protein is highlighted.
the percentage of cumulated occupancies of h-bond interactions occurring in the mutated r145p te model is normalized against the te model .
all cumulated occupancy values of the h-bonds were calculated as described in the methods section.
briefly, h-bond interactions are assigned a value according to the distance between their atomic donors/acceptors during the 10 ns time-course of the md simulation .
higher occupancy values being obtained for shorter and, therefore, more stable interactions.
with logic test:“< <dig> ”, value of  <dig> if true and  <dig> if false; nb.si function: nb.si)
several domains were geographically defined in fig.
3a and b, named a and b boxes for twist <dig>  and c and d boxes for e <dig> , with a and d boxes and the b and c boxes facing each other, respectively .
the residues composing a and b boxes of twist <dig> encompass amino acids “120–127” and “149–157”, respectively, whereas the composition in residues of c and d boxes of e <dig> contain amino acids “565–574” and “588–594”, respectively .
the rmsf data interpretation
we studied the root mean square fluctuation , which represents the flexibility of the protein model.
we initially aligned the dimer with the vmd function rmsd trajectory tool , and then calculated the rmsf with the vmd timeline function :  <dig> and step size : 1).
we obtained one rmsf per amino acid per frame and the average rmsf per amino acid was calculated using microsoft excel.
the rmsd data interpretation
to complete rmsf analyses, 1d-root mean square deviation  calculations were carried out on all of the md simulations, taking into account the constraints of the template as a reference.
we obtained the 1d-rmsd with the vmd function rmsd trajectory tool .
the 1d-rmsd calculations conducted encompassed all of the atoms, except hydrogen .
before the extraction of 1d-rmsd values, we aligned all of the frames on the first frame by considering all of the proteins without hydrogen atoms.
the dssp data interpretation
we then used the online dictionary of secondary structure of proteins  program http://swift.cmbi.ru.nl/gv/dssp/ to calculate dssp parameters, such as secondary structure and solvent accessibility of protein residues.
we analyzed the solvent accessibility value defined for each residue and we compared these values between the te and te r154p md models.
variation of free energy of binding computed by mm-pbsa method
the resulting conformation of the protein dna complex from molecular dynamics simulations with namd was extracted and several files were prepared using the xleap module.
the complex was neutralized with sodium counter ions.
ions and dna were called the receptor and the protein the ligand.
the complexe structure with ions was inserted into a tip3p water pbc box.
the amber  <dig> software was used with the ff14sb forcefield.
the structure was minimized for  <dig> steps and the gently thermalized with an increase temperature from  <dig> to 300 k in 60 ps to avoid destruction of interactions.
the resulting structure was submitted to a 2 ns ntp molecular dynamics using a 300 k and 1 atm on titan x gpu card.
a frame was sampled every 20 ps so  <dig> frames were used for mm-pbsa calculations.
for mm-pbsa calculation the calculation was performed with a total non-polar solvation term free energy modeled with one term , and with igb =  <dig>
alanine scanning method
this methodology computes variations in free energy by replacing each residue by an alanine residue.
the alanine scanning calculation was performed with moe from the chemical computing group  company.
the amber10eth forcefield was chosen, and starting from the resulting structure of the namd molecular dynamics, calculations were performed with lowmode conformer generation .
the interaction energy was computed for each conformation of each mutation with the dna structure.
analysis of the persistence of the interactions arising during md simulations
among the interactions occurring between residue-residue  and residue-base , we focused our current investigation on hydrogen bond  interactions established between donor and acceptor groups.
the h-bond interaction is an intermolecular force involving a hydrogen atom and an electronegative atom, such as oxygen or nitrogen.
h-bonds can be established between donor atoms and acceptor atoms of side chain residues.
we studied the r-r or r-base interactions, which are established between atoms .
the lengths of the h-bond interactions were represented via graphpad prism 5: “smooth, differentiate or integrate curve, with  <dig> neighbors”.
these lengths were previously estimated around 2– <dig>  Å depending on the nature of the acceptor and donor atoms  <cit> .
jeffrey categorizes h-bonds with donor-acceptor distances of  <dig> – <dig>  Å as “strong, mostly covalent”,  <dig> – <dig>  Å as “moderate, mostly electrostatic”,  <dig> – <dig>  Å as “weak, electrostatic”  <cit> .
consequently, we defined the threshold of the h-bond at  <dig>  Å in order to mainly consider covalent h-bonds and to be more stringent.
using a computer software, we made the following hypothesis: any interaction occurring under this threshold was deemed “true” and assigned a score of  <dig>  whereas any interaction occurring above this threshold was deemed “false” and given a score of  <dig>  with logic test:“< <dig> ”, value of  <dig> attributed if true and  <dig> if false; nb.si function: nb.si).
the scores were reckoned and the value obtained was designated as the rate of occupancy of h-bond interactions.
thus, for a given h-bond, the higher the rate of occupancy, the greater the number of interactions occurring under  <dig>  Å, the higher the level of persistence of that h-bond during the md simulation.
in vitro analysis
vector constructs for immunoprecipitation assays
the cdna of twist <dig>  or wild-type cdna of e <dig> were cloned into the pci-neo expression vector with flag- or myc-tag, respectively.
the constructs obtained were named flag-twist <dig> vector , flag-twist <dig> r154p vector , and myc-tag e <dig> vector .
the twist <dig> r154p mutant construct was modified by genscript .
all final constructs were amplified using the maxiprep kit provided by promega and sequenced by genoscreen compagny.fig.
<dig> consequences of impaired twist1/e <dig>  dimerization on the loop structure of the bhlh domain.
a-b box plots representing the average distances  between the od <dig> atom of asparagine  and the nz <dig>  nz <dig> or nz <dig> atoms of the lysine  residues , and between n <dig> and k <dig> residues  during the wild-type twist1/e <dig>   and mutated te r154p  10 ns in silico molecular dynamics  simulations.
c the horizontal bar chart shows the cumulated occupancy values for the h-bond interactions established between the n125-k <dig> residues of twist <dig>  and the n566-k <dig> residues of e <dig>  during the te and te r154p md simulations.
all cumulated occupancy values of the h-bonds were calculated as described in the methods section.
briefly, h-bond interactions are assigned a value according to the distance between their atomic donors/acceptors during the time  .
higher occupancy values being obtained for shorter and, therefore, more stable interactions.
with logic test:“< <dig> ”, value of  <dig> if true and  <dig> if false; nb.si function: nb.si).
d western blot showing the interaction between wild-type twist <dig>  or t <dig> r154p and e <dig>  as assessed by immunoprecipitation assays.
twist <dig> or mutated twist <dig> r154p and e <dig> were transiently produced in hek293t cells.
the twist <dig> protein was immunoprecipitated with a monoclonal α-flag antibody and the presence of endogenous e <dig> protein in the immunoprecipitates  was assessed.
input 10%.
the protein sizes were 99 kda for the hetreodimer te, 73 kda for the e <dig> protein and 25 kda for the twist <dig> protein
vector constructs for streptavidin/biotin assays
the pbabe-neo expression vector was donated by h. land & j. morgenstern & b. weinberg  and was used as a negative control .
the cdna of the tethered te complex composed of  <dig> amino acids, including a flag-tag at the n-terminal position, twist <dig> cdna, an amino acid linker and e <dig> cdna, was cloned into the pbabe-neo expression vector  <cit> .
this construct of the ectopic te protein is approximately 99 kda in size.
the final construct was named the flag-twist1/e <dig> pbabe-neo expression vector .
this te construct was modified by genscript in order to obtained the tethered te r154p mutant complex composed of  <dig> amino acids, including a flag-tag at the n-terminal position, twist <dig> cdna with the substitution of the arginine  <dig> by a proline, an amino acid linker and e <dig> cdna, was cloned into the pbabe-neo expression vector with the same size of te .fig.
<dig> consequences of impaired twist1/e <dig>  dimerization on dna binding.
a 3d in silico representation of the te complex bound to dna, carried out using the vmd  <dig> .
<dig> software.
the proximal flanking  and first  bases of the e-box are represented in grey and green vdw, respectively.
b-c the pie charts show the percentages of cumulated occupancy of h-bonds established between residues of twist <dig>  and e <dig>  and dna bases during the  te and  r154p te md simulations.
occupancies of h-bonds established with the proximal flanking  bases are represented in yellow, while the bar charts highlight the exact residues binding to those bases.
the h-bonds established with the first consensus bases of the e-box  are represented in grey and green for the twist <dig> and the e <dig> residues, respectively.
d-g distribution of “specific” h-bonds established between residues and e-box bases in the  te and  mutated r154p te molecular dynamics simulations.
the pie charts show percentages of the total rate of cumulated occupancy of specific h-bonds established during the md simulations.
consensus bases and variable bases of the e-box sequences are represented in grey and pink, respectively.
the bar charts highlight the specific residues binding to cytosine  and  bases, and show their implication in the dna binding affinity.
the dna sequences are represented for  the te and  mutated r154p te dimers.
the arrows highlight the major base interactions for the te complex  and mutated r154p te complex .
h western blot showing that the tethered twist1/e <dig> r154p dimer  was not able to interact with the te-box unlike the tethered twist1/e <dig> wild-type dimer, as assessed by streptavidin/biotin assays.
tethered te and te r154p dimers were transiently produced in hela cells.
the protein sizes were 99 kda for the thetered dimer te and 25 kda for the twist <dig> protein
immunoprecipitation assays
hek293t cells were provided by the atcc.
hek293t and hela cells were transfected in 10 cm in diameter culture dishes at a density of 80%–90% using the fugene hd transfection reagent  for 48 h. the transfection reagent contained  <dig>  μg of the empty pci-neo expression vector or of the pci-neo expression vector containing cdna coding the flag-twist <dig>  flag-twist <dig> r154p, or myc-e <dig> pci-neo, as well as 10 μl of fugene.
following their transfection, cells were washed with cold pbs  and lysed in ebc solution .
after incubation on ice for 20 min, lysates were collected by scrubbing the plates, sonicated  and centrifuged for 20 min at  <dig>  rpm at 4 °c.
the supernatant was collected and the total protein content was quantified using the bio-rad protein assay .
after clearing by centrifugation, the protein extract was incubated with an anti-flag m <dig> resin .
after intensive washing to eliminate non-specific binding with ebc solution , the resin was resuspended in laemmli buffer and boiled for 5 min.
after elimination of the beads by centrifugation, samples were reduced by adding β-mercaptoethanol and separated by sds-page.
proteins were analyzed with the rabbit polyclonal flag antibody  and the rabbit polyclonal v <dig> e2a antibody  and a polyclonal goat anti-rabbit immunoglobulin conjugated with horseradish peroxidase as the secondary antibody .
the detection was performed by western blot analysis using the luminol reagent .
streptavidin/biotin binding assays
hela cells were transfected in 6 cm in diameter culture dishes at a density of 80%–90% using the fugene hd transfection reagent  for 48 h. the transfection reagent contained  <dig>  μg of the empty pbabe-neo expression vector or of the pbabe-neo expression vector coding the cdna of the flag-twist1/e <dig> pbabe vector or flag-twist1/e <dig> r154p pbabe vector, as well as 21 μl of fugene reagent.
following their transfection, protein extractions were carried out as reported for the immunoprecipitation assay.
the streptavidin/biotin binding assay was conducted to isolate proteins  bound to specific dna sequences .
to do so, total cellular proteins  were incubated with biotin-coupled dna sequences, and the dna bound te complex was then purified by immunoprecipitation  via the recognition of biotin by streptavidin beads.
hence, this approach decreased the risk of isolating non-specifically bound proteins.
briefly, both strands of dna probes corresponding to various e-box sequences were labeled in their 3’ extremity with biotin.
the probe used was the active te-box .
for probe hybridization, an initial denaturing step at  <dig> °c for 5 min was followed by a hybridization step at  <dig> °c for 10 min .
probe hybridizations were then confirmed on 3% agarose gels.
the binding between the dna hybridized probes  and the te complex  was carried out in binding buffer  for 1 h at  <dig> °c.
biotinylated dna probes bound to the te complex were purified using dynabeads® m- <dig> streptavidin .
coupled streptavidin dynabeads® bound to the dna/te complex  were run on a 12% sds-polyacrylamide gel using a migration buffer  at 80 v for 10 min in the stacking gel, followed by the migration phase at 100 v for 120 min.
the liquid transfer onto a pvdf membrane was conducted using a transfer buffer  at 200 ma for 80 min.
the membrane was then saturated with 5% tris-buffered saline  containing  <dig> % tween and  <dig> % milk for 45 min, and incubated with the mouse monoclonal twist <dig> antibody  as a primary antibody, and a polyclonal rabbit anti-mouse immunoglobulin conjugated with horseradish peroxidase as the secondary antibody .
the detection was performed by western blot analysis using the luminol reagent .
RESULTS
the final 3d model used to generate our te dimer was based on the mus musculus neurod1/e <dig> complex .
after aligning the four protein sequences using the clustalw software  <cit> , we mainly observed that neurod <dig> is composed of  <dig> amino acids with a bhlh domain containing amino acids 101– <dig>  whereas twist <dig> is composed of  <dig> amino acids with a bhlh domain encompassing amino acids 108– <dig> .
the crucial, strong physicochemical r <dig> residue of twist <dig> is a serine, an asparagine and a glutamine residue in e <dig>  murine neurod <dig> and murine e <dig>  respectively .
as speculated by spicer et al.
<cit> , we could clearly observe that the substitution of the arginine residue with a proline residue, the lateral side chains of which contain a cycle known to destabilize the alpha helix, in this crucial position at the dimerization interface of te altered the conformation of the dimer .
indeed, while the nh <dig> groups of arginine are positioned to carry out h-bonds with partners or close residues of the hlh loop, and strengthen the conformational structure of the dimer, the biochemical properties of proline impede such an interaction.
next, to characterize the effect of this substitution on molecular mechanisms involved in the dimerization of the te complex  <cit> , we submitted the four in silico models  to 10 ns md simulations.
we then carried out root mean square fluctuation  analyses to measure the structural flexibility of our proteins ; the higher the rmsf value, the greater the flexibility.
we observed an increase in rmsf values in the absence of dna for twist <dig> and e <dig> residues, suggesting that the dna sequence stabilizes the dimers.
nevertheless, no significant rmsf variation between te and te r154p md simulations was observed, indicating the same level of flexibility of these two models .
to complete this analysis, root mean square deviation  calculations were also carried out using the noh parameter .
clearly, the presence of dna during the dynamic simulations induced a decrease in 1d-rmsd values and enabled the stabilization of the dimer .
again, no significant difference could be observed between the te and r154p te models.
when focusing on r-r interactions between the  <dig> basic residues of twist <dig> and e <dig>  , the absence of dna resulted in an increase in the mean r-r distances by almost 5 Å in te and te r154p molecular dynamics simulations .
since such a molecular instability in the dna-free model may have overweighed the structural impact of the r154p point-mutation, or led to a biological artifact, all of the following md simulations included a specific dna e-box sequence  <cit> .
we then evaluated the predictive value of our in silico strategy in assessing the functional disruption generated by the r154p mutation of twist <dig> on protein dimerization and dna binding.
of note, protein dimerization precedes the binding of the complex to dna sequences  <cit> .
we observed that the mean distance between basic domains of te r154p  was greater than that of te  .
we then arbitrarily separated the dimer into  <dig> regions , namely the “top” , “middle”  and “bottom”  .
a clear variation in distances between twist <dig> and e <dig> backbone residues  was observed with the r154p mutation, indicating a distortion of the te structure.
indeed, these distances either increased, namely between the m <dig> and v <dig> residues , and between l <dig> and d <dig> , or slightly decreased, namely between i <dig> and l <dig> residues .
these results strongly suggest that the basic te domains are further apart from the e-box sequence  during the mutated te r154p md simulation compared to the te simulation, thus, impeding proper dna binding .
to complete these predictions, we calculated the dictionary of secondary structure of proteins  parameter, which provides information on secondary structure and solvent accessibility of protein residues.
no variation in the secondary structure prediction between te and te r154p for the arginine or proline residue were unveiled.
nevertheless, the r154p mutation twist <dig> decreased solvent accessibility of twist <dig> residues, mainly the loop residues of twist <dig> including residues i <dig> to l <dig>  in contrast, this point-mutation did not modify the solvent accessibility of e <dig> residues .
having shown a disruption in the ca-ca interactions between twist <dig> and e <dig> residues in the r154p mutated dimer, we investigated the effects of this mutation on the persistence of h-bonds  established between residue-residue  or residue-dna base  during md simulations .
we studied four dimerizing domains along the mutated complex, which are directly involved in dimerization and dna binding, named a and b boxes for twist <dig>  and c and d boxes for e <dig> .
a and d boxes  and b and c boxes  interacted with each other, respectively.
as expected, the r154p mutation had the biggest impact on the persistence of h-bonds established by amino-acid residues situated within the b box, where the r <dig> residue resides, with a 41% decrease in h-bond establishment .
when evaluating the persistence of h-bonds exclusively established between r-r, we observed that these were also impaired in the a box of twist <dig>  and d box of e <dig> .
when we evaluated the interaction r-base, we observed an increase in these interactions, enabling a stabilization of the dimer on dna.
this clear decrease in the stability of these boxes, when comparing the te and te r154p models, confirms that dna acts as an anchor for the c-α chain of hlh domains of twist <dig> .
the destabilizing effect of the r154p substitution on twist <dig> was further explored, by studying the difference between the cumulated interactions of each residue within the a and b boxes of the wild-type and mutated twist <dig>  during md simulations.
these simulations focused exclusively on the a and b boxes, since these domains were reported to experience major alterations .
this strategy revealed that the impact of the r154p mutation is distributed throughout the α-helix.
moreover, the residues that presented the highest rate of variation in their h-bond interactions, such as threonine , glutamine , and asparagine  within the a box , and leucine , lysine , and alanine  within the b box , were considered to be directly implicated in stabilizing the complex.
in addition, critical h-bonds localized in the heart of the dimer were disrupted, namely between the n125-k <dig> residue pair of twist <dig> and the n566-k <dig> residue pair of e <dig> in the r154p twist <dig> variant .
this disruption occurs between the od <dig> atom of the asparagine  residues and either one of the three nz <dig>  nz <dig> or nz <dig> atoms of the lysine  residues that are present under physiological conditions.
indeed, the interaction between the n566-k <dig> residue pair of e <dig> was largely impaired, revealing the clear impact of the twist <dig> mutation on the structure of heart of the te complex, which is at the interface of the functional e-box recognition site .
we confirmed the putative decrease in affinity by calculating the free energy of binding in kcal/mol between the te complex and dna using the mm-pbsa method.
we obtained a dg score of −217 kcal/mol and of −191 kcal/mol in the te wild-type and the te r154p mutant, respectively.
however, the dg computed with the mm-pbsa method implemented in amber  <dig> software is different from the namd method used during our md simulations.
consequently, we carried out the alanine scanning method, similarly to that used in our md models.
this alascan approach enabled us to define the affinity of twist <dig> for e <dig> and reciprocally of e <dig> for twist <dig> in the te or te r154p complexes.
the affinity of twist <dig> for e <dig> was 46 kcal.mol− <dig> and 66 kcal.mol− <dig> in the te complex and in the te r154p complex, respectively.
the affinity of e <dig> for twist <dig> was 55 kcal.mol− <dig> and 51 kcal.mol− <dig> in the te complex and in te r154p complex, respectively.
taken together, the alascan and mm-pbsa studies revealed that twist <dig> has a higher affinity for e <dig> in the te complex, and thus confirmed that the r154p mutation may impact twist1 dimerization and its recognition or binding, once dimerized, to active e-box sequences.
consistently, we showed in vitro that twist <dig> r154p  was not efficiently able to bind e <dig>  as evidenced by immunoprecipitation assays .
these assays revealed the presence of endogenous e <dig> protein  in the empty control lanes  .
furthermore, although a clear te dimer was obtained in the t1/e <dig> lane around 95–100 kda, we also observed additional complexes t1/t <dig>  and e12/e <dig> , no dimer was observed in the t <dig> r154p/e <dig> lane .
consequently, we explored the dna binding affinity of the wild-type or mutated te complexes, to investigate how such a mutation may affect canonical e-box recognition.
based on recent findings  <cit> , we studied the particular involvement of the , ,  and  dna bases in the establishment of h-bond interactions between the wild-type or r154p mutated te complex and the e-box sequence .
we noted an important increase  in the implication of  and  adjacent bases in the r154p variant .
moreover, this increase involved residues s <dig>  r <dig> and r <dig> which were shown to play a pivotal role in the specific dna binding affinity of the te complex  <cit>  .
our previous findings demonstrated that the specific affinity of the te complex to functional e-box sequences was associated with specific h-bond interactions  established with consensus bases , ,  and , as well as variable central bases , ,  and  of the e-box  <cit> .
here, we studied their distribution in the current md simulations.
we clearly noted that specific h-bond interactions were lost in the case of the mutated r154p te complex, especially with variable central bases .
this decrease was largely compensated by an increase  in specific h-bond interactions with consensus bases  and  of the e-box sequences .
in conclusion, these data strongly indicate that the r154p mutant, similarly to the te complex binding to modified e-boxes  <cit> , establishes alternative h-bond interactions with adjacent bases in our in silico models in an attempt to compensate for the decrease in structuration of the heterodimer.
hence, the r154p mutation not only affects the dimerization function but also the dna binding function of the te complex.
this finding was validated in vitro using the strepatvidin/biotin assay.
indeed, although the tethered te r154p dimer seemed less stable than the te dimer , likely due to the impact of the r154p mutation on dimerization, it was unable to bind to the te-box .
thus, the present in silico strategy is able to predict the impact of mutations occurring within the hlh domains of twist <dig>  and possibly of e2a proteins, on the dimerization of the proteins and on dna binding.
discussion
our study provides yet another rationale for using molecular dynamics  simulations as a predictive tool in the context of protein interactions, dimerization and dna binding, and in determining the impact of protein point-mutations on the persistence of the hlh domains of the oncogenic te complex  <cit> .
indeed, this approach previously enabled our team to study the effects of molecular modifications  of twist <dig> on the binding of the te complex to dna  <cit> .
this in silico md approach highlighted  the identification of the function of individual residues and molecular cooperation between residues,  the predominant role of protein side chain residues, close to the heart of the complex, and those necessary for anchoring the dimer to dna sequences, and  the localization of the te dimer on the dna groove, by studying the shift towards adjacent e-box bases  <cit> .
in agreement with sauve’s model  <cit> , we identified three different states of binding of the te bhlh complex to functional and degenerate e-box sequences, based on md simulations  <cit> .
this in silico approach strongly suggested that we were able to define the molecular mechanisms implicated in the binding flexibility of the te complex to e-box sequences of a targeted gene promoter, by predicting the proper transcriptional function.
here, using a twist <dig> protein displaying the r154p mutation, we accurately predicted both an impairment in the protein dimerizing function and a subsequent decrease in the e-box binding affinity.
we highlighted the determinant role of the interhelical loops in maintaining the structure of the twist1-dna complex, by studying two variants with a 21-bp tandem repeat insertion in the twist <dig> gene, leading to the aberrant presence of seven extra amino-acids on positions  <dig> and  <dig> of the interhelical loop in scs patients  <cit> .
these insertions strongly modified the structure of the interhelical loops, and led to fewer contacts between interhelical loops and dna.
these proof of concept experiments were conducted prior to carrying out in vitro assays, namely immunoprecipitation and streptavidin/biotin assays, to validate the functional outcome of substituting residues.
taken together, these results corroborate findings of spicer et al.
<cit> , who suggested that the scs mutation r154p  led to a decrease in dimerization.
this, in turn, may affect the quantity of functional te complexes present in cells, explaining the intermediate loss of luciferase activity following the r154p mutation compared to the te complex  <cit> .
as we reported previously, this alteration impaired the transformation potential of the te complex induced by the cooperation with the h-rasg12v protein  <cit> .
this approach also provided a structural explanation for the loss-of-function associated with twist1-haploinsufficiency observed in scs patients  <cit> .
using md simulations and biochemical assays, we and others also highlighted the pivotal function of the r118h/q/c, r120p, s144r, and k145e/q residues in the molecular binding of the te complex .
mutations affecting these residues, as observed in scs, lead to a decrease in the binding affinity of the dimer with the regulatory e-box sequences, and likely modify the transactivation functions of twist <dig>  <cit> , resulting in an imbalance in the twist1/twist <dig> homodimer and in the te heterodimer during the developmental process  <cit> .
in the present work, we completed this study by showing in silico and in vitro that the h-bond integrity between residues of bhlh was partially impaired, which appears to be the major cause of the decrease in protein dimerization.
to compensate for this, the r154p mutant alternatively establishes h-bond interactions with both non-specific and consensus bases of the e-box, thus modifying the conformation of the heart of the dimer by losing the crucial n125-k <dig> and n566-k588 h-bonds interactions.
it is worth noting that k <dig> mutations are also reported in patients suffering from scs syndrome  <cit> .
several bhlh proteins are known to dimerize with twist <dig>  including the hand proteins and proteins derived from the following genes: the tcf2/heb, the tcf3/e2a  and the tcf4/e2- <dig>  considering the degree of homology in their bhlh domains , applying the current in silico approach to the evaluation of the dimerizing properties of all of these complexes would be highly relevant.
in the future, it may be possible to identify specific transcriptional signatures according to the twist <dig> heterodimers generated, for example twist1/e-proteins versus twist1/twist <dig> or twist1/hands, and to their e-box binding specificities.
CONCLUSIONS
these md approaches allowed us to determine the key residues of twist <dig> and its partners involved in hlh dimerization, which may completely modify the balance of homo- and heterodimeric twist <dig> complexes.
overall, md simulations may constitute a powerful tool to predict the biological impact of these alterations in a cancer cell, in terms of dimerization, but also of binding affinity to active e-box sequences.
additional file
additional file 1: figure s <dig>  consequence of impaired twist1/e <dig>  dimerization on dna binding.
a-c: dictionary of secondary structure of proteins  parameters were calculated for the te and te r154p models, to obtain information on secondary structure and solvent accessibility of twist <dig> and e <dig> residues.
bar chart presenting the variation in solvent accessibility of twist <dig> residues.
3d representation of the conserved twist <dig> /e <dig>  complex displaying residue  <dig> represented in cartoon and residues impacted by dssp calculations represented in cpk.
bar chart presenting the variation in solvent accessibility of e <dig> residues.
electronic supplementary material
the online version of this article  contains supplementary material, which is available to authorized users.

 twist is a transcription factor belonging to the basic helix-loop-helix superfamily. during embryogenesis, it functions as an inducer of the epithelial-to-mesenchymal transition, a transdifferentiation process promoting the transient conversion of epithelial cells into mesenchymal cells. the twist protein is highly conserved from drosophila to humans, where it plays a key role during embryonic morphogenesis, mesoderm patterning and development. it is involved in cell-type determination and differentiation during myogenesis, cardiogenesis, neurogenesis, hematopoiesis and osteogenesis. the aberrant expression of twist has recently been implicated in cancer development, by fostering tumorigenesis and promoting the invasion-metastasis cascade. one of the major functions of proteins belonging to the bhlh superfamily, which includes twist twist e e hand and hand, is their interaction, once dimerized, with e-box sequences. twist proteins can form either homodimeric or heterodimeric complexes, mainly in association with e2a proteins. this dimerization is a prerequisite for the recognition of e-box sequences, and involves basic domains and key residues of the bhlh domains. these complexes can bind to numerous variable canonical core recognition e-box sequences with different levels of affinity, and display distinct and sometimes even antagonistic functions during embryonic development. the differences in the composition of the e-box sequences, in effect, largely modulate the in vivo function of cellular transactivation by closely related transcription factors. the saethre-chotzen syndrome is an autosomal dominant craniosynostosis characterized by facial and limb deformities caused by loss-of-function mutations of the twist gene on chromosome 7p. over twist mutations have been described in scs patients, a majority of which are present in the bhlh domains, mediating protein dimerization, and involving a single base pair substitution. these substitutions either create premature termination codons, which lead to truncated proteins, or substitute highly conserved residues in the bhlh region. these mutations affect the dimerizing ability and the dna-binding properties of twist deletions and insertions of nucleotides have also been reported in scs patients. one such mutation, termed r154g, is the naturally-occurring, rare substitution of the arginine amino acid with a glycine residue on the twist protein. arginine binds phosphate anions with a high affinity, and is often present in the active sites of proteins that bind phosphorylated substrates. as such, arginine residues play crucial roles in maintaining the overall balance of the charges within a protein. the arginine residue plays a role in maintaining the overall charge balance of a protein. in addition, the nh groups of arginine are positioned so as to carry out h-bonds with partners or close residues of the protein, and strengthen the conformational structure of the dimer, while the biochemical properties of proline impede such an interaction. this arginine residue is not conserved in other bhlh proteins, which contain a glutamine, serine or asparagine residue at this position, residues that provide only limited physicochemical changes compared to an arginine residue. however, the r residue is instrumental in maintaining proper dimerizing functions of the twist protein, as demonstrated by spicer et al., who performed a proline point-mutation to ensure stronger physicochemical changes, since the lateral cycle usually disrupts the alpha helix of the hlh, and reported an impaired dimerization. this was later confirmed by our team in vitro in mammary epithelial cells by immunoprecipitation experiments.fig. comparison of the persistence of the wild-type twist1/e dimer and mutated r154p te dimer with and without dna. a primary sequence alignment of the bhlh domains of the neurod twist and e2a proteins. residues within the interhelical loops are underlined, h and m stand for human and murine, respectively. the arrows indicate the localization of the r residue and its equivalent on the other proteins. b-c 3d representation of the conserved twist /e complex in the te and te r154p dimers in frontal and lateral views. the localization of the arginine and the proline residues in the twist1/e and twist1/e r154p complexes on the twist ribbon is highlighted in cpk. d-e the root mean square fluctuation of twist and e amino acids were estimated during 10 ns in silico molecular dynamics simulations using the vmd. software. the graphical representation showed the calculated rmsf in angstroms for each twist or e residue in the mutated r154p te model with or without dna anchor, and the te model with or without a dna anchor. f-g box plots representing the root mean square deviation by considering all of the atoms except hydrogen during 10 ns in silico molecular dynamics simulations. these distances were estimated during the md simulations using the vmd. software. the noh 1d-rmsd is shown in the mutated r154p te model with or without dna anchor, and the te model with or without a dna anchor our present aim was thus to investigate whether our previously published in silico approach was able to predict the decrease in twist1/e dimerization observed in vitro and to decipher the molecular mechanisms involved, by comparing the dimerizing ability of the wild-type twist1/e complex with that of the mutated r154p te complex. furthermore, we examined whether these complexes bound to functional e-box sequences of targeted gene promoters using md simulations, as previously described. this study was based both on the use of a recently published 3d dynamic structural model of the te dimer, initially generated using the murine neurod1/e crystal structure, and on functional in vitro assays conducted on cancer cells to validate these computer-based models. methods in silico analysis selection of x-ray structure and in silico model building when initiating our study, we first selected the most appropriate 3d comparative model of the twist1/e complex from the structures available in the protein data bank. this choice mainly resided in the level of homology of these structures with twist and e and in the composition of the dna sequence in the x-ray structure. indeed, at the level of protein identity two candidates were identified, namely the human scl/e complex and murine neurod1/e complex, displaying 58% and 48% homology with twist respectively, and 100% and 86% homology with e respectively. we next focused on the dna sequence used in the generation of the model. indeed, all four models were generated using a dna sequence to stabilize the structures and facilitate the crystallization process. having previously shown that the composition of dna bases of the e-box plays a crucial role for dna binding by bhlh dimers and for their transactivation activity, and that the e-box sequence used to generate the neurod1/e structure is a highly specific e-box sequence of the te complex, we finally selected this model for our study.table 3d bhlh structures available in the protein data bank table containing four protein complexes structurally close to twist1/e. the information included in this table enabled us to select the most appropriate crystal structure, based primarily on the percentage of identity, the dna sequence in x-ray structure and the resolution of structure. the closest match was neurod1/e47 in effect, although the “cagatg” e-box sequence contained in the scl/e structure could be a potential dna binding site of twist as predicted by de masi et al., and confirmed by emsa experimentation in particular for the dna binding of twist1/e dimer, the specificity of this box for twist homo- or heterodimers has not yet been determined. lastly, the crystallization resolution of the neurod1/e structure was the highest between these two models at  Å. the mouse neurod and e 3d structure was thus obtained from pdb files, and the dna sequence used to generate the model was kept in place, so as to preserve the proper position of the dimer in the dna groove, and avoid altering residue-base interactions. sequences, restricted to the bhlh domains, were aligned using the clustalw software. the alignments were then manually refined, taking residue properties into account. two x-ray structures of the mouse neurod1/e complex have previously been reported, which differ slightly. this divergence being fully corrected by a series of minimization steps followed by 10 ns molecular dynamics simulations, the two models were considered equivalent, generating comparable homology models. sequence alignments were submitted to the swiss-model workspace to generate models of heterodimeric twist complexes by homology with the neurod template /e. minimizations were carried out with the sybyl-x software package, using the tripos method with the amber force field and a dielectric constant of in order to refine and correct the positions of residue side chains. as mentioned above, the conserved twist1/e dimer model used throughout this study included a dna sequence named the e-box. the r154p mutated te complex was generated by substituting the arginine residue by a proline residue, using the mutation function of the sybyl-x software package. the resulting in silico models studied herein were, therefore, the te complex bound or not to the e-box sequence, as well as the r154p mutated te complex bound or not to the e-box sequence. all of the models were generated following the same algorithm and using the same parameters. molecular dynamics simulations the established homology models were visualized with the vmd. software. the resulting model was inserted into a parapipedic tip3p solvent box by means of the add solvation box module of the vmd. software. a distance of 15 Å was set between the surface 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 b software for steps and molecular dynamics simulations, and was computed on a xeon core cpu cluster supercomputer. simulations were carried out at constant temperature and pressure and by implementing the widely used charmm force fields. the time frame was set at 1-fs and the langevin and pme algorithms were applied. a conformation was sampled every 10-ps. in the first instance, the effect of the r154p mutation of twist on the molecular interactions of the te dimer with the e-box sequence was investigated in comparison with the effect of the wt twist protein, over a 10 ns experimental time-course. in the second instance, the impact of the anchor provided by the dna sequence on the binding efficacy of the wt or r154p mutated te dimer was studied by conducting md simulations over a time course of 10 ns. definition of the domains studied several domains were arbitrarily assigned to regions of the te dimer in figs.  and indeed, in fig. 2a and b, we studied the position of the te dimer in the dna groove by studying the distance between basic domains of twist and e in the te and te r154p md simulations. we considered that the basic domain of twist is composed of amino acids: t q r v m a n v r e r q r t q s l n and e126; whereas that of e is composed of the following amino acids: e r r v a n n a r e r l r v r d i and n in the fig. 2c, d, e and f, we defined three domains as “bottom”, “middle” and “top”. the former encompasses methionine of twist and valine of e localized at the bottom of te dimer and near the dna sequence, while “middle” designates residues leucine of twist and aspartic acid of e localized in the middle of te dimer, and finally, the “top” is constituted of isoleucine of twist and leucine of e localized at the top of te dimer.fig. comparison of the persistence of the wild-type twist1/e dimer and mutated r154p te dimer. a 3d representation of the conserved twist /e complex. description of the position of the basic domain residues is represented as a cartoon and cpk in grey for twist and in green for e. b the ca-ca distances between pairs of basic domain residues on twist and e were estimated during 10 ns in silico molecular dynamics simulations using the vmd. software. the graphical representation shows the calculated mean distances between pairs of twist1/e residues in angstroms in the mutated r154p te model with or without dna anchor, and the te model with or without a dna anchor. c 3d representation of the conserved twist /e complex bound to dna along with the e-box sequence, carried out using the vmd. software. the residues are numbered in each strand of the 5′-catctg-3′ e-box core. the three pairs of residues are shown, namely m112-v l124-d and i134-l which were located at the bottom, in the middle and at the top of the dimer, respectively. d-f box plots representing the distances between pairs of residues of the dimer as a function of time, during the te and te r154p 10 ns in silico molecular dynamics simulations. these distances were estimated during the md simulations using the vmd. software. the lengths of h-bond interactions were represented via graphpad prism 5: “smooth, differentiate or integrate curve, with neighbors”. interactions between the m of twist and v of e residue pair, close to the bottom of the dimer, l of twist and d of e residue pair, close to the middle of the dimer, and i of twist and l of e residue pair, close to the top of the dimer fig. consequences of impaired twist1/e dimerization on dna binding. 3d representation of the conserved twist /e complex. description of the position of the two series of residues boxes a and d, and b and c boxes, in the te dimer are represented as a cartoon and cpk. the first dimerization blocs composed of boxes a and d, b and c are represented by cylindrical grey and green solid surfaces, on the twist and e proteins, respectively. the localization of r on the twist ribbon is highlighted in cpk. c evaluation of the impact of the r154p mutation on the number of h-bonds, in each individual box a to d by studing the percentage of variation of h-bond interactions between residues during te and e r154p md simulations, established between residue-residue and residue-dna or exclusively between residues. d bar chart representing the variation of h-bond interactions between residues of the te versus r154p te complex. the sum of the interactions occurring between residues within the box a and box b of the wild type or mutated twist protein is highlighted. the percentage of cumulated occupancies of h-bond interactions occurring in the mutated r145p te model is normalized against the te model. all cumulated occupancy values of the h-bonds were calculated as described in the methods section. briefly, h-bond interactions are assigned a value according to the distance between their atomic donors/acceptors during the 10 ns time-course of the md simulation. higher occupancy values being obtained for shorter and, therefore, more stable interactions. with logic test:“< ”, value of if true and if false; nb.si function: nb.si) several domains were geographically defined in fig. 3a and b, named a and b boxes for twist and c and d boxes for e, with a and d boxes and the b and c boxes facing each other, respectively. the residues composing a and b boxes of twist encompass amino acids “120–127” and “149–157”, respectively, whereas the composition in residues of c and d boxes of e contain amino acids “565–574” and “588–594”, respectively. the rmsf data interpretation we studied the root mean square fluctuation, which represents the flexibility of the protein model. we initially aligned the dimer with the vmd function rmsd trajectory tool, and then calculated the rmsf with the vmd timeline function: and step size: 1). we obtained one rmsf per amino acid per frame and the average rmsf per amino acid was calculated using microsoft excel. the rmsd data interpretation to complete rmsf analyses, 1d-root mean square deviation calculations were carried out on all of the md simulations, taking into account the constraints of the template as a reference. we obtained the 1d-rmsd with the vmd function rmsd trajectory tool. the 1d-rmsd calculations conducted encompassed all of the atoms, except hydrogen. before the extraction of 1d-rmsd values, we aligned all of the frames on the first frame by considering all of the proteins without hydrogen atoms. the dssp data interpretation we then used the online dictionary of secondary structure of proteins program http://swift.cmbi.ru.nl/gv/dssp/ to calculate dssp parameters, such as secondary structure and solvent accessibility of protein residues. we analyzed the solvent accessibility value defined for each residue and we compared these values between the te and te r154p md models. variation of free energy of binding computed by mm-pbsa method the resulting conformation of the protein dna complex from molecular dynamics simulations with namd was extracted and several files were prepared using the xleap module. the complex was neutralized with sodium counter ions. ions and dna were called the receptor and the protein the ligand. the complexe structure with ions was inserted into a tip3p water pbc box. the amber software was used with the ff14sb forcefield. the structure was minimized for steps and the gently thermalized with an increase temperature from to 300 k in 60 ps to avoid destruction of interactions. the resulting structure was submitted to a 2 ns ntp molecular dynamics using a 300 k and 1 atm on titan x gpu card. a frame was sampled every 20 ps so frames were used for mm-pbsa calculations. for mm-pbsa calculation the calculation was performed with a total non-polar solvation term free energy modeled with one term, and with igb =  alanine scanning method this methodology computes variations in free energy by replacing each residue by an alanine residue. the alanine scanning calculation was performed with moe from the chemical computing group company. the amber10eth forcefield was chosen, and starting from the resulting structure of the namd molecular dynamics, calculations were performed with lowmode conformer generation. the interaction energy was computed for each conformation of each mutation with the dna structure. analysis of the persistence of the interactions arising during md simulations among the interactions occurring between residue-residue and residue-base, we focused our current investigation on hydrogen bond interactions established between donor and acceptor groups. the h-bond interaction is an intermolecular force involving a hydrogen atom and an electronegative atom, such as oxygen or nitrogen. h-bonds can be established between donor atoms and acceptor atoms of side chain residues. we studied the r-r or r-base interactions, which are established between atoms. the lengths of the h-bond interactions were represented via graphpad prism 5: “smooth, differentiate or integrate curve, with neighbors”. these lengths were previously estimated around 2–  Å depending on the nature of the acceptor and donor atoms. jeffrey categorizes h-bonds with donor-acceptor distances of –  Å as “strong, mostly covalent”, –  Å as “moderate, mostly electrostatic”, –  Å as “weak, electrostatic”. consequently, we defined the threshold of the h-bond at  Å in order to mainly consider covalent h-bonds and to be more stringent. using a computer software, we made the following hypothesis: any interaction occurring under this threshold was deemed “true” and assigned a score of whereas any interaction occurring above this threshold was deemed “false” and given a score of with logic test:“< ”, value of attributed if true and if false; nb.si function: nb.si). the scores were reckoned and the value obtained was designated as the rate of occupancy of h-bond interactions. thus, for a given h-bond, the higher the rate of occupancy, the greater the number of interactions occurring under  Å, the higher the level of persistence of that h-bond during the md simulation. in vitro analysis vector constructs for immunoprecipitation assays the cdna of twist or wild-type cdna of e were cloned into the pci-neo expression vector with flag- or myc-tag, respectively. the constructs obtained were named flag-twist vector, flag-twist r154p vector, and myc-tag e vector. the twist r154p mutant construct was modified by genscript. all final constructs were amplified using the maxiprep kit provided by promega and sequenced by genoscreen compagny.fig. consequences of impaired twist1/e dimerization on the loop structure of the bhlh domain. a-b box plots representing the average distances between the od atom of asparagine and the nz nz or nz atoms of the lysine residues, and between n and k residues during the wild-type twist1/e and mutated te r154p 10 ns in silico molecular dynamics simulations. c the horizontal bar chart shows the cumulated occupancy values for the h-bond interactions established between the n125-k residues of twist and the n566-k residues of e during the te and te r154p md simulations. all cumulated occupancy values of the h-bonds were calculated as described in the methods section. briefly, h-bond interactions are assigned a value according to the distance between their atomic donors/acceptors during the time. higher occupancy values being obtained for shorter and, therefore, more stable interactions. with logic test:“< ”, value of if true and if false; nb.si function: nb.si). d western blot showing the interaction between wild-type twist or t r154p and e as assessed by immunoprecipitation assays. twist or mutated twist r154p and e were transiently produced in hek293t cells. the twist protein was immunoprecipitated with a monoclonal α-flag antibody and the presence of endogenous e protein in the immunoprecipitates was assessed. input 10%. the protein sizes were 99 kda for the hetreodimer te, 73 kda for the e protein and 25 kda for the twist protein vector constructs for streptavidin/biotin assays the pbabe-neo expression vector was donated by h. land & j. morgenstern & b. weinberg and was used as a negative control. the cdna of the tethered te complex composed of amino acids, including a flag-tag at the n-terminal position, twist cdna, an amino acid linker and e cdna, was cloned into the pbabe-neo expression vector. this construct of the ectopic te protein is approximately 99 kda in size. the final construct was named the flag-twist1/e pbabe-neo expression vector. this te construct was modified by genscript in order to obtained the tethered te r154p mutant complex composed of amino acids, including a flag-tag at the n-terminal position, twist cdna with the substitution of the arginine by a proline, an amino acid linker and e cdna, was cloned into the pbabe-neo expression vector with the same size of te.fig. consequences of impaired twist1/e dimerization on dna binding. a 3d in silico representation of the te complex bound to dna, carried out using the vmd. software. the proximal flanking and first bases of the e-box are represented in grey and green vdw, respectively. b-c the pie charts show the percentages of cumulated occupancy of h-bonds established between residues of twist and e and dna bases during the te and r154p te md simulations. occupancies of h-bonds established with the proximal flanking bases are represented in yellow, while the bar charts highlight the exact residues binding to those bases. the h-bonds established with the first consensus bases of the e-box are represented in grey and green for the twist and the e residues, respectively. d-g distribution of “specific” h-bonds established between residues and e-box bases in the te and mutated r154p te molecular dynamics simulations. the pie charts show percentages of the total rate of cumulated occupancy of specific h-bonds established during the md simulations. consensus bases and variable bases of the e-box sequences are represented in grey and pink, respectively. the bar charts highlight the specific residues binding to cytosine and bases, and show their implication in the dna binding affinity. the dna sequences are represented for the te and mutated r154p te dimers. the arrows highlight the major base interactions for the te complex and mutated r154p te complex. h western blot showing that the tethered twist1/e r154p dimer was not able to interact with the te-box unlike the tethered twist1/e wild-type dimer, as assessed by streptavidin/biotin assays. tethered te and te r154p dimers were transiently produced in hela cells. the protein sizes were 99 kda for the thetered dimer te and 25 kda for the twist protein immunoprecipitation assays hek293t cells were provided by the atcc. hek293t and hela cells were transfected in 10 cm in diameter culture dishes at a density of 80%–90% using the fugene hd transfection reagent for 48 h. the transfection reagent contained  μg of the empty pci-neo expression vector or of the pci-neo expression vector containing cdna coding the flag-twist flag-twist r154p, or myc-e pci-neo, as well as 10 μl of fugene. following their transfection, cells were washed with cold pbs and lysed in ebc solution. after incubation on ice for 20 min, lysates were collected by scrubbing the plates, sonicated and centrifuged for 20 min at  rpm at 4 °c. the supernatant was collected and the total protein content was quantified using the bio-rad protein assay. after clearing by centrifugation, the protein extract was incubated with an anti-flag m resin. after intensive washing to eliminate non-specific binding with ebc solution, the resin was resuspended in laemmli buffer and boiled for 5 min. after elimination of the beads by centrifugation, samples were reduced by adding β-mercaptoethanol and separated by sds-page. proteins were analyzed with the rabbit polyclonal flag antibody and the rabbit polyclonal v e2a antibody and a polyclonal goat anti-rabbit immunoglobulin conjugated with horseradish peroxidase as the secondary antibody. the detection was performed by western blot analysis using the luminol reagent. streptavidin/biotin binding assays hela cells were transfected in 6 cm in diameter culture dishes at a density of 80%–90% using the fugene hd transfection reagent for 48 h. the transfection reagent contained  μg of the empty pbabe-neo expression vector or of the pbabe-neo expression vector coding the cdna of the flag-twist1/e pbabe vector or flag-twist1/e r154p pbabe vector, as well as 21 μl of fugene reagent. following their transfection, protein extractions were carried out as reported for the immunoprecipitation assay. the streptavidin/biotin binding assay was conducted to isolate proteins bound to specific dna sequences. to do so, total cellular proteins were incubated with biotin-coupled dna sequences, and the dna bound te complex was then purified by immunoprecipitation via the recognition of biotin by streptavidin beads. hence, this approach decreased the risk of isolating non-specifically bound proteins. briefly, both strands of dna probes corresponding to various e-box sequences were labeled in their 3’ extremity with biotin. the probe used was the active te-box. for probe hybridization, an initial denaturing step at °c for 5 min was followed by a hybridization step at °c for 10 min. probe hybridizations were then confirmed on 3% agarose gels. the binding between the dna hybridized probes and the te complex was carried out in binding buffer for 1 h at °c. biotinylated dna probes bound to the te complex were purified using dynabeads® m- streptavidin. coupled streptavidin dynabeads® bound to the dna/te complex were run on a 12% sds-polyacrylamide gel using a migration buffer at 80 v for 10 min in the stacking gel, followed by the migration phase at 100 v for 120 min. the liquid transfer onto a pvdf membrane was conducted using a transfer buffer at 200 ma for 80 min. the membrane was then saturated with 5% tris-buffered saline containing % tween and % milk for 45 min, and incubated with the mouse monoclonal twist antibody as a primary antibody, and a polyclonal rabbit anti-mouse immunoglobulin conjugated with horseradish peroxidase as the secondary antibody. the detection was performed by western blot analysis using the luminol reagent.  the final 3d model used to generate our te dimer was based on the mus musculus neurod1/e complex. after aligning the four protein sequences using the clustalw software, we mainly observed that neurod is composed of amino acids with a bhlh domain containing amino acids 101– whereas twist is composed of amino acids with a bhlh domain encompassing amino acids 108–. the crucial, strong physicochemical r residue of twist is a serine, an asparagine and a glutamine residue in e murine neurod and murine e respectively. as speculated by spicer et al., we could clearly observe that the substitution of the arginine residue with a proline residue, the lateral side chains of which contain a cycle known to destabilize the alpha helix, in this crucial position at the dimerization interface of te altered the conformation of the dimer. indeed, while the nh groups of arginine are positioned to carry out h-bonds with partners or close residues of the hlh loop, and strengthen the conformational structure of the dimer, the biochemical properties of proline impede such an interaction. next, to characterize the effect of this substitution on molecular mechanisms involved in the dimerization of the te complex, we submitted the four in silico models to 10 ns md simulations. we then carried out root mean square fluctuation analyses to measure the structural flexibility of our proteins; the higher the rmsf value, the greater the flexibility. we observed an increase in rmsf values in the absence of dna for twist and e residues, suggesting that the dna sequence stabilizes the dimers. nevertheless, no significant rmsf variation between te and te r154p md simulations was observed, indicating the same level of flexibility of these two models. to complete this analysis, root mean square deviation calculations were also carried out using the noh parameter. clearly, the presence of dna during the dynamic simulations induced a decrease in 1d-rmsd values and enabled the stabilization of the dimer. again, no significant difference could be observed between the te and r154p te models. when focusing on r-r interactions between the basic residues of twist and e, the absence of dna resulted in an increase in the mean r-r distances by almost 5 Å in te and te r154p molecular dynamics simulations. since such a molecular instability in the dna-free model may have overweighed the structural impact of the r154p point-mutation, or led to a biological artifact, all of the following md simulations included a specific dna e-box sequence. we then evaluated the predictive value of our in silico strategy in assessing the functional disruption generated by the r154p mutation of twist on protein dimerization and dna binding. of note, protein dimerization precedes the binding of the complex to dna sequences. we observed that the mean distance between basic domains of te r154p was greater than that of te. we then arbitrarily separated the dimer into regions, namely the “top”, “middle” and “bottom”. a clear variation in distances between twist and e backbone residues was observed with the r154p mutation, indicating a distortion of the te structure. indeed, these distances either increased, namely between the m and v residues, and between l and d, or slightly decreased, namely between i and l residues. these results strongly suggest that the basic te domains are further apart from the e-box sequence during the mutated te r154p md simulation compared to the te simulation, thus, impeding proper dna binding. to complete these predictions, we calculated the dictionary of secondary structure of proteins parameter, which provides information on secondary structure and solvent accessibility of protein residues. no variation in the secondary structure prediction between te and te r154p for the arginine or proline residue were unveiled. nevertheless, the r154p mutation twist decreased solvent accessibility of twist residues, mainly the loop residues of twist including residues i to l in contrast, this point-mutation did not modify the solvent accessibility of e residues. having shown a disruption in the ca-ca interactions between twist and e residues in the r154p mutated dimer, we investigated the effects of this mutation on the persistence of h-bonds established between residue-residue or residue-dna base during md simulations. we studied four dimerizing domains along the mutated complex, which are directly involved in dimerization and dna binding, named a and b boxes for twist and c and d boxes for e. a and d boxes and b and c boxes interacted with each other, respectively. as expected, the r154p mutation had the biggest impact on the persistence of h-bonds established by amino-acid residues situated within the b box, where the r residue resides, with a 41% decrease in h-bond establishment. when evaluating the persistence of h-bonds exclusively established between r-r, we observed that these were also impaired in the a box of twist and d box of e. when we evaluated the interaction r-base, we observed an increase in these interactions, enabling a stabilization of the dimer on dna. this clear decrease in the stability of these boxes, when comparing the te and te r154p models, confirms that dna acts as an anchor for the c-α chain of hlh domains of twist. the destabilizing effect of the r154p substitution on twist was further explored, by studying the difference between the cumulated interactions of each residue within the a and b boxes of the wild-type and mutated twist during md simulations. these simulations focused exclusively on the a and b boxes, since these domains were reported to experience major alterations. this strategy revealed that the impact of the r154p mutation is distributed throughout the α-helix. moreover, the residues that presented the highest rate of variation in their h-bond interactions, such as threonine, glutamine, and asparagine within the a box, and leucine, lysine, and alanine within the b box, were considered to be directly implicated in stabilizing the complex. in addition, critical h-bonds localized in the heart of the dimer were disrupted, namely between the n125-k residue pair of twist and the n566-k residue pair of e in the r154p twist variant. this disruption occurs between the od atom of the asparagine residues and either one of the three nz nz or nz atoms of the lysine residues that are present under physiological conditions. indeed, the interaction between the n566-k residue pair of e was largely impaired, revealing the clear impact of the twist mutation on the structure of heart of the te complex, which is at the interface of the functional e-box recognition site. we confirmed the putative decrease in affinity by calculating the free energy of binding in kcal/mol between the te complex and dna using the mm-pbsa method. we obtained a dg score of −217 kcal/mol and of −191 kcal/mol in the te wild-type and the te r154p mutant, respectively. however, the dg computed with the mm-pbsa method implemented in amber software is different from the namd method used during our md simulations. consequently, we carried out the alanine scanning method, similarly to that used in our md models. this alascan approach enabled us to define the affinity of twist for e and reciprocally of e for twist in the te or te r154p complexes. the affinity of twist for e was 46 kcal.mol− and 66 kcal.mol− in the te complex and in the te r154p complex, respectively. the affinity of e for twist was 55 kcal.mol− and 51 kcal.mol− in the te complex and in te r154p complex, respectively. taken together, the alascan and mm-pbsa studies revealed that twist has a higher affinity for e in the te complex, and thus confirmed that the r154p mutation may impact twist1 dimerization and its recognition or binding, once dimerized, to active e-box sequences. consistently, we showed in vitro that twist r154p was not efficiently able to bind e as evidenced by immunoprecipitation assays. these assays revealed the presence of endogenous e protein in the empty control lanes. furthermore, although a clear te dimer was obtained in the t1/e lane around 95–100 kda, we also observed additional complexes t1/t and e12/e, no dimer was observed in the t r154p/e lane. consequently, we explored the dna binding affinity of the wild-type or mutated te complexes, to investigate how such a mutation may affect canonical e-box recognition. based on recent findings, we studied the particular involvement of the,, and dna bases in the establishment of h-bond interactions between the wild-type or r154p mutated te complex and the e-box sequence. we noted an important increase in the implication of and adjacent bases in the r154p variant. moreover, this increase involved residues s r and r which were shown to play a pivotal role in the specific dna binding affinity of the te complex. our previous findings demonstrated that the specific affinity of the te complex to functional e-box sequences was associated with specific h-bond interactions established with consensus bases,, and, as well as variable central bases,, and of the e-box. here, we studied their distribution in the current md simulations. we clearly noted that specific h-bond interactions were lost in the case of the mutated r154p te complex, especially with variable central bases. this decrease was largely compensated by an increase in specific h-bond interactions with consensus bases and of the e-box sequences. in conclusion, these data strongly indicate that the r154p mutant, similarly to the te complex binding to modified e-boxes, establishes alternative h-bond interactions with adjacent bases in our in silico models in an attempt to compensate for the decrease in structuration of the heterodimer. hence, the r154p mutation not only affects the dimerization function but also the dna binding function of the te complex. this finding was validated in vitro using the strepatvidin/biotin assay. indeed, although the tethered te r154p dimer seemed less stable than the te dimer, likely due to the impact of the r154p mutation on dimerization, it was unable to bind to the te-box. thus, the present in silico strategy is able to predict the impact of mutations occurring within the hlh domains of twist and possibly of e2a proteins, on the dimerization of the proteins and on dna binding. discussion our study provides yet another rationale for using molecular dynamics simulations as a predictive tool in the context of protein interactions, dimerization and dna binding, and in determining the impact of protein point-mutations on the persistence of the hlh domains of the oncogenic te complex. indeed, this approach previously enabled our team to study the effects of molecular modifications of twist on the binding of the te complex to dna. this in silico md approach highlighted the identification of the function of individual residues and molecular cooperation between residues, the predominant role of protein side chain residues, close to the heart of the complex, and those necessary for anchoring the dimer to dna sequences, and the localization of the te dimer on the dna groove, by studying the shift towards adjacent e-box bases. in agreement with sauve’s model, we identified three different states of binding of the te bhlh complex to functional and degenerate e-box sequences, based on md simulations. this in silico approach strongly suggested that we were able to define the molecular mechanisms implicated in the binding flexibility of the te complex to e-box sequences of a targeted gene promoter, by predicting the proper transcriptional function. here, using a twist protein displaying the r154p mutation, we accurately predicted both an impairment in the protein dimerizing function and a subsequent decrease in the e-box binding affinity. we highlighted the determinant role of the interhelical loops in maintaining the structure of the twist1-dna complex, by studying two variants with a 21-bp tandem repeat insertion in the twist gene, leading to the aberrant presence of seven extra amino-acids on positions and of the interhelical loop in scs patients. these insertions strongly modified the structure of the interhelical loops, and led to fewer contacts between interhelical loops and dna. these proof of concept experiments were conducted prior to carrying out in vitro assays, namely immunoprecipitation and streptavidin/biotin assays, to validate the functional outcome of substituting residues. taken together, these results corroborate findings of spicer et al., who suggested that the scs mutation r154p led to a decrease in dimerization. this, in turn, may affect the quantity of functional te complexes present in cells, explaining the intermediate loss of luciferase activity following the r154p mutation compared to the te complex. as we reported previously, this alteration impaired the transformation potential of the te complex induced by the cooperation with the h-rasg12v protein. this approach also provided a structural explanation for the loss-of-function associated with twist1-haploinsufficiency observed in scs patients. using md simulations and biochemical assays, we and others also highlighted the pivotal function of the r118h/q/c, r120p, s144r, and k145e/q residues in the molecular binding of the te complex. mutations affecting these residues, as observed in scs, lead to a decrease in the binding affinity of the dimer with the regulatory e-box sequences, and likely modify the transactivation functions of twist, resulting in an imbalance in the twist1/twist homodimer and in the te heterodimer during the developmental process. in the present work, we completed this study by showing in silico and in vitro that the h-bond integrity between residues of bhlh was partially impaired, which appears to be the major cause of the decrease in protein dimerization. to compensate for this, the r154p mutant alternatively establishes h-bond interactions with both non-specific and consensus bases of the e-box, thus modifying the conformation of the heart of the dimer by losing the crucial n125-k and n566-k588 h-bonds interactions. it is worth noting that k mutations are also reported in patients suffering from scs syndrome. several bhlh proteins are known to dimerize with twist including the hand proteins and proteins derived from the following genes: the tcf2/heb, the tcf3/e2a and the tcf4/e2- considering the degree of homology in their bhlh domains, applying the current in silico approach to the evaluation of the dimerizing properties of all of these complexes would be highly relevant. in the future, it may be possible to identify specific transcriptional signatures according to the twist heterodimers generated, for example twist1/e-proteins versus twist1/twist or twist1/hands, and to their e-box binding specificities.  these md approaches allowed us to determine the key residues of twist and its partners involved in hlh dimerization, which may completely modify the balance of homo- and heterodimeric twist complexes. overall, md simulations may constitute a powerful tool to predict the biological impact of these alterations in a cancer cell, in terms of dimerization, but also of binding affinity to active e-box sequences. additional file additional file 1: figure s consequence of impaired twist1/e dimerization on dna binding. a-c: dictionary of secondary structure of proteins parameters were calculated for the te and te r154p models, to obtain information on secondary structure and solvent accessibility of twist and e residues. bar chart presenting the variation in solvent accessibility of twist residues. 3d representation of the conserved twist /e complex displaying residue represented in cartoon and residues impacted by dssp calculations represented in cpk. bar chart presenting the variation in solvent accessibility of e residues. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. 
some loss-of-function mutations of the twist gene have been shown to cause an autosomal dominant craniosynostosis, known as the saethre-chotzen syndrome. the online version of this article contains supplementary material, which is available to authorized users. in a previous study, we highlighted the predictive value of in silico molecular dynamics simulations in deciphering the molecular function of twist residues. our study demonstrates that md simulations provide a structural explanation for the loss-of-function associated with the scs twist mutation and provides a proof of concept of the predictive value of these md simulations. the bhlh transcription factor twist plays a key role in the embryonic development and in tumorigenesis. here, since the substitution of the arginine amino acid by a glycine residue is responsible for the scs phenotype and the substitution of arginine by a proline experimentally decreases the dimerizing ability of twist we investigated the molecular impact of this point mutation using md approaches. although the functional impacts of many twist mutations have been experimentally reported, little is known on the molecular mechanisms underlying their loss-of-function. consistently, md simulations highlighted a clear decrease in the stability of the α-helix during the dimerization of the mutated r154p twist1/e dimer compared to the wild-type te complex, which was further confirmed in vitro using immunoassays.
 the bhlh transcription factor twist plays a key role in the embryonic development and in tumorigenesis. some loss-of-function mutations of the twist gene have been shown to cause an autosomal dominant craniosynostosis, known as the saethre-chotzen syndrome. although the functional impacts of many twist mutations have been experimentally reported, little is known on the molecular mechanisms underlying their loss-of-function. in a previous study, we highlighted the predictive value of in silico molecular dynamics simulations in deciphering the molecular function of twist residues.  here, since the substitution of the arginine amino acid by a glycine residue is responsible for the scs phenotype and the substitution of arginine by a proline experimentally decreases the dimerizing ability of twist we investigated the molecular impact of this point mutation using md approaches. consistently, md simulations highlighted a clear decrease in the stability of the α-helix during the dimerization of the mutated r154p twist1/e dimer compared to the wild-type te complex, which was further confirmed in vitro using immunoassays.  our study demonstrates that md simulations provide a structural explanation for the loss-of-function associated with the scs twist mutation and provides a proof of concept of the predictive value of these md simulations. this in silico methodology could be used to determine reliable pharmacophore sites, leading to the application of docking approaches in order to identify specific inhibitors of twist complexes. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords twist1/e12dimerizationbhlh transcription factormolecular dynamicsin silico analysisissue-copyright-statement© the author 2017 