TWIST1 is essential in embryological morphogenesis, mesoderm patterning and development. The protein is highly conserved from Drosophila to humans. In vertebrates, TWIST1 is involved in cell type determination and differentiation during myogenesis, cardiogenesis, neurogenesis , hematopoiesis  and osteogenesis . TWIST1 is a basic helix-loop-helix (bHLH) transcription factor (TF) in which the basic DNA-binding region is followed by a dimerization region composed of two amphipathic α-helices separated by a loop domain. TWIST1 forms either homo- or heterodimers with other bHLH proteins and binds to short conserved sequences called E-boxes (5´-CANNTG–3´) in promoter regions, regulating the transcription of target genes .
The dimer partner choice is a critical factor in determining TWIST1 activity in both flies and vertebrates [5, 6]. In mammals, the transcription of thrombospondin is induced by heterodimers of TWIST1 with E2A (also known as TCF3; it presents two isoforms, E12 and E47), whereas homodimers of TWIST1 up-regulate the transcription of FGFR2 and periostin. In vitro assays have shown that TWIST1/E2A heterodimers bind DNA more efficiently than their homodimers , and this association also protects TWIST1 from ubiquitin-dependent proteasome degradation . The TWIST1/E2A heterodimer also represses osteoblast differentiation by downregulating the expression of CDKN1A (p21), an inhibitor of cyclin-dependent kinases . It has been shown that heterodimers of MyoD with E12 or E47 bind to the E-box sequence more efficiently than E12 or even E47 homodimers . As only the heterodimers of the myogenic bHLH protein with the ubiquitous E2A protein are able to activate muscle-specific gene expression and differentiation, it is very important to ensure that only these heterodimers, and not E2A protein homodimers, bind to the relevant E-box sites. The myogenic bHLH proteins do not form homodimers efficiently. To compete with the E2A protein homodimers, the heterodimers must have a higher affinity for the binding site. However, this does not mean that E2A protein homodimers are of no use. The E2A proteins in B cells may be unique in their ability to bind DNA as homodimers. In muscle cells and pancreatic cells, they clearly prefer to bind DNA as heterodimers [10–13].
Null mutations of twist1 in Drosophila result in embryonic lethality because of the complete absence of mesoderm, and homozygous knock-out mice die at E10.5-11, presenting a failure of neural tube closure and defects in the head mesenchyme, branchial arches, somites and limb buds . Mice that are heterozygous for twist1 null mutations display a phenotype that is similar to a human hereditary disorder called Saethre-Chotzen Syndrome (SCS – also known as acrocephalosyndactyly type III). Humans with twist1 gene germ-line haploinsufficiency suffer from premature fusion of cranial sutures, skull deformations, limb abnormalities and facial dysmorphism .
More than 70 different mutations in the TWIST1 gene have been identified in unrelated SCS patients and cluster in the bHLH coding sequence, either truncating or disrupting the transcription factor [16, 17]. Approximately 75% of these mutations are single base pair substitutions that either create premature termination codons or substitute highly conserved residues in the bHLH region. The first type of mutation is represented mainly by nonsense mutations that are upstream to or within the bHLH motif. These mutations produce truncated proteins that rapidly degrade. The second type of mutations are missense mutations that involve the helix I or II region, creating proteins that fail to heterodimerize and which then become abnormally located in the cytoplasm . Three missense mutations described by El Ghouzzi , Arg118Cys (R118C – helix I), Ser144Arg (S144R - loop) and Lys145Glu (K154E – helix II), are important because they lead to a loss of DNA binding for the TWIST/E12 heterodimer and, as a result, impair TWIST1 activity.
The three-dimensional (3D) structure of the TWIST1 protein has not yet been solved experimentally, and as the structure and function of a protein are intimately correlated, the elucidation of the 3D structure of TWIST1 could allow function prediction studies and the possibility of studying mutation effects, dynamic behavior under different conditions, and rational drug design. Given that only a limited number of proteins have had their 3D structures solved, theoretical methods, such as ab initio or comparative modeling, would appear to be fast and reliable methods for addressing this issue.
Because of the importance of TWIST1 in the regulation of embryonic development, its substantial relationship with SCS and the lack of an experimentally solved structure for this protein, we performed comparative modeling for the TWIST1 bHLH region for both the homodimer and heterodimer with E47. These are important for DNA binding in the promoter region of target genes, and we evaluated their behavior in aqueous solution using molecular dynamics simulations. Three mutations that promote DNA binding failure, R118C, S144R and K145E, were also studied.