Crystal Structures of Ternary Complexes of MEF2 and NKX2-5 Bound to DNA Reveal a Disease Related Protein-Protein Interaction Interface.

MEF2 and NKX2-5 transcription factors interact with each other in cardiogenesis and are necessary for normal heart formation. Despite evidence suggesting that these two transcription factors function synergistically and possibly through direct physical interactions, molecular mechanisms by which they interact are not clear. Here we determined the crystal structures of ternary complexes of MEF2 and NKX2-5 bound to myocardin enhancer DNA in two crystal forms. These crystal structures are the first example of human MADS-box/homeobox ternary complex structures involved in cardiogenesis. Our structures reveal two possible modes of interactions between MEF2 and NKX2-5: MEF2 and NKX bind to adjacent DNA sites to recognize DNA in cis; and MEF2 and NKX bind to different DNA strands to interact with each other in trans via a conserved protein-protein interface observed in both crystal forms. Disease-related mutations are mapped to the observed protein-protein interface. Our structural studies provide a starting point to understand and further study the molecular mechanisms of the interactions between MEF2 and NKX2.5 and their roles in cardiogenesis.

Synthesis, Identification, and Structure-Activity Relationship Analysis of GATA4 and NKX2-5 Protein-Protein Interaction Modulators.

Transcription factors GATA4 and NKX2-5 directly interact and synergistically activate several cardiac genes and stretch-induced cardiomyocyte hypertrophy. Previously, we identified phenylisoxazole carboxamide 1 as a hit compound, which inhibited the GATA4-NKX2-5 transcriptional synergy. Here, the chemical space around the molecular structure of 1 was explored by synthesizing and characterizing 220 derivatives and structurally related compounds. In addition to the synergistic transcriptional activation, selected compounds were evaluated for their effects on transcriptional activities of GATA4 and NKX2-5 individually as well as potential cytotoxicity. The structure-activity relationship (SAR) analysis revealed that the aromatic isoxazole substituent in the southern part regulates the inhibition of GATA4-NKX2-5 transcriptional synergy. Moreover, inhibition of GATA4 transcriptional activity correlated with the reduced cell viability. In summary, comprehensive SAR analysis accompanied by data analysis successfully identified potent and selective inhibitors of GATA4-NKX2-5 transcriptional synergy and revealed structural features important for it.

Can All-Atom Molecular Dynamics Simulations Quantitatively Describe Homeodomain-DNA Binding Equilibria?

We systematically investigate the applicability of a molecular dynamics-based setup for the calculations of standard binding free energies of biologically relevant protein-DNA complexes. The free energies are extracted from a potential of mean force calculated using umbrella sampling simulations. Two protein-DNA systems derived from a homeodomain transcription factor complex are studied in order to investigate the binding of both disordered and globular proteins. Free energies and trajectories obtained using two modern molecular mechanical force fields are compared to each other and to experimental data. The temperature dependence of the calculated standard binding free energies is investigated by performing all simulations over a range of temperatures. We show that the values of standard binding free energies obtained from these simulations are overestimated compared to experimental results. Significant differences are observed between the two protein-DNA systems and between the two force fields, which are explained by different propensities to form inter- and intramolecular contacts. The number of protein-DNA contacts increases with increasing temperature, in agreement with the experimentally known temperature dependence of enthalpies of binding. However, conclusions about the temperature dependence of the standard binding free energies cannot be made with confidence, as the differences among the values are on the order of statistical uncertainty.

Prevalence and spectrum of NKX2.5 mutations in patients with congenital atrial septal defect and atrioventricular block.

Congenital atrial septal defect (ASD) and progressive atriventricular block (AVB) are the two most common phenotypes linked to NK2 homeobox 5 (NKX2.5) mutations in animals and humans. However, the prevalence and spectrum of NKX2.5 mutation in patients with ASD and AVB remain to be elucidated. In the present study, the coding exons and flanking introns of the NKX2.5 gene, which encodes a homeobox­containing transcription factor essential for development of the heart, were sequenced in a cohort of 62 unrelated patients with ASD and AVB, and subsequently in a mutation carrier's available family members. As controls, 300 unrelated, ethnically­matched healthy individuals were recruited, who were also genotyped for NKX2.5. The functional consequence of the mutant NKX2.5 was evaluated in contrast to its wild­type counterpart using a dual­luciferase reporter assay system. As a result, a novel heterozygous NKX2.5 mutation, p.Q181X, was identified in an index patient with ASD and AVB, with a prevalence of ~1.61%. Genetic analysis of the proband's pedigree revealed that the mutation co­segregated with ASD and AVB with complete penetrance. The nonsense mutation, which eliminated partial homeobox and the carboxyl terminus, was absent in the 600 control chromosomes. Functional evaluation showed that the NKX2.5 mutant had no transcriptional activity. Furthermore, the mutation disrupted the synergistic activation between NKX2.5 and GATA binding protein 4, another cardiac core transcription factor associated with ASD. The results of the present study expand the spectrum of NKX2.5 mutations linked to ASD and AVB, and indicated that NKX2.5 loss­of­function mutations are an uncommon cause of ASD and AVB in humans.

A R/K-rich motif in the C-terminal of the homeodomain is required for complete translocating of NKX2.5 protein into nucleus.

NKX2.5 plays important roles in heart development. Being a transcription factor, NKX2.5 exerts its biological functions in nucleus. However, the sequence motif that localize NKX2.5 into nucleus is still not clear. Here, we found a R/K-rich sequence motif from Q187 to R197 (QNRRYKCKRQR) was required for exclusive nuclear localization of NKX2.5. Eight truncated plasmids (E109X, Q149X, Q170X, Q187X, Q198X, Y256X, Y259X, and C264X) which were associated with congenital heart disease (CHD) were constructed. Compared with the wild type NKX2.5, the proteins E109X, Q149X, Q170X, Q187X without intact homeodomain (HD) showed no transcriptional activity while Q198X, Y256X, Y259X and C264X with intact HD showed 50 to 66% transcriptional activity. E109X, Q149X, Q170X, Q187X without intact HD localized in the cytoplasm and nucleus simultaneously and Q198X, Y256X, Y259X and C264X with intact HD localized completely in nucleus. These results inferred the indispensability of 187QNRRYKCKRQR197 in exclusive nucleus localization. Additionally, this sequence motif was very conservative among human, mouse and rat, indicating this motif was important for NKX2.5 function. Thus, we concluded that R/K-rich sequence motif 187QNRRYKCKRQR197 played a central role for NKX2.5 nuclear localization. Our findings provided a clue to understand the mechanisms between the truncated NKX2.5 mutants and CHD.

A Comprehensive In Silico Analysis on the Structural and Functional Impact of SNPs in the Congenital Heart Defects Associated with NKX2-5 Gene-A Molecular Dynamic Simulation Approach.

Congenital heart defects (CHD) presented as structural defects in the heart and blood vessels during birth contribute an important cause of childhood morbidity and mortality worldwide. Many Single nucletotide polymorphisms (SNPs) in different genes have been associated with various types of congenital heart defects. NKX 2-5 gene is one among them, which encodes a homeobox-containing transcription factor that plays a crucial role during the initial phases of heart formation and development. Mutations in this gene could cause different types of congenital heart defects, including Atrial septal defect (ASD), Atrial ventricular block (AVB), Tetralogy of fallot and ventricular septal defect. This highlights the importance of studying the impact of different SNPs found within this gene that might cause structural and functional modification of its encoded protein. In this study, we retrieved SNPs from the database (dbSNP), followed by identification of potentially deleterious Non-synonymous single nucleotide polymorphisms (nsSNPs) and prediction of their effect on proteins by computational screening using SIFT and Polyphen. Furthermore, we have carried out molecular dynamic simulation (MDS) in order to uncover the SNPs that would cause the most structural damage to the protein altering its biological function. The most important SNP that was found using our approach was rs137852685 R161P, which was predicted to cause the most damage to the structural features of the protein. Mapping nsSNPs in genes such as NKX 2-5 would provide valuable information about individuals carrying these polymorphisms, where such variations could be used as diagnostic markers.