Nuclear Receptor-Like Structure and Interaction of Congenital Heart Disease-Associated Factors GATA4 and NKX2-5

Abstract

AimsTranscription factor GATA4 is a dosage sensitive regulator of heart development and alterations in its level or activity lead to congenital heart disease (CHD). GATA4 has also been implicated in cardiac regeneration and repair. GATA4 action involves combinatorial interaction with other cofactors such as NKX2-5, another critical cardiac regulator whose mutations also cause CHD. Despite its critical importance to the heart and its evolutionary conservation across species, the structural basis of the GATA4-NKX2-5 interaction remains incompletely understood.Methods and ResultsA homology model was constructed and used to identify surface amino acids important for the interaction of GATA4 and NKX2-5. These residues were subjected to site-directed mutagenesis, and the mutant proteins were characterized for their ability to bind DNA and to physically and functionally interact with NKX2-5. The studies identify 5 highly conserved amino acids in the second zinc finger (N272, R283, Q274, K299) and its C-terminal extension (R319) that are critical for physical and functional interaction with the third alpha helix of NKX2-5 homeodomain. Integration of the experimental data with computational modeling suggests that the structural arrangement of the zinc finger-homeodomain resembles the architecture of the conserved DNA binding domain of nuclear receptors.ConclusionsThe results provide novel insight into the structural basis for protein-protein interactions between two important classes of transcription factors. The model proposed will help to elucidate the molecular basis for disease causing mutations in GATA4 and NKX2-5 and may be relevant to other members of the GATA and NK classes of transcription factors.

Targeting GATA4 for cardiac repair

“…Nuclear receptors have two zinc fingers packed together with a conserved set of residues mediating stability of the domain. Our model predicts that the interaction between GATA4 and NKX2‐5 share a conserved architecture with the DNA binding domain of nuclear receptors . Another recent study employing molecular dynamics simulations suggests an alternative protein–protein interaction model for GATA4‐NKX2‐5 complex .…”

“…Another recent study employing molecular dynamics simulations suggests an alternative protein–protein interaction model for GATA4‐NKX2‐5 complex . Conversely, experimental studies do not find support for this theoretical observation …”

“…Structural and functional data of key cardiac TF complexes are of major importance to enhance the understanding of molecular mechanisms and TF functions. Our previous study with GATA4–NKX2‐5 proteins showed that several single point mutations of GATA4 are capable of interfering or activating the GATA4–NKX2‐5 interaction, indicating that this PPI can be targeted by direct inhibition, activation or allosteric modulation. We established homology models of GATA4 and NKX2‐5 and evaluated the point mutations having critical effects on the GATA4–NKX2‐5 interaction.…”

“…Since all mutations were located outside the antibody recognition site, western blots quantified the mutant protein levels produced in mammalian COS‐1 cells. The GATA4–NKX2‐5 interaction was assessed via coimmunoprecipitation with N‐terminal FLAG‐NKX2‐5 and analyzed by western blots using GATA4 and NKX2‐5 antibodies . Moreover, the DNA binding capacity of GATA4 mutations was evaluated.…”

Functionally significant, novelGATA4variants are frequently associated with Tetralogy of Fallot

“…Besides this, mutation c.682T>A; p.Trp228Arg also causes conformational changes from Arg284 to Glu287 amino acids (Figure G). Arg284 is considered as a critical residue for NKX2‐5 binding (Kinnunen et al., ). The in silico predictions are supported by luciferase assay as well as EMSA, where mutant has clearly showed decreased transactivation and reduced DNA‐binding affinity as compared to wild‐type protein.…”