Crystallization and preliminary X-ray analysis of the cardiac transcription factor complex of NKX2.5 and TBX5 with DNA

Pradhan, Lagnajeet; Gopal, Sunil; Nam, Hyun Joo

doi:10.1107/s2053230x14006761

Cited by 2 publications

(7 citation statements)

References 16 publications

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“…In solution, the ternary complexes, NKX2.5 HD/TBX5 TBD/ANF-252 and NKX2.5 HD/TBX5 1−239 /ANF-252, were readily formed and purified. 23 The crystals of the ternary complex, NKX2.5 HD and TBX5 TBD in complex with ANF-252 DNA, were obtained from initial crystallization trials. However, crystals for NKX2.5 HD, TBX5 1−239 , and the DNA complex were not produced from repeated trials.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…Crystallization of the ternary complex of NKX2.5 HD, TBX5 TBD, and DNA has been discussed previously. 23 For crystallization of TBX5 1−239 , the purified complex of NKX2.5 HD, TBX5 1−239 , and ANF-252 DNA were screened by the sparse matrix with crystallization drops containing 1 μL of a protein/DNA sample and 1 μL of a crystallization solution at room temperature. Hexagonal crystals were obtained from well solutions containing 0.1 M sodium cacodylate (pH 6.4), 10 mM MgSO 4 , and 1.8 M (NH 4 ) 2 SO 4 .…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

“…Data collection and structure determination of the NKX2.5 HD, TBX5 TBD, and ANF-252 DNA ternary complex have been discussed elsewhere. 23 The TBX5 1−239 crystals were cryoprotected in HEPES (pH 7.0), 100 mM KCl, 10 mM CaCl 2 , 30% PEG 400, and 10 mM DTT and flash-frozen in liquid nitrogen. The X-ray diffraction data were collected at the A1 beamline of the Cornell High Energy Synchrotron Source (CHESS).…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

“…Cloning of the NKX2.5 HD and TBX5 TBD expression constructs and purification of the proteins were previously reported. − The DNA encoding amino acids 1–239 of TBX5 protein (TBX5 1–239 ) was cloned between the NdeI and BamHI sites of the pET28 vector (Novagene). The expressed protein contains an N-terminal six-histidine tag and a thrombin cleavage site derived from the vector.…”

Section: Methodsmentioning

confidence: 99%

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Intermolecular Interactions of Cardiac Transcription Factors NKX2.5 and TBX5

Pradhan

Gopal

et al. 2016

Biochemistry

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Heart development in mammalian systems is controlled by combinatorial interactions of master cardiac transcription factors such as TBX5 and NKX2.5. They bind to promoters/enhancers of downstream targets as homo- or heteromultimeric complexes. They physically interact and synergistically regulate their target genes. To elucidate the molecular basis of the intermolecular interactions, a heterodimer and a homodimer of NKX2.5 and TBX5 were studied using X-ray crystallography. Here we report a crystal structure of human NKX2.5 and TBX5 DNA binding domains in a complex with a 19 bp target DNA and a crystal structure of TBX5 homodimer. The ternary complex structure of NKX2.5 and TBX5 with the target DNA shows physical interactions between the two proteins through Lys158 (NKX2.5), Asp140 (TBX5), and Pro142 (TBX5), residues that are highly conserved in TBX and NKX families across species. Extensive homodimeric interactions were observed between the TBX5 proteins in both crystal structures. In particular, in the crystal structure of TBX5 protein that includes the N-terminal and DNA binding domains, intermolecular interactions were mediated by the N-terminal domain of the protein. The N-terminal domain of TBX5 was predicted to be "intrinsically unstructured", and in one of the two molecules in an asymmetric unit, the N-terminal domain assumes a β-strand conformation bridging two β-sheets from the two molecules. The structures reported here may represent general mechanisms for combinatorial interactions among transcription factors regulating developmental processes.

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Section: ■ Results and Discussionmentioning

confidence: 99%

Section: ■ Materials and Methodsmentioning

confidence: 99%

Section: ■ Materials and Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Intermolecular Interactions of Cardiac Transcription Factors NKX2.5 and TBX5

Pradhan

Gopal

et al. 2016

Biochemistry

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“…The TN domain was suggested to mediate the repressive activity of NKX2‐5 (Elliott, Kirk, Schaft, & Harvey, 2010). The HD is a conserved helix‐loop‐helix domain with three α helices (Pradhan, Gopal, & Nam, 2014) that recognizes and binds to DNA and can homo‐ or hetero‐dimerize (Elliott et al, 2010). The NK2‐SD was suggested to function as an intramolecular transactivation regulator (Watada, Mirmira, Kalamaras, & German, 2000).…”

Section: Introductionmentioning

confidence: 99%

An update on genetic variants of theNKX2‐5

et al. 2020

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NKX2‐5 is a homeodomain transcription factor that plays a crucial role in heart development. It is the first gene where a single genetic variant (GV) was found to be associated with congenital heart diseases in humans. In this study, we carried out a comprehensive survey of NKX2‐5 GVs to build a unified, curated, and updated compilation of all available GVs. We retrieved a total of 1,380 unique GVs. From these, 970 had information on their frequency in the general population and 143 have been linked to pathogenic phenotypes in humans. In vitro effect was ascertained for 38 GVs. The homeodomain had the biggest cluster of pathogenic variants in the protein: 49 GVs in 60 residues, 23 in its third α‐helix, where 11 missense variants may affect protein–DNA interaction or the hydrophobic core. We also pinpointed the likely location of pathogenic GVs in four linear motifs. These analyses allowed us to assign a putative explanation for the effect of 90 GVs. This study pointed to reliable pathogenicity for GVs in helix 3 of the homeodomain and may broaden the scope of functional and structural studies that can be done to better understand the effect of GVs in NKX2‐5 function.

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Crystallization and preliminary X-ray analysis of the cardiac transcription factor complex of NKX2.5 and TBX5 with DNA

Cited by 2 publications

References 16 publications

Intermolecular Interactions of Cardiac Transcription Factors NKX2.5 and TBX5

Intermolecular Interactions of Cardiac Transcription Factors NKX2.5 and TBX5

An update on genetic variants of theNKX2‐5

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