title: Structural basis of TBX5-DNA recognition: the T-box domain in its DNA-bound and -unbound form.
abstract: TBX5, a member of the T-box transcription factor family, plays an important role in heart and limb development. More than 60 single point or deletion mutations of human TBX5 are associated with Holt-Oram syndrome that manifests itself as heart and limb malformations in 1 out of 100,000 live births. The majority of these mutations are located in the TBX5 T-box domain. We solved the crystal structures of the human TBX5 T-box domain in its DNA-unbound form and in complex with a natural DNA target site allowing for the first time the comparison between unbound and DNA-bound forms. Our analysis identifies a 3(10)-helix at the C-terminus of the T-box domain as an inducible recognition element, critically required for the interaction with DNA, as it only forms upon DNA binding and is unstructured in the DNA-unbound form. Using circular dichroism, we characterized the thermal stability of six TBX5 mutants containing single point mutations in the T-box domain (M74V, G80R, W121G, G169R, T223M, and R237W) and compared them with wild-type protein. Mutants G80R and W121G show drastically reduced thermal stability, while the other mutants only show a marginal stability decrease. For all TBX5 mutants, binding affinities to specific and nonspecific DNA sequences were determined using isothermal titration calorimetry. All TBX5 mutants show reduced binding affinities to a specific DNA target site, although to various degrees. Interestingly, all tested TBX5 mutants differ in their ability to bind unspecific DNA, indicating that both sequence-specific and unspecific binding might contribute to the misregulation of target gene expression.
authors.affiliation: European Molecular Biology Laboratory, Structural and Computational Biology Unit, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
authors.forename: Christian U
authors.initials: CU
authors.lastname: Stirnimann
authors.forename: Denis
authors.initials: D
authors.lastname: Ptchelkine
authors.forename: Clemens
authors.initials: C
authors.lastname: Grimm
authors.forename: Christoph W
authors.initials: CW
authors.lastname: Müller
mesh_terms: D000015:Abnormalities, Multiple / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005326:Fetal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006330:Heart Defects, Congenital / Q000235:genetics / Q000378:metabolism; D006801:Humans; D017880:Limb Deformities, Congenital / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D020825:T-Box Domain Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000098908:Brachyury Protein
chemical_list: D004247:DNA; D005326:Fetal Proteins; D020825:T-Box Domain Proteins; D000098908:Brachyury Protein; C103172:T-box transcription factor 5