Loss of function and inhibitory effects of human CSX/NKX2.5 homeoprotein mutations associated with congenital heart disease
J. Clin. Invest. Hideko Kasahara, et al. 106:299 doi:10.1172/JCI9860 [Go to this article.]

Figure 3
DNA binding affinity of group 1, 2, and 5 mutant proteins versus wild-type CSX/NKX2.5. (a) Sequence of two consensus CSX/NKX2.5 binding sites (–242 bp and –87 bp sites) in rat ANF promoter; the paired binding sites in –242 bp site, and a single binding site in –87 bp site. (b) –242 bp site was used for the DNA binding assay (lane 1) mixed with threefold serially increased CSX/NKX2.5 fusion proteins (0.018–4.4 μg/mL of MBP-CSX/NKX2.5 fusion protein) (lanes 2–7). Wild-type CSX/NKX2.5 bound as a monomer (M) as well as a dimer (D) depending on the protein concentration. No shifted bands were observed in groups 1 and 5 (M149, M170, and M112). (c) The EMSA of the group 2 mutant proteins that have a single missense mutation in the HD. ¹⁷⁸Thr-Met (M178) mutation was located just before the third helix, and ¹⁸⁸Asn-Lys (M188), ¹⁸⁹Arg-Gly (M189), and ¹⁹¹Tyr-Cys (M191) were located in the third helix. Two conserved amino acid mutations were identified: ¹⁸⁸Asn (⁵¹Asn in HD) is conserved in all the HD protein that is directly bound to the major groove of DNA, and ¹⁹¹Tyr (⁵⁴Tyr in HD) is conserved in all NK2 class homeoprotein. All four mutant proteins show dramatically reduced DNA binding affinity compared with the wild-type CSX/NKX2.5. D, dimer; M, monomer; F, free probe. Mutation sites of group 2 are indicated with white asterisks.