Choreoathetosis, hypothyroidism, and pulmonary alterations due to human NKX2-1 haploinsufficiency
J. Clin. Invest. Heiko Krude, et al. 109:475 doi:10.1172/JCI14341 [Go to this article.]

Figure 2
Position of aa exchange due to the NKX2-1 mutations and structural consequences for the DNA interaction. (a) Alignment of the nine known mammalian NK2 HDs (3, 27); Tyr54, which is characteristic for NK2 HDs, is shown in white, mutations detected in our patients are shown in white i underlined, the known missense mutations of the NKX2-5 HD are shown in gray (20). (b) Three-dimensional model of NK2 HD-DNA complex. Position of Val45Phe mutation in patient 2 is shown in black. (c) EMSA performed with in vitro–expressed HD protein show a clear loss of binding capacity in the mutant. Lane 1: without protein, lanes 2–5: wild-type protein (0.5, 1, 2, and 5 μl purified protein) lane 6: unrelated oligo; lanes 7 and 8: competition with 100-fold excess of unlabeled oligo-C (lane 7) and unrelated oligo (lane 8) (amount of protein applied corresponding to lane 3); lanes 9–11: mutant protein (2, 5, and 10 μl of purified protein); lane 12: mutant protein with unrelated and unlabeled oligo; black arrow marks bound oligo, white arrow marks free oligo. (d) Western blots using anti-Flag–antibody are aligned with lanes 2–5 and 9–11 of (c) showing the equivalence in amounts of HD protein used.(e) The truncated HD due to the 2595insGG nonsense mutation in patient 3 is shown in the predicted conformation relative to the DNA-binding groove lacking helix 3.