title: Structural basis of an embryonically lethal single Ala --> Thr mutation in the vnd/NK-2 homeodomain.
abstract: The structural and DNA binding behavior is described for an analog of the vnd/NK-2 homeodomain, which contains a single amino acid residue alanine to threonine replacement in position 35 of the homeodomain. Multidimensional nuclear magnetic resonance, circular dichroism, and electrophoretic gel retardation assays were carried out on recombinant 80-aa residue proteins that encompass the wild-type and mutant homeodomains. The mutant A35T vnd/NK-2 homeodomain is unable to adopt a folded conformation free in solution at temperatures down to -5 degreesC in contrast to the behavior of the corresponding wild-type vnd/NK-2 homeodomain, which is folded into a functional three-dimensional structure below 25 degreesC. The A35T vnd/NK-2 binds specifically to the vnd/NK-2 target DNA sequence, but with an affinity that is 50-fold lower than that of the wild-type homeodomain. Although the three-dimensional structure of the mutant A35T vnd/NK-2 in the DNA bound state shows characteristic helix-turn-helix behavior similar to that of the wild-type homeodomain, a notable structural deviation in the mutant A35T analog is observed for the amide proton of leucine-40. The wild-type homeodomain forms an unusual i,i-5 hydrogen bond with the backbone amide oxygen of residue 35. In the A35T mutant this amide proton resonance is shifted upfield by 1.27 ppm relative to the resonance frequency for the wild-type analog, thereby indicating a significant alteration of this i,i-5 hydrogen bond.
authors.affiliation: Laboratory of Biophysical Chemistry, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: B
authors.initials: B
authors.lastname: Xiang
authors.forename: S
authors.initials: S
authors.lastname: Weiler
authors.forename: M
authors.initials: M
authors.lastname: Nirenberg
authors.forename: J A
authors.initials: JA
authors.lastname: Ferretti
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000196:embryology / Q000235:genetics; D005804:Genes, Lethal; D018398:Homeodomain Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D017510:Protein Folding; D013696:Temperature; D014157:Transcription Factors
chemical_list: D029721:Drosophila Proteins; D018398:Homeodomain Proteins; D014157:Transcription Factors; C094797:vnd protein, Drosophila; D004247:DNA