Heterodimerization of the yeast homeodomain transcriptional regulators alpha 2 and a1: secondary structure determination of the a1 homeodomain and changes produced by alpha 2 interactions.
The homeodomain proteins, a1 and alpha 2, act cooperatively to regulate cell-type specific genes in yeast. The basis of this cooperativity is an interaction between the two proteins, forming a heterodimer that binds DNA tightly and specifically. A fragment containing the homeodomain of a1, a1(66-126), has been studied by NMR spectroscopy to gain secondary structure information and to characterize the changes in a1 upon heterodimerization with alpha 2. Heteronuclear (1H-15N) NMR methods were used to assign backbone resonances of the 61 amino acid fragment. The a1(66-126) secondary structure was determined using NOE connectivities, 3JHN alpha coupling constants and hydrogen exchange kinetic data. NMR data identify three helical segments separated by a loop and a tight turn that are the characteristic structural elements of homeodomain proteins. The a1 fragment was titrated with alpha 2(128-210), the homeodomain-containing fragment of alpha 2, to study changes in a1(66-126) spectra produced by alpha 2 binding. The a1(66-126) protein was labeled with 15N and selectively observed using isotope-edited NMR experiments. NMR spectra of bound a1(66-126) indicate that residues in helix 1, helix 2, and the loop connecting them are directly involved in the binding of the alpha 2 fragment. Relatively minor effects on the resonances from residues in helix 3, the putative DNA-binding helix, were noted upon alpha 2 binding. We have thus located a region of the a1 homeodomain important for specific protein recognition.
Baxter
S M
SM
Institute of Molecular Biology, University of Oregon, Eugene 97404.
Gontrum
D M
DM
Phillips
C L
CL
Roth
A F
AF
Dahlquist
F W
FW
D000595:Amino Acid Sequence; D015966:Gene Expression Regulation, Fungal; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000235:genetics; D006860:Hydrogen Bonding; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011485:Protein Binding; D017433:Protein Structure, Secondary; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide / Q000737:chemistry*; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics; D014157:Transcription Factors / Q000737:chemistry*
D018398:Homeodomain Proteins; D046911:Macromolecular Substances; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide; D011994:Recombinant Proteins; D014157:Transcription Factors