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 al, a1(66-126), has been studied by NMR spectroscopy to gain secondary structure information and to characterize the changes in al upon heterodimerization with alpha 2. Heteronuclear (H-1-N-15) 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, (3)J(HN 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 al 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 N-15 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 al homeodomain important for specific protein recognition.