Subunit interaction in the CCAAT-binding heteromeric complex is mediated by a very short alpha-helix in HAP2.

Abstract

We did the domain of HAP2 that mediates subunit asiation In the heteromeric CCAAT-binding complex, first by genetic mutational analysis and then by structural studies. The mutational data suggest that a very short region in HAP2 mediates protein-protein association and that the structure of this domain is likely to be an a-helix. The CD analyses ofa 15-residue synthetic oligopeptide covering this region confirm this surmise. The oligopeptide indeed formed an unusuafly thermal stable a-helix in aqueous solution. Eight amino acids that lie along one face of this helix, including three arginines, are found to be critical for protein-protein association. The partner that interacts with this helical motif is likely to be another subunit in the HAP complex, since the CCAATbinding factor is shown to contain one molecule of HAP2. Our results suggest that very short regions in proteins can encode precise structures and mediate stable and specific proteinprotein recognition and interactions.The specificity and stability of protein-protein interactions are critical determinants in the assembly of macromolecular complexes consisting of protein subunits. One well-studied example of these complexes involves the transcription factors Fos and Jun (for review, see refs. 1 and 2). Jun-Jun homodimers and Fos-Jun heterodimers will form readily and are active in cells, whereas Fos-Fos dimers do not form (3-5). Inspection of a family of transcription factors with DNA-binding domains like those of Fos and Jun led to the leucine zipper hypothesis (6).A heteromeric transcriptional activator that was identified early on is the yeast (Saccharomyces cerevisiae) CCAATbox binding factor HAP2/3/4 (7-9). Both HAP2 and HAP3were present in a gel-shift complex with a CCAAT boxcontaining probe (9), and HAP2 and HAP3 remained stably associated in the absence of DNA (8) vivo (10, 14).Here we describe the dissection of the SAD of HAP2 by genetic mutational and biophysical structural analyses. Our findings delimit the region and pinpoint which residues in HAP2 are critical for its function. The mutational data suggest that a very short region in HAP2 is likely to form an a-helix which mediates assembly. Our findings show that 8 aa along one face of this helix dictate the specificity and at least part of the stability of interaction between HAP2 and its interacting partner. Further, a 15-aa synthetic peptide covering this region showed a typical a-helical circular dichroism (CD) spectrum. We conclude that very short peptide regions can encode precise structures and mediate stable and specific protein-protein recognition and interactions in macromolecular complexes. MATERIALS AND METHODSStrains, Plasmids, and f-Galactodase Assays. The S. cerevisiae strain BWG 1-7a (MAT a leu2-3,112 his4-519 adel-100 ura3-52) or isogenic derivatives were used in all experiments described. HAP2 complementation was assayed by using strain JO1-la (14). Vectors pRB1155 (16), pJO323 (14), and pLGA265UP1 (17); plasmid pXY3236 (10); and the assay for /3-galactosidase ac...

The NF-YB/NF-YC Structure Gives Insight into DNA Binding and Transcription Regulation by CCAAT Factor NF-Y

“…1A). The N terminus of NF-YA1 forms an ␣-helix in solution and only residues on one side of this helix are functionally important (23). In NF-YB, mutations on helix ␣2 (E90R and S97R) were shown to influence NF-YA binding (16,22).…”

Multiple Genes Encoding the Conserved CCAAT-Box Transcription Factor Complex Are Expressed in Arabidopsis

“…In organisms other than plants, this relative position is occupied by an Arg residue. Mutation of this residue in the yeast HAP2 protein to Pro, Leu, or Gly leads to a greatly reduced ability to associate with the HAP3,5 dimer and to form a functional complex (Xing et al, 1994). Each AtHAP2 contains a short aliphatic residue at this position, either Gly or Ala, which by comparison with the mutated yeast protein should inhibit their ability to form a functional HAP complex and complement the yeast hap2 mutant.…”

“…Although many of the residues for subunit association characterized in yeast mutation studies (Xing et al, 1994) have been conserved, there is a greater variation between the Arabidopsis homologs than between animal and yeast HAP2 (Fig. 6).…”

Interfacial assembly of proteins and peptides: recent examples studied by neutron reflection

“…The technique has been used for characterizing interfacial adsorption from both biomimetic and designed peptides. The 15-mer peptide YYY15 (YVNAKQYYR-ILKRRY) was directly copied from the native sequence of a DNA-binding domain within a heteromeric transcriptional activator, HAP2, identified from yeast Saccharomyces cerevisiae, with tyrosine (Y) present at the 1st, 8th and 15th amino acid positions (Xing et al 1994). Its point mutations at these three Y positions to tryptophans (W) (denoted as WWW15) led to the same length sequence except the replacement of the three Ys with the three Ws.…”