Characterization of critical interactions between Ndt80 and MSE DNA defining a novel family of Ig-fold transcription factors.
The Ndt80 protein of the yeast Saccharomyces cerevisiae is the founding member of a new sub-family of proteins in the Ig-fold superfamily of transcription factors. The crystal structure of Ndt80 bound to DNA shows that it makes contacts through several loops on one side of the protein that connect beta-strands which form the beta-sandwich fold common to proteins in this superfamily. However, the DNA-binding domain of Ndt80 is considerably larger than many other members of the Ig-fold superfamily and it appears to make a larger number of contacts with the DNA than these proteins. To determine the contribution of each of these contacts and to examine if the mechanism of Ndt80 DNA binding was similar to other members of the Ig-fold superfamily, amino acid substitutions were introduced at each residue that contacts the DNA and assayed for their effect on Ndt80 activity. Many of the mutations caused significant decreases in DNA-binding affinity and transcriptional activation. Several of these are in residues that are not found in other sub-families of Ig-fold proteins. These additional contacts are likely responsible for Ndt80's ability to bind DNA as a monomer while most other members require additional domains or cofactors to recognize their sites.
Fingerman
Ian M
IM
Waksman Institute and Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 08854, USA.
Sutphen
Kristina
K
Montano
Sherwin P
SP
Georgiadis
Millie M
MM
Vershon
Andrew K
AK
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000235:genetics* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D007136:Immunoglobulins / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D020218:Response Elements / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity; D013499:Surface Properties; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism*
D004268:DNA-Binding Proteins; D007136:Immunoglobulins; C097071:NDT80 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D004247:DNA; D000409:Alanine