Structural analysis of yeast HSF by site-specific crosslinking.
We have introduced cysteine substitutions into the yeast HSF1 gene at a variety of locations. Most have no phenotypic effect, and therefore provide site-specific probes for thiol-specific reagents. Crosslinking of single mutants identifies locations where equivalent regions of individual monomers can approach each other in the HSF trimer. Crosslinking of double mutants indicates regions that can approach closely within a single subunit. Results for the DNA binding domain and trimerization domain are consistent with known structural information, and provide essential controls on the validity of the technique. In contrast to these two domains, the N-terminal and C-terminal domains, wherein lie the transcriptional activators, are highly flexible, and do not appear to be in stable contact with any other portions of the protein. None of these patterns are affected by the conformational change that is induced by superoxide or heat shock. We suggest a new model for the mechanism of HSF regulation that accomodates the structural information provided by these studies.
Bonner
J J
JJ
Department of Biology, Indiana University, Bloomington, IN 47405, USA. jbonner@bio.indiana.edu
Chen
D
D
Storey
K
K
Tushan
M
M
Lea
K
K
D019943:Amino Acid Substitution; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004220:Disulfides / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D008301:Maleimides; D008954:Models, Biological; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010641:Phenotype; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D015203:Reproducibility of Results; D029701:Saccharomyces cerevisiae Proteins; D013329:Structure-Activity Relationship; D013481:Superoxides / Q000494:pharmacology; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015003:Yeasts
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D004220:Disulfides; D005656:Fungal Proteins; C067168:HSF1 protein, S cerevisiae; D006360:Heat-Shock Proteins; D008301:Maleimides; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D013481:Superoxides; D004247:DNA; D003545:Cysteine