Structural analysis of yeast HSF by site-specific crosslinking

Area of Science:

• Molecular Biology
• Protein Structure and Dynamics
• Yeast Genetics

Background:

• Heat Shock Factor 1 (HSF1) is a crucial transcription factor regulating cellular stress responses.
• Understanding HSF1's structural dynamics is key to deciphering its regulatory mechanisms.

Purpose of the Study:

• To investigate the structural organization of yeast HSF1 using site-specific cysteine substitutions.
• To map protein-protein interaction sites within HSF1 monomers and trimers.

Main Methods:

• Introduction of cysteine mutations into the yeast HSF1 gene.
• Chemical crosslinking of single and double mutants to probe protein proximity.
• Analysis of structural changes under stress conditions (superoxide, heat shock).

Main Results:

• Most cysteine substitutions had no phenotypic effect, serving as specific probes.
• Crosslinking identified regions of monomer proximity in the HSF1 trimer and within subunits.
• The DNA binding and trimerization domains showed expected structural proximity.
• N-terminal and C-terminal domains, including transcriptional activators, are highly flexible and not in stable contact.

Conclusions:

• Yeast HSF1's N- and C-terminal domains are conformationally flexible.
• Structural flexibility is maintained even during stress-induced conformational changes.
• A revised model for HSF1 regulation is proposed, incorporating these flexibility insights.