Conformational analysis of heat shock factor 1 and its C -terminal domain.

Abstract

Heat Shock Transcription Factor 1 (HSF1) is a key regulator of the expression of heat shock proteins during the heat shock response. It functions as a connector between the upstream stress signals and the downstream cellular responses needed for cell survival. HSF1 is activated by heat and other pathological or physiological stresses, undergoing homotrimerization to acquire the ability to bind to DNA, and structural and chemical modifications to achieve transcriptional competence. In aged animals, HSF1 function is impaired, leading to a decline in HSP70 gene expression and a defect in responding to stress. The C-terminal domain (CT), which encompasses the regulatory and activation domains of HSF1, is predicted to contain natively unfolded regions by PONDR<super> RTM</super> disorder analysis. Natively unfolded domains play an important role in transcription factors, allowing them to undergo posttranslational modifications and interactions with a myriad of partners essential for the transcriptional process. Experimental evidence presented in this dissertation indicates that CT is natively unfolded under physiological conditions since it exhibits the following structural characteristics: (1) a very low abundance of secondary and tertiary structure as observed by circular dichroism, (2) no hydrophobic core as monitored by a TNS binding assay, (3) a large hydrodynamics radius as measured by SEC-HPLC, and (4) high structural flexibility as probed by limited proteolysis. Interestingly, CT can be induced to fold when the negatively charged amino acid residues are neutralized by acidic pH or the cationic detergent DTA. The folded CT is very compact, containing secondary structure and hydrophobic clusters. Interaction of CT and its physiological partner, the core domain of the TATA-binding protein (cTBP), indicates a moderate structural alteration upon complex formation. CT interacts at the hydrophobic sites of cTBP, with a dissociation constant of 0.91 muM, as determined using competitive TNS-binding assay. The induced-fit binding between CT and cTBP suggests a structural plasticity of the natively unfolded structure of CT. The structural flexibility of CT could also imply its adaptability to transiently interact with various transcriptional effectors/regulators.