Intermolecular Interactions between Cysteine and Aromatic Amino Acids with a Phenyl Moiety in the DNA-Binding Domain of Heat Shock Factor 1 Regulate Thermal Stress-Induced Trimerization.

In this study, we investigated the trimerization mechanism and structure of heat shock factor 1 (HSF1) using western blotting, tryptophan (Trp) fluorescence spectroscopy, and molecular modeling. First, we examined the DNA-binding domains of human (Homo sapiens), goldfish (Carassius auratus), and walleye pollock (Gadus chalcogrammus) HSF1s by mutating key residues (36 and 103) that are thought to directly affect trimer formation. Human, goldfish, and walleye pollock HSF1s contain cysteine at residue 36 but cysteine (C), tyrosine (Y), and phenylalanine (F), respectively, at residue 103. The optimal trimerization temperatures for the wild-type HSF1s of each species were found to be 42, 37, and 20 °C, respectively. Interestingly, a mutation experiment revealed that trimerization occurred at 42 °C when residue 103 was cysteine, at 37 °C when it was tyrosine, and at 20 °C when it was phenylalanine, regardless of the species. In addition, it was confirmed that when residue 103 of the three species was mutated to alanine, trimerization did not occur. This suggests that in addition to trimerization via disulfide bond formation between the cysteine residues in human HSF1, trimerization can also occur via the formation of a different type of bond between cysteine and aromatic ring residues such as tyrosine and phenylalanine. We also confirmed that at least one cysteine is required for the trimerization of HSF1s, regardless of its position (residue 36 or 103). Additionally, it was shown that the trimer formation temperature is related to growth and survival in fish.

pH Dependence of HSF1 trimerization is shaped by intramolecular interactions.

Heat shock factor 1 (HSF1) primarily regulates various cellular stress responses. Previous studies have shown that low pH within the physiological range directly activates HSF1 function in vitro. However, the detailed molecular mechanisms remain unclear. This study proposes a molecular mechanism based on the trimerization behavior of HSF1 at different pH values. Extensive mutagenesis of human and goldfish HSF1 revealed that the optimal pH for trimerization depended on the identity of residue 103. In particular, when residue 103 was occupied by tyrosine, a significant increase in the optimal pH was observed, regardless of the rest of the sequence. This behavior can be explained by the protonation state of the neighboring histidine residues, His101 and His110. Residue 103 plays a key role in trimerization by forming disulfide or non-covalent bonds with Cys36. If tyrosine resides at residue 103 in an acidic environment, its electrostatic interactions with positively charged histidine residues prevent effective trimerization. His101 and His110 are neutralized at a higher pH, which releases Tyr103 to interact with Cys36 and drives the effective trimerization of HSF1. This study showed that the protonation state of a histidine residue can regulate the intramolecular interactions, which consequently leads to a drastic change in the oligomerization behavior of the entire protein.