Structure-Function

The structure of the DNA binding domain of the Kluyveromyces lactis Heat Shock Factor (HSF) has been determined both in solution (Harrison et. al. 1994) and complexed with DNA (Littlefield et. al. 1999). Both of the structures reveal that the HSF DNA binding domain is a diverged member of the helix-turn-helix transcription factor family. Even though the HSF's structure resembles a canonical HTH motif it contains several distinct features.

In addition to the two alpha helices seen in all HTH structures, the HSF structure contains an extra alpha helix, a four stranded beta sheet, and a protruding structure termed a "wing". The 5'-nGAAn-3' penta-nucleotide sequence that HSF binds preferentially occurs in repeats called heat shock elements, allowing for several HSF monomers to bind as a complex. The greater the number of penta-nucleotide sequences in a heat shock element the higher the affinity for HSF, suggesting that cooperative interactions between HSF monomers may increase their affinity for DNA. Additionally, it has been shown that the affinity of HSF for DNA increases with increasing temperature.

One structural feature that may account for the thermal dependence of DNA binding by HSF is a kink in alpha helix 2. This bent helix contains an extra residue which results in a small bulge that deforms the helix structure. The increased temperature during heat shock may alleviate a steric hinderance caused by the deformed alpha helix, resulting in a better "fit" of the DNA binding domain into the major groove of the double helix.

The structure of HSF complexed with DNA has also revealed that the number of interactions made between the recognition helix of HSF and the nucleotide sequence it binds are minimal in comparison to other transcription factors. In fact, only two rsidues (Ser247 and Arg 250) make direct contact with nucleotides in the major groove. the rest of the DNA interactions are made through a complex network of water molecules and likely provide only minimal stability. Taken as a whole,the interactions between the HSF DNA binding domain and DNA only result in a small amount of stability which may be sensitive to the biochemical environment the protein is exposed to.

It has been shown biochemically that HSF prefers to bind DNA as a trimer unlike most DNA binding proteins which bind as a dimer. The protein-protein interactions that allow HSF to bind DNA as a trimer have been a mystery for some time but the structure of HSF has revealed that there are two main surfaces of interaction which permit trimers of HSF to form. The first surface is located within helix 2 and consists primarily of two arginine residues which make hydrogen bond interactions acrossed the DNA double helix. Also, the interactions between the helix 2's exclude water from the cleft tht seperates teh two monomers. This exclusion of water may also be facilitated by changes in the biochemical environemnt such as heat shock or rapid pH change. The second surface of protein-protein interaction came as a surprise because it includes the "wing" structure which has previously been shown in other "winged" helix-turn-helix structures to be involved in DNA binding instead of oligomer formation. Extensive hydrogen bonding is predicted to occur between the "wing" of one HSF monomer and the turn of the second HSF monomer. This interaction occurs not only between monomers that are directly acrossed from one another but also between monomers that are situated diagonally from one another when bound to an heat shock element. Together these two surfaces of protein-protein interaction make it possible for monomers to interact with more than one other monomer at a time and predict that the formation of trimers or larger oligomers may be more stable than dimers.

The structure of the DNA binding domain of HSF complexed with DNA supports the observations that HSF binds only weakly to DNA (an interaction which may be modulated by temperature) and that trimers of HSF monomers can form through interactions at two distinct surfaces.