Like many other living proteins, Hsp90 is a shape shifter: A reversible chemical bond (ATP hydrolysis, the fuel of life) activates these biomolecules as it alters their physical structure. But even though Hsp90 is one of the most abundant proteins in our cells – and one that is crucial to many cellular processes – scientists are still unsure exactly how its active parts get reorganized in response to the ATP binding and hydrolysis. A group at the Weizmann Institute of Science has now succeeded in observing this process, and they accomplished it with a clever switch in the chemical reaction. The results of their study, which may aid further research into both protein activities and drugs to target this protein, were published in the Proceedings of the National Academy of Sciences (PNAS), USA.
Hsp90 serves as a cradle-to-grave assistant to the other proteins: As a “chaperone,” it helps new proteins fold; as a “heat shock protein” (the Hsp in its name), it stabilizes other proteins against stress; and ultimately, it aids in protein degradation. Under certain circumstances, it also helps cancer cells grow. This versatile protein is made of two individual, identical protein molecules (that is, a molecular structure known as a “dimer”), each “string” of the dimer having three parts. The structure of one end is relatively fixed; but on the opposite end, two parts open to separate, and then close up together. The energy-supplying ATP is needed for closing: In the process of hydrolysis, ATP binds to the protein along with a magnesium ion. This causes the ATP to lose one of its phosphate groups, becoming ADP as it does so. The ADP remains in contact with the protein, and the flexible end of the dimer presumably stays closed and “on.”
Scientists thus understood that a closed position was necessary for Hsp90 to begin working and recruiting its client proteins, but they had further questions about the way the protein continues to function: Did it stay closed or did it open up again immediately following the chemical reaction? These questions go to the heart of how this protein functions when it goes to work on other proteins, as well as how it might be targeted by drugs to reduce its activity. Previous studies attempting to answer this question had produced conflicting or inconclusive results.
Prof. Daniella Goldfarb of the Institute’s Chemical and Biological Physics Department is one of the leading experts in a method used to investigate the dynamics of large, complex proteins such as Hsp90. The method involves attaching tiny magnetic tags to two points on a protein. These tags contain electrons whose spins can be observed with a technique known as electronic paramagnetic resonance (EPR). The distance between the two tags is measured in a number of such pairs yielding well-rounded picture of the proteins’ movement. More importantly, the method enables researchers to investigate these proteins noninvasively, within living cells.
Ultimately, the Weizmann team was able to answer the outstanding question
The problem for those researching Hsp90 with the usual tags was that the links holding them to the protein molecule were themselves a bit floppy, making it hard to differentiate between movement that was due to the labels and that due to the protein itself. Even the new metallic tags that Goldfarb has been experimenting with in her group could not be efficiently attached to the protein.
That’s when Goldfarb and Dr. Angeliki Giannoulis, a postdoctoral fellow in Goldfarb’s group, had a bright idea: Switch the magnesium atoms in the proteins that are essential for the hydrolysis reaction with those of another element – manganese. Manganese (and manganese, alone) can step into magnesium’s place without disrupting the protein’s functioning. The manganese ion, although it would perform the same function as the magnesium, has an electron-spin signal, which magnesium lacks, and thus the EPR equipment would be able to pick it out. Since it would be embedded in the protein structure, there would be no superfluous movement.
The first step was to modify Hsp90 proteins so the scientists could attach the standard tags. For help in this initial task, Giannoulis turned to members of the Institute’s Structural Proteomics Unit, led by Dr. Shira Albeck. Then Giannoulis labeled and switched the magnesium for manganese. She and the team in Goldfarb’s lab also fine-tuned their EPR equipment for picking up the spin signals from the manganese. Goldfarb: “Observing manganese interactions presented some challenges, but we were able to detect two well-defined closed states of the Hsp90 proteins’ ends, one with ATP and the other with ADP. Our method bypassed the problems associated with linking the tags, and thanks to the advanced equipment in our lab and the high-field measurements we carried out, we were able to clearly plot the varying distances between the two parts of the protein.”
Ultimately, the Weizmann team was able to answer the outstanding question, providing proof that Hsp90 does indeed remain closed after the initial chemical reaction, but in a slightly different conformation.
Since the chemical reactions involving ATP and magnesium are among the most common in living organisms, the method may prove useful for uncovering the dynamics of other proteins. Goldfarb hopes the new findings may advance the understanding of the dynamics of the crucial protein Hsp90 and assist those who are designing drugs to limit its activities in cancer cells as well as other cells.
Prof. Daniella Goldfarb is the incumbent of the Erich Klieger Professorial Chair in Chemical Physics.
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