Daniel Gewirth aligns a protein crystal mounted in a laboratory x-ray diffraction instrument at the Hauptman-Woodward Institute in Buffalo, NY. X-ray diffraction is used to determine the high resolution structure of proteins. Initial diffraction data is often collected using commercial diffraction equipment such as that shown above, while the highest resolution data is typically collected using much more intense x-rays generated by synchrotrons at National Laboratory facilities. Both types of facilities were used for his work on GRP94. (Source: Daniel Gewirth)

"These have been the golden months of HSP90 structural biology," says Daniel Gewirth, Ph.D. senior research scientist at the Hauptman-Woodward Medical Research Institute and associate professor at the State University of New York (SUNY) in Buffalo. HSP90 is a member of the HSP family most commonly found in human cells. Since April 2006 structures of three members of the HSP90 family have been determined. The first one from yeast was published by a group in the UK. The second one was determined by a group at the University of California in San Francisco and the third one was published by Gewirth's group in the October 12, 2007 edition of Molecular Cell.

The human cell consists of different isoforms of HSP90 and what Gewirth's group found was the structure of the mammalian inducible form called GRP94, seen in the endoplasmic reticulum. "The actual sequence is from dog," says Gewirth. "It turns out that among the 804 amino acids that constitute GRP94 only six of them vary between human and dog and those six differences are entirely conservative. So they are essentially identical and there are no differences in any of the functionally relevant domains."

There had been conflicting theories in the literature about GRP94 and its relation to the HSP90 family. "When we started this project the understanding in the field was that GRP94 was not a member of the HSP90 family because it did not hydrolyze ATP," says Gewirth. So they (researchers) set out to approach the problem both structurally and functionally. "Like with most structural biology projects, we adopted a divide and conquer strategy." But finding the optimal conditions for crystallization turned out to be quite a challenge. "It turns out that the real trick was to have the correct protein construct but finding out which part to trim off took five years," says Gewirth. Finally, in May 2006 they got their first structure.

"What we found knocked our socks off," says Gewirth. They had expected to find the protein in a sort of folded over, doubly dimerized form but instead they found that the protein was not dimerized at all at the N-terminal domain. "That was contrary to the dogma in the field and so that had us scratching our heads for a very long time," he says. Even though they had solved the structure they had to perform several control experiments to make sure that the results were not an artifact. By Fall 2006, they had completed all their functional and biochemical assays and were finally able to publish their findings on the structure and function of GRP94, which now places it firmly in the HSP90 family.

Although structures from three different systems-the yeast, bacteria and mammal have now emerged, they have key differences. The main difference is how the three domains in HSP90, which are very well conserved structurally, are arranged relative to one another in the intact molecule. The one from yeast shows the molecule heavily dimerized, while the one from bacteria shows a confirmation that is hard to understand functionally. Gewirth's structure shows yet another confirmation, which probably indicates a resting state or a state that the protein is most commonly found in. "These three pictures together have presented a challenge to create an integrated functional picture of how these proteins work," says Gewirth.

Gewirth's work has stimulated a lot of interest since it elucidates, for the very first time, the structure of the mammalian form of GRP94. Like all HSP90s, GRP94 is a molecular chaperone and has a specialized clientele that includes all the toll-like receptors, which are responsible for innate immunity. With the structure now known researchers can start to modulate the innate immune system by designing inhibitors of GRP94. The structure could also help in modulating disease pathways in other areas like septic shock, atherosclerosis, cystic fibrosis and Alzheimer's disease.

Mail-order Crystallography

Improvements in molecular biology, cloning techniques and advancements in computer technology have made protein expression and data acquisition, both fast and reliable. The availability and access to synchrotron x-ray sources has also accelerated the field. "When I started out in this business 25 years ago, there was one synchrotron where you could collect data and it broke down a lot," says Gewirth. "Now mail-order crystallography is coming into vogue where you don't even have to travel to the synchrotron. You can send your samples in and it can be robotically mounted onto the beam to collect the data."

However, one of the challenges that still remain to be tackled is getting the right crystal. According to Gewirth the failure rate in crystallography is nearly 85%. "I think what you are going to see over the next ten years is a much better understanding of how to reliably form protein crystals." Once that happens he thinks structural biology will be used even more extensively to solve biological problems. "So I think it's really a brave new world. If you're a technical person you've got to ride the wave and if you're a biological person you've got to think of interesting projects."