March 24, 2000

Study tracks calcium’s protein interactions

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Walter Chazin, Ph.D.

Study tracks calcium's protein interactions

In cells, calcium makes things happen. It participates in a variety of different signaling pathways by interacting — like a baseball in a glove — with a family of calcium binding proteins.

Researchers in Vanderbilt University's Center for Structural Biology have now discovered that the two calcium binding "gloves" in a protein called calbindin-D9k communicate with each other, even though they are far apart in the protein. The results, reported this month in Nature Structural Biology, are important to understanding how calcium binding proteins work.

"If we know what distinguishes the three-dimensional structure and dynamics of different calcium binding proteins, we can begin to rationally target (with therapeutic drugs) a single calcium signaling pathway in a cell," said Walter J. Chazin, Ph.D., who joined the Vanderbilt faculty last year as director of the new Center for Structural Biology.

Chazin has been interested in calcium binding proteins for more than 15 years. As a group, the proteins offer an interesting challenge to structural biologists — they all bind calcium and have similar structural features, but they do different things. Some participate in various signaling pathways, others are responsible for calcium uptake, transport, buffering, and general calcium homeostasis in the cell.

"The question is: how do you generate all this diversity in functional activity, given a common structural framework?," Chazin said.

Chazin's group has approached this question using a structural biology tool called nuclear magnetic resonance (NMR) spectroscopy to determine the structure and dynamics of calcium binding proteins, whose structure can vary greatly depending on whether or not calcium is bound.

"In order to really understand calcium binding proteins, you need to understand their different forms, in particular the 'off state' in the absence of calcium, and the 'on state' in the presence of calcium," Chazin said.

In 1994, Chazin and colleagues reported the structure of calbindin-D9k in both the absence and presence of calcium, the first time this was done for a calcium binding protein.

But because calbindin has two sites that bind calcium, it can actually exist in four different states: no calcium bound (off state), one calcium atom bound in either of the two sites, and two calcium atoms bound (on state). The current studies determine the structure of calbindin when calcium binds in only one of the two available sites, adding to Chazin's earlier work to complete the picture of calbindin's four different structural states.

The investigators were surprised to find that when calcium binds in only one of the two sites, the other site — with no calcium in it — assumes a structure that looks like calcium is bound. In other words, the second "glove" acts like it has also caught a calcium ball.

"When calcium binds in the one site, we see structural effects all the way over on the other side of the protein," Chazin said. "Our results show the power and the effectiveness of site-site communication. This is one of the few very clear demonstrations of a very important long range effects in a protein."

Site-site communication is the likely molecular explanation for a property called "cooperativity" that is important to calcium binding proteins, especially those involved in signaling pathways.

Cooperativity enhances the ability of these proteins to go from completely "off" to completely "on" when the calcium concentration changes.

"The change in calcium concentration in a cell in response to a signal outside is rather subtle," Chazin said, "and these proteins need to be very sensitive to this very small change. With cooperativity, you get the 'all or nothing' response that you need for effective calcium signaling."

Calbindin-D9k is the first calcium binding protein for which structures of all its possible states have been determined.

"Part of the beauty of this study is that we now have the full characterization of the calbindin calcium binding pathway," Chazin said. "So now we can really start taking it apart, using computational studies and additional experiments to examine the critical features of the protein."

Chazin's colleagues who participated in the studies while he was at The Scripps Research Institute in La Jolla, Calif., include Lena Mäler, Ph.D., now an assistant professor of Biophysics at Stockholm University in Sweden, and John Blankenship, a rotating graduate student.

A critical role involving highly sophisticated NMR experiments was played by long-time collaborator Mark Rance, Ph.D., at the University of Cincinnati Medical School. The research was supported by the National Institutes of Health.