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A key protein linked to neurodegenerative diseases, such as Huntington's and Alzheimer's diseases, has been characterised using NMR by US researchers. John Cavanagh, Douglas Kojetin, David Kordys, and Richele Thompson of North Carolina State University teamed with colleagues Ronald Venters of Duke University and Rajiv Kumar of the Mayo Clinic and Foundation have obtained a structure for the protein, calbindin-D28K. This protein modulates calcium levels by sequestering calcium from areas that have too much or serves as an on/off switch for further chemical reactions. It is found in the kidneys, pancreas, ocular nerve and large amounts are present in the brain. It is its role in the brain as a "bodyguard" against the action of the enzyme caspase-3 that links it to neurodegenerative diseases. By binding to and inhibiting caspase-3, calbindin D28K prevents the formation of damaging plaque and tangle formation in the brain, which are hallmarks of neurodegenerative disease. The structure of this key protein has remained elusive, until now. "If you don't know the shape of the protein, you can't figure out how it works," Cavanagh says. "It took a long time, about five years, but we've characterized the structure of this protein and found where it binds caspase-3. Insight into how it binds to caspase-3 might lead to a way of exploiting those interactions to develop therapeutics." One of the obstacles the team was to overcome was finding a way to induce cells to produce adequate quantities of calbindin-D28K for study. Once they had enough a second hurdle remained - many parts of the protein are very similar and so are extremely difficult to distinguish from one another. This is where high-resolution NMR came to the rescue. The NMR solution structure of calbindin-D28K shows it to have a single, globular fold with six distinct helical calcium-binding sub-domains. It is these sub-domains that endow the protein with its calcium ion binding ability. This is the first detailed insight into the workings of this essential protein. "This structure is one of the largest high-resolution NMR structures and the largest monomeric EF-hand protein to be solved to date," the researchers say. NMR does not provide all the answers, however, and Cavanagh will not be satisfied until he has high-resolution structures for the calcium- free structure of the protein, which will be very different. This shape-shifting could provide clues as to the flexibility of the roles calbindin D28K can play in the body. "This could be why the protein plays so many different roles," Cavanagh adds, "Proteins that change shape usually serve as on/off switches, but this protein also grabs calcium and takes it elsewhere. Now we're working to determine the structure of this protein when it has no calcium." Related links: |
.jpg) Cavanagh, unravelling calcium protein with NMR
 Doing a calbindin flip
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