"The active site looks nothing like any other kinase active site it's a unique architecture," Sanders said.
The researchers also performed exhaustive mutagenesis studies in which they characterized mutations at each amino acid in DAGK and used the data to map the active site of the enzyme onto the structure. They identified two sets of mutations that resulted in non-functional DAGK. One set altered the active site so that it no longer did its job, and the second set caused the protein to fold incorrectly (misfolding).
Sanders said the team was surprised to find that nearly all of the mutations that caused misfolding were in the active site. The expectation, he explained, is that mutations in the active site would cause a loss of function but would not usually affect protein folding, whereas key residues for folding would be located elsewhere in the protein to underpin the scaffold for the active site.
"Our study shows that you can't make that assumption," he said.
Sanders cautions that investigators cannot simply predict the impact of a mutation based on it being located in the active site. The finding has implications for personalized medicine, which aims to use the predicted impact of disease-causing mutations to make therapy decisions.
"The therapeutic strategy for addressing catastrophic misfolding versus simple loss of function may be very different," Sanders said.
Sanders and his team, who got interested in protein folding because of their work with DAGK, are now pursuing structural studies of misfolded membrane proteins that cause diseases including peripheral neuropathy (Charcot-Marie-Tooth Disease), diabetes insipidus and Alzheimer's disease.
"For proteins that misfold because of mutations, we're using NMR tools to understand exactly what the mutations do to the proteins in terms of structure and st
|Contact: Leigh MacMillan|
Vanderbilt University Medical Center