3D Structure of Twinkle Protein Solved for the First Time

Researchers at the National Institutes of Health have developed a three-dimensional structure that allows them to see how and where disease mutations on the Twinkle protein cause mitochondrial disease. This protein is involved in helping cells use the energy that our bodies convert from food. Until this 3D structure is developed, researchers have only models and cannot determine how these mutations cause disease. Mitochondrial diseases are a group of inherited diseases that affect 1 in 5,000 people and have few treatments.

"For the first time, we can map mutations that cause these devastating diseases," said lead author Dr. Amanda A. Riccio, a researcher at the Mitochondrial DNA Replication Group at the National Institute for Environmental Health Sciences (NIEHS), part of the NIH. "Clinicians can now see where these mutations are located and can use this information to help identify the cause and help families make choices, including deciding to have more children."

The new findings are particularly important for the development of targeted therapies for patients with mitochondrial diseases, such as progressive external eye paralysis, which leads to loss of muscle function associated with eye and eyelid movements; Penot syndrome, a rare genetic disorder that may lead to hearing loss; hereditary neurological diseases—infantile-onset spinocerebellar ataxia; and hepato-cerebral mitochondrial DNA (mtDNA) failure syndrome, a genetic disorder that can lead to liver failure and neurological complications in infants.

This study shows how NIEHS researchers were the first to accurately map clinically relevant variants of Twinkle helicase, an enzyme that unwinds the double helix structure of mitochondrial DNA. The Twinkle structure and all coordinates are now freely available to all researchers at the Protein Data Bank in open data.

"Researchers have not figured out the structure of Twinkle for many years. This is a very difficult protein to process," said Dr. William C. Copeland, head of the mitochondrial DNA replication group and corresponding author of the paper. "By stabilizing proteins and using the best equipment in the world, we were able to construct the last deleted fraction of the human mitochondrial DNA replicon."

The researchers used cryo-electron microscopy (Cryo-EM) to allow them to see the complex structure of the interior of the protein and hundreds of amino acid or amino acid residues, and how they interact.

Mitochondria, which are responsible for energy production, are especially prone to mutations. Mutations in mtDNA disrupt their ability to efficiently produce energy for cells. Unlike other specialized structures in cells, mitochondria have their own DNA. In the nucleus, there are two copies of each chromosome, but in mitochondria there may be thousands of copies of mtDNA. Having a large number of mitochondrial chromosomes allows cells to tolerate some mutations, but accumulating too many copies of the mutation can lead to mitochondrial disease.

For the study, the researchers used a clinical mutation, W315L, to resolve this structure, which is known to cause progressive external ophthalmoplegia. Using Cryo-EM, they were able to observe thousands of protein particles appearing in different orientations. The final structure shows the circular arrangement of the polyprotein. They also used mass spectrometry to verify the structure, followed by computer simulations to understand why mutations cause disease.

In the blink of an eye, they were able to map up to 25 disease-causing mutations. They found that many of the disease mutations are located right at the junction of two protein subunits, suggesting that mutations in this region weaken the subunits' interactions so that the helicase cannot function.

"The arrangement of Twinkle is much like a puzzle. Clinical mutations may change the shape of Twinkle fragments, which may no longer fit together properly for the intended function," explained Riccio.

"The beauty of Dr. Riccio and his team's work is that this structure allows you to see so many disease mutations clustered in one place," said author Matthew J. Longley, Ph.D., a NIEHS investigator. "It is very rare for a paper to explain so many clinical mutations. Thanks to this work, we are one step closer to having information that can be used to develop treatments for these debilitating diseases."

Randi Warren from Creative Biostructure.