Scientists at the NIH say they have developed a three-dimensional (3D) structure that allows them to see how and where disease mutations on the twinkle protein, which is involved in helping cells use energy our bodies convert from food, can lead to treatments for mitochondrial diseases.

Prior to the development of this 3D structure, researchers only had models and were unable to determine how these mutations contribute to disease. Mitochondrial diseases are a group of inherited conditions that affect 1 in 5,000 people and have very few treatments.

The NIH study “Structural insight and characterization of human Twinkle helicase in mitochondrial disease” appears in PNAS.

“Twinkle is the mammalian helicase vital for replication and integrity of mitochondrial DNA. Over 90 Twinkle helicase disease variants have been linked to progressive external ophthalmoplegia and ataxia neuropathies among other mitochondrial diseases. Despite the biological and clinical importance, Twinkle represents the only remaining component of the human minimal mitochondrial replisome that has yet to be structurally characterized,” write the investigators.

“Here, we present 3-dimensional structures of human Twinkle W315L. Employing cryo-electron microscopy (cryo-EM), we characterize the oligomeric assemblies of human full-length Twinkle W315L, define its multimeric interface, and map clinical variants associated with Twinkle in inherited mitochondrial disease. Cryo-EM, crosslinking-mass spectrometry, and molecular dynamics simulations provide insight into the dynamic movement and molecular consequences of the W315L clinical variant.

“Collectively, this ensemble of structures outlines a framework for studying Twinkle function in mitochondrial DNA replication and associated disease states.”

“For the first time, we can map the mutations that are causing a number of these devastating diseases,” said lead author Amanda A. Riccio, PhD, and researcher in the National Institute of Environmental Health Sciences (NIEHS) Mitochondrial DNA Replication Group. “Clinicians can now see where these mutations lie and can use this information to help pinpoint causes and help families make choices, including decisions about having more children.”

Findings may lead to targeted treatments

The new findings will be particularly relevant for developing targeted treatments for patients who suffer from mitochondrial diseases such as progressive external ophthalmoplegia, a condition that can lead to loss of muscle functions involved in eye and eyelid movement, Perrault syndrome, a rare genetic disorder that can cause hearing loss, infantile-onset spinocerebellar ataxia, a hereditary neurological disorder, and hepatocerebral mitochondrial DNA (mtDNA) depletion syndrome, a hereditary disease that can lead to liver failure and neurological complications during infancy.

The NIH paper describes how the NIEHS researchers were the first to accurately map clinically relevant variants in the twinkle helicase, which unwinds the mitochondrial double helix. The twinkle structure and all the coordinates are now available in the open data Protein Data Bank that is freely available to all researchers.

“The structure of twinkle has eluded researchers for many years. It’s a difficult protein to work with,” noted William C. Copeland, PhD, who leads the Mitochondrial DNA Replication Group and is the corresponding author on the paper. “By stabilizing the protein and using the best equipment in the world we were able to build the last missing piece for the human mitochondrial DNA replisome.”

The researchers used cryo-electron microscopy (CryoEM), which allowed them to see inside the protein and the intricate structures of hundreds of amino acids or residues and how they interact. The final structure shows a multi-protein circular arrangement. They also used mass spectrometry to verify the structure and then did computer simulations to understand why the mutation results in disease.

Within twinkle, the team was able to map up to 25 disease-causing mutations. They found that many of these disease mutations map right at the junction of two protein subunits, suggesting that mutations in this region would weaken how the subunits interact and make the helicase unable to function.

“The arrangement of twinkle is a lot like a puzzle. A clinical mutation can change the shape of the twinkle pieces, and they may no longer fit together properly to carry out the intended function,” Riccio explained.

“What is so beautiful about Riccio and the team’s work is that the structure allows you to see so many of these disease mutations assembled in one place,” said Matthew J. Longley, PhD, an author and NIEHS researcher. “It is  unusual to see one paper that explains 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.”

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