Dissecting mysterin or the power of cryo-EM
Moyamoya disease, a blood vessel disorder that often leads to strokes, is associated with mutations in the enzyme RNF213, a ubiquitin ligase also known as “mysterin”. Researchers at the IMP provide the first detailed 3D structure of the respective mouse protein – a gigantic molecule – using cryo electron microscopy. Their findings were published in eLife on 23 June 2020.
Seeing strokes in children of elementary school age seems to go against all clinical experience. Yet, a brain stroke is usually the first symptom in a disease that most commonly affects young adults and children and is known as Moyamoya. The Japanese expression can be roughly translated as a “puff of smoke” and describes the appearance of tiny and fragile blood vessels that have been formed to circumvent a constricted artery in the brain. Such narrowed arteries, which often result in thrombosis, are a characteristic feature of Moyamoya disease. More than 50,000 individuals suffer from the disorder, which most frequently occurs in East Asia.
The exact causes of the disease are not clear, but the protein RNF213 seems to play a major role, since most patients have mutations in their RNF213 gene. In total, more than 16 million people in East Asia (over 2% of the population) are believed to carry such a mutation, and although they may not develop Moyamoya they could pass on the predisposition. It is therefore highly relevant for the medical community to find out more about the RNF213 protein and its role in vascular disease.
A protein of gigantic size
Previous studies had suggested that the enzyme functions as a metabolic gatekeeper. It was linked to various cellular processes including hypoxia, lipid metabolism, and the formation of blood vessels. But despite this broad biological relevance, it was not at all clear what molecular function RNF213 performs or how it is associated with the Moyamoya disease.
Studying RNF213 is tricky. It is a molecule of gigantic size, larger than 99.9 percent of all known proteins, and exceedingly complex. Until recently, no structural or biochemical data were available on which a detailed functional characterisation could have been built. Combining the expertise of two structural biology labs and tapping into the resources of advanced scientific infrastructure, researchers at the IMP are now able to fill this gap. They applied an integrative biochemical and structural approach to reconstitute RNF213, address its structure and mechanism and analyse the molecular basis of its disease-causing mutations.
A powerless motor as a powerful switch
In their current publication in eLife, the team around Tim Clausen and David Haselbach describe the 3D molecular structure of mouse RNF213 that is composed of three major parts – an elongated scaffold resembling a molecular arm, a central core formed by a ring of ATPase units and an E3 ligase module for tagging client proteins with a ubiquitin modifier. Combining a ubiquitin-marking entity with an ATPase motor in a single protein is unique, making RNF213 a special factor in the quality-control machinery maintaining cellular homeostasis. Against all odds, and to everyone’s surprise, the cryo-EM analysis revealed that the RNF213’s ATPase core displays similarity to the motor protein dynein. However, in contrast to the powerful dynein transporter, the ATP-burning motor of RNF213 is weak. The ATPase engine is thus strongly tuned down such that it seems to function as a molecular on/off switch, presumably keeping the ubiquitin ligase under control.
A new category of a ubiquitination protein
Another surprising result of the study is the realisation that the E3 module of RNF213 represents a new type of ubiquitination enzyme. The roughly six hundred E3 ligases that are known in humans can be grouped into three different classes, depending on how ubiquitin is transferred to client proteins. RNF213 does not display structural similarity to any of them. The authors therefore propose it as the founding member of a new, distinct class of E3 ligases. “With so much structural knowledge already available, it is becoming increasingly unlikely to discover a new protein fold”, says David Haselbach. “We are therefore very excited and lucky to be able to describe a completely new category of proteins.”
When looking at the disease relevance of their findings, the authors were able to show that the founder Moyamoya mutation neither damages the structure, nor the ATPase or E3 activities of RNF213. They conclude that the disease-causing mutation may hinder interactions with partner proteins in the cell, rather than disrupting itself.
In sum, the data show a new kind of an E3 protein, broadening our understanding of how ubiquitin signalling in the cell works. The authors provide the first detailed molecular description of RNF213, revealing the overall architecture of the giant E3 ligase in atomic detail. This structural framework will pave the way to further investigations of the role RNF213 plays in Moyamoya, metabolism, and otherwise in health and disease. Tim Clausen adds “As RNF213 is only one of the many mysterins in the cell, we hope that our work may stimulate structural studies of other obscure yet fascinating proteins. It was great fun, as it was an amazing team effort to dissect the mystic RNF213 giant and, ultimately, open up a new chapter in ubiquitin biology.”
Illustration:
The Cryo-EM structure of RNF213 reveals its organisation with a ring-shaped part in the middle containing an ATPase motor and the E3 enzyme module at one end (Image by Juraj Ahel, IMP).
Original Publication:
Juraj Ahel, Anita Lehner, Antonia Vogel, Alexander Schleiffer, Anton Meinhart, David Haselbach and Tim Clausen. Moyamoya disease factor RNF213 is a giant E3 ligase with a dynein-like core and a distinct ubiquitin-transfer mechanism.
eLife 2020;9:e56185 doi: 10.7554/eLife.56185