SAN DIEGO—Researchers who study the primary causes of Parkinson’s disease have been focusing on mutations of the protein known as leucine-rich repeat kinase 2 (LRRK2). But understanding how LRRK2 disrupts normal functioning has been difficult, due to a lack of information on the protein’s structure. Since LRRK2 is a major drug target, efforts to decipher LRRK2’s architecture have gone as far as launching samples into space, as a way of using microgravity conditions to help crystalize protein samples — all without success.
But now, researchers are beginning to get a peek inside. Using leading-edge technologies, University of California, San Diego scientists have produced the first visualizations of LRRK2 inside its natural cellular environment, and the first high-resolution blueprint of the protein. They used these depictions to describe how LRRK2 binds to microtubules, and acts as a roadblock for molecular motors that move along the microtubules. The findings are described in two papers published in Cell and Nature.
“These two papers take giant steps towards developing more effective therapeutics for Parkinson’s disease, which impacts so many lives,” said Kit Pogliano, Ph.D., dean of Biological Sciences and professor of Molecular Biology. “Combining cryo-electron microscopy with live cell imaging allows researchers to see proteins working inside cells and to more rapidly determine how potential drugs affect their function. This will accelerate drug discovery and provide new hope to those suffering from this debilitating disease.”
As described in the Cellpaper, UC San Diego researchers led by Elizabeth Villa, Ph.D., and her colleagues used cryo-electron tomography (cryo-ET), a modality of cryo-electron microscopy (cryo-EM), to view LRRK2 in its natural environment within cells. Villa’s team imaged frozen cells that contained the molecules under study, taking pictures at different angles — somewhat like a CAT scan. They were able to describe its structure at a level previously unseen.
“We lifted the hood to look inside the cell at frozen molecules caught in the act of interacting with each other in different places in the cell. We used electron and ion beams like a light saber to blast away parts of the cell. In the middle we left a window that contains the molecules that we are interested in looking at,” noted Villa.
The researchers also used light microscopy to find the molecules within cells, and advanced computational modeling tools to develop a high-resolution integrative model of a mutant LRRK2. Their data revealed that LRRK2 bound to microtubules and predicted that LRRK2’s kinase resembled an “overdrive” state known to occur in Parkinson’s.
“This combination of powerful new techniques was applied for the first time in this research and made it possible to get the first glimpses into the structure of a mutant LRRK2, with the added benefit of its cellular context,” Villa added. “To our knowledge it’s the highest resolution structure of a human protein that was determined inside cells before using conventional biochemical tools. We are bringing structure to cell biology.”
In order to understand how LRRK2 works at a chemical level and to design therapeutics, an even higher resolution structure was required to reveal the position of atoms and how they interact with potential drugs. In the Naturestudy, co-senior authors Samara Reck-Peterson, Ph.D., and Andres Leschziner, Ph.D., took a deeper look at LRRK2’s structure and function. They also teamed up with Villa’s group to determine how LRRK2 interacts with microtubules.
Using cryo-EM, Leschziner’s team captured a detailed image of LRRK2’s structure, down to the atomic level. Stefan Knapp, a professor at Goethe University in Frankfurt, Germany, and his group were also instrumental: they determined how to make LRRK2 tractable for structural work. The structure comprises the business end of the protein — which includes the part that tags other proteins with phosphates. The locations of all major Parkinson’s disease-causing mutations are found in their structure.
Next, the Leschziner group combined their structure with Villa’s and created a model that explains how LRRK2 binds to microtubules.
“You can think of the kinase part of LRRK2 as a bit like Pacman; it can be either open or closed,” said Leschziner. “Our modeling suggested that, when bound to microtubules, the kinase needs to be in a closed state, indicating that the shape of the kinase may regulate the binding of LRRK2 to microtubules. Teaming up with the Reck-Peterson lab, we decided to test this model directly.”
Reck-Peterson and her team are interested in the molecular motors that transport cargo along microtubules, and how defects in this transport cause human neurodevelopmental and neurodegenerative diseases. The researchers theorized that LRRK2’s interaction with microtubules might be detrimental for the molecular machines that move on them and carry essential cargos from place to place in cells.
Her team discovered that LRRK2 does create roadblocks that stop these molecular machines. Researchers also showed that some drugs that target LRRK2’s kinase can enhance this effect, while others diminish it.
Leschziner and Reck-Peterson are not yet sure if these roadblocks play a role in Parkinson's disease, but their findings already have implications for the design of drugs that work by inhibiting LRRK2. This work points to the possibility that kinase inhibitors that close LRRK2’s kinase domain might lead to the unwanted effect of blocking the molecular motors’ movement.
“It's not yet clear what role LRRK2-microtubule binding plays in Parkinson’s disease,” pointed out Reck-Peterson. “But what we have now are cellular and molecular blueprints, and that's what is needed to figure out what LRRK2 does and to fine-tune therapeutic drugs that target LRRK2.”
