Molecular motors are complex devices made up of many different parts that consume energy to perform various cellular activities. In short, molecular machines transform energy into useful work. Understanding the mechanical aspects underlying these engines begins with generating a detailed description of their overall architecture and atomic organization. However, to uncover the central mechanisms that power these engines, it is essential to decode all of the molecular dynamics in atomic detail.
Now the research team of Thomas C. Marlovits from the Center for Structural Systems Biology CSSB at DESY and the University Medical Center Hamburg-Eppendorf (UKE) in Hamburg reveals the architecture, complete functional cycle and mechanism of such molecular motor: They report in the journal Nature how a “RuvAB branch migration complex” converts chemical energy into mechanical work to effect DNA recombination and repair. DNA recombination is one of the most fundamental biological processes in living organisms. It is the process by which chromosomes “swap” DNA either to generate genetic diversity, by creating new offspring, or to maintain genetic integrity, by repairing breaks in existing chromosomes. During DNA recombination, four arms of DNA separate from their double helix formations and come together at an intersection known as the Holliday junction. Here, the DNA arms exchange strands in a process called active branch migration.
The essential energy needed for this branch migration comes from a molecular machinery that scientists have identified as the RuvAB branch migration complex. This complex assembles around the Holliday junction and is composed of two motors labeled RuvB AAA+ ATPases, which power the reaction, and a RuvA stator. The research team has now provided a complex blueprint that explains how the RuvB AAA+ motors work under the regulation of the RuvA protein to effect synchronized DNA movement.
The migrations of the active branches energized by the RuvB AAA+ motor molecule are very fast and very dynamic. To determine the different stages of this process, the scientists used time-resolved cryo-electron microscopy to observe the engine machinery idling. “We basically fed the RuvB AAA+ engine a slower-burning fuel that allowed us to capture the biochemical reactions as they happened,” says Marlovits.
The scientist captured more than ten million images of the motor machinery interacting with the Holliday junction. Jiri Wald (CSSB, UKE and member of the Vienna BioCenter PhD Program), the first author of the paper, sifted through the immense amount of data and carefully classified the subtle changes occurring in each image. Using DESY’s high-performance computing facility, scientists were then able to put all the pieces of the puzzle together to generate a high-resolution movie detailing how the RuvAB complex works at the molecular level.
“We were able to visualize seven distinct engine states and demonstrate how the interconnected elements work together in a cyclical fashion,” says Wald. “We also demonstrated that the RuvB motor converts energy into a lever motion that generates the force that drives branch migration. We were amazed by the finding that the motors use a basic lever mechanism to move the substrate Overall, the sequential mechanism, coordination and way of generating force of the RuvAB engine shares conceptual similarities with combustion engines.”
AAA+ motors are often used in other biological systems, such as protein transport. Therefore, this detailed model of the RuvB AAA+ motor can be used as a template for similar molecular motors. “We understand how the engine works and now we can put that engine into another system with some minor adaptations,” says Marlovits. “We are essentially presenting the basic principles of AAA+ engines.”
Future work by the Marlovits group will explore ways to interfere with the operation of AAA+ engines. This could serve as the basis for the development of a new generation of drugs, which would disrupt the mechanisms of such a motor in pathogens and thus stop the spread of infection. “We are excited to explore the possibilities that exist now that we have a blueprint for the RuvB AAA+ engine,” notes Wald.
Scientists from CSSB, UKE, Molecular Biotechnology Institute, Molecular Pathology Research Institute, both in Vienna, Austria, and DESY contributed to this research.
CSSB is a joint initiative of ten research partners from northern Germany, including three universities and six research institutes, dedicated to research on infection biology.
DESY is one of the world’s leading particle accelerator centers and studies the structure and function of matter – from the interaction of tiny elementary particles and the behavior of new nanomaterials and vital biomolecules to the great mysteries of the universe. . The particle accelerators and detectors that DESY develops and builds at its sites in Hamburg and Zeuthen are unique research tools. They generate the most intense X-radiation in the world, accelerating particles to record energies and opening new windows on the universe. DESY is a member of the Helmholtz Association, Germany’s largest scientific association, and receives its funding from the German Federal Ministry of Education and Research (BMBF) (90%) and the German federal states of Hamburg and Brandenburg (10%).