Cells have a fascinating feature of orderly organizing their interiors by using tiny protein machines called molecular motors that generate directed movements. Most of them use a common type of fuel, a type of chemical energy, called ATP to function. Now, researchers from the Max Planck Institute for Molecular Cell Biology and Genetics (MPI-CBG), the Physics of Life Excellence Group (PoL) and the Center for Biotechnology (BIOTEC) at the TU Dresden in Dresden, Germany, and the National Center for Biological Sciences (NCBS) in Bangalore, India, discovered a novel molecular system that uses alternative chemical energy and employs a novel mechanism to perform mechanical work. By repeatedly contracting and expanding, this molecular motor works similar to a classical Stirling engine and helps distribute charge to membrane-bound organelles. It is the first motor that uses two components, two proteins of different sizes, Rab5 and EEA1, and is driven by GTP instead of ATP. The results are published in the journal Physics of Nature.
Motor proteins are remarkable molecular machines within a cell that convert chemical energy, stored in a molecule called ATP, into mechanical work. The most prominent example is myosin, which helps our muscles move. By contrast, GTPases, which are small proteins, have not been seen as molecular force generators. An example is a molecular motor made up of two proteins, EEA1 and Rab5. In 2016, an interdisciplinary team of cell biologists and biophysicists in the groups of MPI-CBG directors Marino Zerial and Stephan Grill and their colleagues, including PoL and BIOTEC research group leader Marcus Jahnel, discovered that the small protein GTPase Rab5 could trigger a contraction in EEA1. These rope-like attachment proteins can recognize and bind to the Rab5 protein present in a vesicle membrane. Binding of the much smaller Rab5 sends a message along the elongated structure of EEA1, increasing its flexibility, similar to how cooking softens spaghetti. Such a change in flexibility produces a force that pulls the vesicle toward the target membrane, where docking and fusion occurs. However, the team also hypothesized that EEA1 could switch between a flexible and rigid state, similar to a mechanical motor movement, simply by interacting with Rab5 alone.
This is where the current research is located, which takes shape through the doctoral work of the first two authors of the study. Joan Antoni Soler of Marino Zerial’s research group at MPI-CBG and Anupam Singh of Shashi Thutupalli’s group, a biophysicist at the Simons Center for the Study of Living Machines at NCBS in Bangalore, set out to experimentally observe this motor in action.
With an experimental design to investigate EEA1 protein dynamics in mind, Anupam Singh spent three months at the MPI-CBG in 2019. “When I met Joan, I explained to her the idea of measuring EEA1 protein dynamics. But these experiments required specific modifications to the protein that would allow its flexibility to be measured based on its structural changes,” says Anupam. Joan Antoni Soler’s background in protein biochemistry was perfect for this challenging task. “I was delighted to learn that the approach to characterize the EEA1 protein could answer whether EEA1 and Rab5 form a two-component motor, as previously suspected. I realized that the difficulties in obtaining the correct molecules could be resolved by modifying the EEA1 protein to allow that the fluorophores adhere to specific regions of the protein.This modification would facilitate the characterization of the protein’s structure and the changes that can occur when it interacts with Rab5”, explains Joan Antoni.
Armed with the right protein molecules and valuable support from co-author Janelle Lauer, a senior postdoctoral researcher in Marino Zerial’s research group, Joan and Anupam were able to characterize EEA1 dynamics thoroughly using the advanced laser scanning microscopes provided by the optical microscopy facilities. at the MPI-CBG and the NCBS. Surprisingly, they found that the EEA1 protein could undergo multiple flexibility transition cycles, from rigid to flexible and back again, driven solely by the chemical energy released by its interaction with GTPase Rab5. These experiments showed that EEA1 and Rab5 form a GTP-driven two-component motor. To interpret the results, Marcus Jahnel, one of the corresponding authors and leader of the PoL and BIOTEC research group, developed a new physical model to describe the coupling between chemical and mechanical steps on the motorcycle. Together with Stephan Grill and Shashi Thutupalli, the biophysicists were also able to calculate the thermodynamic efficiency of the new motor system, which is comparable to conventional ATP-driven motor proteins.
“Our results show that the EEA1 and Rab5 proteins function together as a two-component molecular motor system that can transfer chemical energy into mechanical work. As a result, they may play active mechanistic roles in membrane trafficking. It is possible that force- The generator molecular motor mechanism can be conserved in other molecules and be used by various other cellular compartments,” summarizes the Marino Zerial study. Marcus Jahnel adds: “I’m delighted that we can finally test the idea of an EEA1-Rab5 motor. It’s great to see it confirmed by these new experiments. Most molecular motors use a common type of cellular fuel called ATP. Small GTPases consume another type of fuel, GTP, and have been thought of primarily as signaling molecules. The fact that they can also drive a molecular system to generate forces and move things puts these abundant molecules in an interesting new light.” Stephan Grill is equally enthused: “It’s a new class of molecular motors! This one doesn’t move like the kinesin motor that carries cargo along microtubules, but does the work while staying in place. It’s a bit like the tentacles of an octopus”.
“The model we use is inspired by that of the classic Stirling engine cycle. While the traditional Stirling engine generates mechanical work by expanding and compressing the gas, the described two-component engine uses protein as a work substrate, and flexibility changes of proteins result in force generation. As a result, this type of mechanism opens up new possibilities for the development of synthetic protein motors,” adds Shashi Thutupalli.
Overall, the authors hope that this new interdisciplinary study may open up new avenues of research in both molecular cell biology and biophysics.
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