Flipping a nano-scale molecular switch may regulate the cell-binding function of a protein involved in healing and other fundamental biological activities. Computer simulations show that, like untying a shoelace, tugging on a strand of the protein fibronectin unravels a loop critical to cell recognition but otherwise leaves the protein intact for reactivation.
Reporting in the Feb. 16 issue of the Proceedings of the National Academy of Sciences, researchers at the University of Washington and the University of Illinois at Urbana-Champaign describe this as the first illustration of how the body may use tension-activated switch mechanisms to regulate biological function.
"Understanding how nature has evolved these systems gives us insight into basic cell biology as well as elegant design principles for mechanical switches in biotechnology devices," says senior author Viola Vogel, associate professor of bioengineering and director of the Center for Nanotechnology at the UW. "Since nano-scale tools have only recently been developed for analyzing the mechanics of single molecules, we are entering a new era of understanding how molecular mechanics control biological activity."
Vogel's research, funded by the National Institutes of Health, is particularly interested in fibronectin, a glycoprotein that is a major building block in the cell-surface network known as the extracellular matrix. The extracellular matrix regulates adhesion, communication, gene expression and other interactions between the cell and its environment. Thus, it is of keen interest to scientists studying basic cell biology as well as engineers designing artificial devices to integrate naturally into the body.
Fibronectin is comprised of a chain of repeating modules, only one of which contains the specific RGD tri-peptide loop responsible for cell adhesion. This module - labeled FnIII10 - consists of seven connected beta strands folded back and forth in layers. The cell-binding RGD loop extends above the surface of the molecule from its slot between the last two strands of the module. Curious about why nature had devised such a large and complex molecule to perform a function seemingly controlled by this relatively tiny loop, Vogel sought to unravel its internal mechanical operations.
X-ray diffraction analysis reveals the 3-D structure of individual molecules in exquisite detail. And researchers using atomic force microscopy in concert with optical tweezers can measure the forces required to pull molecules apart. But to discover what is happening inside the molecule as it unfolds, Vogel turned to physics professor Klaus Schulten at the University of Illinois's Beckman Institute for Advanced Science and Technology, who pioneered a set of computational approaches for simulating molecular dynamics. With a 3-D blueprint of a molecule, Schulten's simulations produce detailed pictures of a molecule's structural response to external forces.
Vogel working with Schulten, his graduate student Hui Lu and post-doctoral fellow Barry Isralewitz, simulated the forced unfolding of the muscle protein titin. Results mirrored the forces required to pull apart titin in laboratory experiments, thus verifying the accuracy of the simulation.
Next, the researchers turned their attention to Fibronectin. Vogel's graduate student Andre Krammer essentially instructed the simulation software to tug on the ends of the FnIII10 module. The researchers observed that the module stretched but remained intact until the tension reached a critical threshold. At that point, the bottom strand of the module broke free from the bonds holding it in place and pulled the RGD loop toward the interior of the molecule as it unraveled and straightened out. This suggests that, with the loop no longer exposed, Fibronectin's affinity for cell-binding is effectively switched off. Furthermore, unless additional tension was applied, the module otherwise remained intact during the simulation and, thus, is able to rapidly reassemble and restore its cell-binding affinity.
"Our experiment provides the first atomic-scale description of a tension-activated switch for regulating a protein's cell-binding affinity," Vogel says. "This gives totally new insight into how nature may utilize tension to enable a cell to detach itself from the extracellular matrix and move on. The regulation of cell motility is important in, among other things, the development of embryos, wound healing and metastasis of cancer cells.
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