Cornell researchers have uncovered a built-in molecular "gate" that controls the production of the molecule nitric oxide, a crucial signaling molecule throughout biology that in humans helps regulate blood pressure, brain signaling, and immune defenses. But when levels go unchecked, it can damage cells and disrupt normal signaling.
How cells manage that balance, knowing when to make nitric oxide and when to shut it down, has long remained unclear. In a study published Feb. 20 in Science Advances, Cornell researchers revealed, for the first time, how a complex nitric oxide-producing enzyme physically rearranges itself to control that balance.
"We discovered a previously unknown, built-in calcium-sensing mechanism that acts like a movable gate, redirecting electrons either toward nitric oxide production or toward nitric oxide breakdown," said corresponding author Brian Crane, director of the Weill Institute for Cell and Molecular Biology and the George W. and Grace L. Todd Professor in the Department of Chemistry and Chemical Biology in the College of Arts and Sciences.
According to Crane, scientists have known for decades that the enzyme that produces nitric oxide, nitric oxide synthase (NOS), is regulated by calcium and undergoes large structural motions, but exactly how those movements controlled nitric oxide production was unclear.
"Our central question was how electron flow is physically gated to turn nitric oxide production on or off," said Dhruva Ajit Nair, Ph.D. student within the Crane Lab and first author on the study.
That question proved difficult to answer because NOS is highly dynamic and constantly moving at the atomic level, while traditional imaging methods work best on proteins that sit still in one shape.
To overcome that challenge, the researchers combined cryo-electron microscopy (cryo-EM) with small-angle X-ray scattering, used to determine the size, shape, structure, and distribution of nano-scale particles, allowing them to visualize not just one structure, but an ensemble of shapes the enzyme adopts as it works.
"Our results show how a large, flexible enzyme changes its shape to control when and where the different types of chemistry occur," Nair said. "For the first time, we directly visualized all the domains that are configured and how a calcium-responsive protein component moves across the larger enzyme."
That movement acts as a gate for electron flow, Nair said, blocking electron transfer needed to make nitric oxide in one position and, in another position, blocking a separate active site involved in nitric oxide breakdown. Calcium levels determine which path the electrons take, effectively switching the enzyme's function.
"This changed our understanding of nitric oxide enzymes from simple on/off switches to dynamic systems that redistribute electrons depending on cellular conditions," Crane said.
The discovery has important implications for human health. Abnormal nitric oxide production has been linked to cardiovascular disease, neurodegeneration and autoimmune disorders.
The newly discovered gate suggests a new, and potentially more precise, alternative to current therapeutic strategies involving protein-based binders that mimic calcium-responsive domains to better regulate nitric oxide levels.
By revealing how protein motion and gating, not just chemistry, controls nitric oxide production, the work provides a new framework for understanding the regulation of one of biology's most important signaling molecules, Crane said.
The research was supported by a grant from the National Science Foundation. The team also drew on specialized facilities including cryo-electron microscopy resources at the National Center for CryoEM Access and Training and Cornell Center for Materials Research, mass spectrometry support from Cornell's Proteomics and Metabolomics Facility, and high-energy X-ray capabilities at the Cornell High Energy Synchrotron Source (CHESS).
Stephen D'Angelo is the communications manager for biological systems at Cornell Research and Innovation.
