By KRISTA CONGER

Solving the three-dimensional structure of proteins is a bit like cracking the Mayan code: difficult yet rewarding. Each solution contributes to an overall understanding of how a cell functions. Now a Stanford University Medical Center laboratory has solved the structure of an important family of cellular receptors that eluded scientists for years.

The receptors display an unprecedented way of interacting with natriuretic peptides -- small molecules that play a critical role in regulating fluid volume and blood pressure. Deciphering the receptor’s mechanism will help researchers design more effective drugs to treat high blood pressure and heart and kidney failure, and offer clues as to how other receptors may work to transmit messages from the outside to the inside of a cell.

Receptors -- proteins that straddle the cellular membrane -- recognize and relay outside messages to the cell’s interior, allowing it to nimbly respond to changing external conditions. Each receptor recognizes and binds to only a few specific protein partners, called ligands. Ligands are secreted by cells or tissues in the body to act as molecular messengers.

The researchers -- led by Christopher Garcia, PhD, assistant professor of microbiology and immunology, and structural biology -- found that the natriuretic peptide receptor, or NPR, changes its shape when it binds to its partner protein. When not bound to its ligand, the NPR is held in an inactive conformation by a unique "molecular spring" created by a short, stretchable segment of the protein. This method of activation is unlike another receptor that Garcia’s lab also studies, gp130, which becomes active when ligand binding induces a clustering of two receptor and ligand pairs.

"These two receptors are two new, very different paradigms," said Garcia. The receptors’ structures netted two recent Science papers for the Garcia lab: NPR’s unique structure was published last Friday, and the gp130 receptor structure was published on March 16 of this year.

The three natriuretic peptides that have been identified are short chains of amino acids that bind to NPR family members. They are secreted by the heart and the endothelial cells. They serve to lower blood pressure and reduce fluid volume in the body by signaling the blood vessel walls to dilate and the kidneys to increase the excretion of urine and salts. One of the peptides, BNP, has recently been approved by the Food and Drug Administration to reduce the amount of fluid that builds up around the lungs of patients with congestive heart failure.

The peptides work by binding to and activating members of the NPR family on cells in the arterial walls and kidneys. In the absence of peptide binding, the receptors can exist singly or as multimers on the cell membrane. But until now the structure of the active complex has eluded researchers, who wondered just how many receptors or peptides must fit together in each group to goad the cell into action.

The NPR receptors, unlike many other receptors, cross the cell membrane only once, making it possible to manufacture just the extracellular portion of the receptor to use in ligand binding studies. Postdoctoral researcher Xiao-lin He, PhD, and research associate Dar-chone Chow, PhD, worked together with research assistant Monika Martick to synthesize enough of the protein to crystallize it both with and without the peptide. X-rays generated at the Stanford Synchrotron Radiation Laboratory were then used to determine the three-dimensional structure of the complexes.

The researchers saw that two receptor molecules lean inward to grasp one peptide, which forms a hooplike structure sitting vertically between the two receptors like a coin in a coin slot. In the absence of the peptide, the peptide-binding domains on the receptors are held apart by the tension of a molecular spring -- a segment of the receptor protein that can stretch and contract. Stretching the spring breaks some chemical bonds between the spring and a carbohydrate on the receptor. In the presence of the peptide, the attraction of the receptor to the ligand provides enough energy to break the bonds and allow the conformational change.

"We haven’t seen anything like this spring structure before," said Garcia. "It’s holding the receptor monomer in the inactive conformation." The result also explains why the presence of the carbohydrate group is a necessary component of an active receptor.

Deciphering the cystallographic image was complicated by the fact that the symmetrical pair of receptors was binding to an asymmetrical peptide that could snuggle into the slot in either orientation.

"We were really baffled by this in the beginning," said Garcia. "This has held the field back for many years." To be sure of their results, the researchers checked them with calorimetry, a technique that measures the heat released when macromolecules interact in solution. The calorimetry studies confirmed the crystallographic data: two receptors were interacting with only one monomer in an asymmetrical fashion.

"This result was a surprise and unprecedented, so we verified it by two independent methods," said Garcia. "And the ring structure of the peptide bears no resemblance to what had been predicted." The researchers speculate that the peptide’s structure is a receptor-induced conformational change.

Garcia and others in his laboratory are excited about their novel finds. Each new bit of information about the receptor and its interaction with the natriuretic peptides will help researchers understand how to develop drugs to manipulate their function to treat such diseases as heart failure and high blood pressure. For instance, said Garcia, hyperactivating the receptor with a drug that mimics the peptides has already been shown to effectively reduce the amount of fluid in the body. Conversely, inhibiting the action of this receptor or others like it, such as the bacterial enterotoxin receptor, may prove therapeutic for other diseases. Bacterial enterotoxins cause diarrheal disease, a worldwide human health problem.

Now the team has turned its attention to determining how the natriuretic signal is amplified in the interior of the cell. Lena Brevnova, PhD, in the lab is also working to further clarify the interactions between the gp130 receptor and its numerous ligands.