Scientists at the Salk Institute for Biological Studies have used artificial amino acids to reengineer proteins and to determine the detailed molecular structure of a key cellular switch and its ligand. The switch, corticotrophin releasing factor type 1 (CRF1R), belongs to a class of cellular receptors whose structures historically have been hard to determine. These receptors regulate processes throughout the body and are involved in many diseases.

The researchers believe their new approach to mapping how proteins interact with each other could aid in the design of new drugs for diseases such as diabetes and osteoporosis.

The team published their paper (“Genetically Encoded Chemical Probes in Cells Reveal the Binding Path of Urocortin-I to CRF Class B GPCR”) in Cell online.

“The data [we obtained] were analyzed in the context of the recently resolved crystal structure of CRF1R transmembrane domain and existing extracellular domain structures, yielding a complete conformational model for the peptide-receptor complex,” wrote the investigators. “Structural features of the receptor-ligand complex yield molecular insights on the mechanism of receptor activation and the basis for discrimination between agonist and antagonist function.”

“Only when you know how the ligand binds to the receptor can you design drugs that target these processes,” said senior study author Lei Wang, Ph.D., an associate professor in Salk’s Jack H. Skirball Center for Chemical Biology.

Typically, researchers determine the three-dimensional arrangement of atoms in a protein molecule by crystalizing the protein and measuring how x-rays diffract off the crystals. But the receptor class the Salk scientists studied—known as class B GPCRs—are tricky to coax into crystal form, since they are only stable when embedded in the cellular membranes that enclose a cell’s cytoplasm and nucleus.

As a result, getting a complete picture of their structure, let alone the structure of the receptor combined with its bound ligand, hasn’t been possible.

Dr. Wang’s group turned to a new approach to try and figure out what CRF1R’s binding pocket looked like. Using genetic engineering, the scientists added a unique new amino acid, one of the building blocks of proteins, to spots all along CRF1R.

“When you shine light on this artificial amino acid, it grabs nearby molecules,” explained Irene Coin, Ph.D., a postdoctoral fellow in Dr. Wang’s lab team. “It’s like a sticky hand.”

When the artificial amino acid, Azi, was added to any spot where the CFR1R ligand attached to the receptor, the sticky hand grabbed the ligand, which was urocortin-1, and kept it bound to the receptor. If Azi was integrated into a place where urocortin-1 didn’t associate, however, it would have nothing to grab.

A second probe, which was more selective than the “sticky hand” in the receptor, was then used. This time, the probe would only capture one particular amino acid (cysteine).

“We inserted cysteines along the ligand to figure out which parts of the receptor were close to precise spots of the ligand,” said Dr. Wang. It took more than a hundred different combinations to get a perfect match, where the artificial amino acids in the receptor lined up with the cysteines in the urocortin-1.

When they know which amino acids in a ligand interact with which amino acids in a receptor, scientists can begin designing ways to block the interaction between the pair, by creating new molecules that attach to the receptor, for instance. So Wang sees the new structure—and the new approach for determining it—as a key step forward for designing drugs that target class B GPCRs.

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