Salk scientists crack riddle of important drug target

Salk scientists crack riddle of important drug target

New method for determining structure of key cellular receptors could speed drug development

LA JOLLA, CA—A new approach to mapping how proteins interact with each other, developed at the Salk Institute for Biological Studies, could aid in the design of new drugs for diseases such as 糖尿病 and osteoporosis. By reengineering proteins using artificial amino acids, the Salk scientists determine the detailed molecular structure of a cellular
switch and its ligand, the molecule that turns it on. The switch—corticotrophin releasing factor type 1
(CRF1R)—belongs to a class of cellular receptors whose structures are notoriously hard to determine. These receptors regulate processes throughout the body and are involved in many diseases.

“Only when you know how the ligand binds to the receptor can you design drugs that target these processes,” says senior study author Lei Wang, an associate professor in Salk’s 杰克 H. 斯基尔鲍尔化学与蛋白质组学中心 and holder of the Frederick B. Rentschler Developmental Chair. Wang and his team describe the new structure and method in a 细胞 paper published online November 27, 2013.

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 G-protein coupled receptors (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.

Wang’s team turned to a new approach to try and figure out what CRF1R’s binding pocket—the area where the ligand attaches—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,” explains Irene Coin, a postdoctoral fellow in Wang’s 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, a molecule called 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. By detecting whether CFR1R and urocortin-1 become irreversibly attached, the researchers would know whether the Azi had been integrated into part of the binding pocket or not.

Repeating this technique throughout the CFR1R molecule revealed that the receptor’s binding pocket consisted of at least 35 amino acids. But that didn’t give the researchers the full picture they wanted of the interaction.

“This first, sticky hand probe had given us information about the shape of the binding pocket,” says Wang. “But we still didn’t know how the ligand is oriented inside that pocket.”

So they used a second probe—one which was more selective than the “sticky hand” in the receptor. 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”, says 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.

“We discovered that the ligand lies in the receptor’s binding pocket like a too-tall person in a bathtub,” Coin says. “One end of the ligand is like the head sticking out, and on the other end, the feet are dangling out.”

“This shape makes sense in light of previous data,” says Wang. “Because we already knew that you can add a lot of molecules onto the feet of the ligand and it doesn’t affect the receptor-ligand interaction.”

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.

“The beauty of this method is that it can be a general method to map other binding pockets and protein interactions as well,” Wang adds. “Its use isn’t limited to only GPCRs.”

Other researchers on the paper were Tingting Sun and Zheng Xiang of the Salk Institute, Vsevolod Katritch, Fai Yiu Siu, and Raymond C. Stevens of the Scripps Research Institute, and Michael Beyermann of the Leibniz-Institute of Molecular Pharmacology.

该项工作获得了以下机构的资助： 美国国立卫生研究院, ， 那个 California Institute of Regenerative Medicine and a Marie Curie fellowship from the European Commission.

关于索尔克生物研究所：

索尔克生物研究所是世界顶尖的基础研究机构之一，其国际知名的教职人员在一个独特、协作和富有创造性的环境中，深入探究生命科学的基本问题。索尔克科学家们致力于发现和指导未来几代研究人员，通过研究神经科学、遗传学、细胞和植物生物学以及相关学科，在癌症、衰老、阿尔茨海默氏症、糖尿病和传染病的认识方面做出了开创性的贡献。.

学院取得了许多成就，获得了包括诺贝尔奖和美国国家科学院院士在内的无数荣誉。该研究所由脊髓灰质炎疫苗先驱 Jonas Salk 博士于 1960 年创立，是一家独立的非营利组织和建筑地标。.