Team may have found key to cell response

May 29, 1992

By Lynn Yarris, [email protected]

A simple scissors-type motion that changes the orientation of one small clump of atoms relative to another may be the key to how living cells and microorganisms sense and respond to their environment, according to a recent study by a team of LBL and UC Berkeley researchers.

The team has made the first determinations of the three- dimensional structures of the sensing region or "domain" of a special type of protein -- called a "transmembrane receptor" -- that receives information from the environment outside the cell and transmits it inside the cell for processing.

Sung Hou Kim, director of LBL's Structural Biology Division (formerly Chemical Biodynamics) and professor of chemistry with UC Berkeley, working with Daniel Koshland Jr., of UCB's Biochemistry Department, and other collaborators, used x-ray crystallography to study the structure of a receptor on a bacterium "Salmonella typhimurium") that detects the presence of an amino acid, "aspartate," which is as an essential nutrient for the bacterium.

"The receptor perceives the presence of aspartate and signals the cell to move toward an increasing concentration of the nutrient," says Kim. "We have determined the three-dimensional structure of the aspartate-binding region of the receptor both with and without the aspartate attached."

Knowledge of the structures of the two forms of the receptor is not only a major step towards understanding the mechanisms of transmembrane signalling, Kim says, but also gives helpful information for the development of "target-seeking" microorganisms that can be used to detect certain toxic chemicals and to treat diseases.

Although it is believed that in most cases, transmembrane receptor information is transferred from the outside of the cell to the inside of the cell through conformational changes in the protein, the mechanism behind this signalling process has been obscure in part because it is difficult to crystallize any transmembrane receptor.

The aspartate receptor, which has been extensively studied, is a member of a large family of bacterial protein receptors and is similar to mammalian receptors for such things as low-density lipoproteins, insulin, and both epidermal and platelet-derived growth factors.

After crystallizing the sensing domain of aspartate receptors, the LBL-UCB researchers had the material irradiated with x-ray beams at the Photon Factory in Tsukuba, Japan, and the Stanford Synchrotron Radiation Laboratory. Through computer analysis of the x-ray data, three-dimensional structures of the receptor's sensing domain were obtained at a resolution of 2.0 angstroms. (One angstrom is about four billionths of an inch.) These structures were then used to construct a molecular model of the entire receptor complex, including the transmembrane domain that is connected to the "effector" domain within the cell's cytoplasm.

The sensing domain of the aspartate receptor was revealed to be a dimer, a molecule consisting of two subunits. Each of these subunits consists of four structural elements called "alpha helices" -- two long and two short -- that form a football-shaped bundle. These helices are the key to the signalling mechanism since they continue through the membrane.

"The binding of aspartate must affect the helices over a distance exceeding 120 angstroms, from the binding site at the top of the receptor (which can be located more than 60 angstroms outside of the membrane surface) down to the point of signal action in the cytoplasm," says Kim.

From the x-ray crystallographic patterns, it was determined that the aspartate binding site is at the interface between the two subunits of the dimer. Aspartate binds between two alpha helices of one unit and one alpha helix of the other in a highly charged pocket that engulfs almost the entire surface area of the captured molecule.

"The effect of aspartate binding is to bring the binding-pocket residues from both subunits closer together," says Kim. "Aspartate locks the dimer into a tighter complex by both the gain of attractive and loss of repulsive electrostatic interactions."

This conformational change in the extracellular domain is transmitted through the receptor's transmembrane domain and into the effector domain which, in response, changes its conformation. This second conformational change sends a signal through the cytoplasm that causes the bacterium's flagella to rotate in such a way that the organism swims toward the source of the aspartate.

Although there are significant differences between the aspartate receptor and mammalian transmembrane receptors, Kim and his colleagues believe there are enough structural similarities to suggest that the conformational change observed in this receptor is a signalling mechanism common to many different types of receptors.

In addition to Kim and Koshland, other members of the research team that did this work included Michael Milburn, Gilbert Prive, Daniel Milligan, William Scott, Joanne Yeh, and Jarmila Jancarik. The research was first reported in the Nov. 29, 1991 issue of Science Magazine.