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CELL BIOLOGY
Protein-conducting Channel Debuts in Crystal StructureProteins may have replaced genes as the new celebrities in biology, but their status fundamentally depends on getting where they need to go--often across or into membranes. Proteins must leave the cell to digest food, fight disease, and regulate reproductive cycles. Other proteins must station themselves in the membrane to monitor changes in the environment, trigger the immune system, and transmit nerve signals.
The first high-res image of a protein-conducting channel shows that a single complex forms an hourglass-shaped pore with a plug (red, on left), which uncorks for proteins (red, on right). (Image courtesy of William Clemons) New evidence shows that all these proteins must negotiate a surprisingly small opening in the membrane, according to the first high-resolution images of a protein-conducting channel, from the lab of Howard Hughes investigator Tom Rapoport, HMS professor of cell biology. The X-ray structure was published in the Dec. 3 advance online edition of Nature (DOI: 10.1038/nature02218). "We were all prepared for a very large channel," said Hughes investigator Randy Schekman of the University of California, Berkeley. "This new paper is going to shake things up."
The paper marks a scientific milestone. Until now, only smaller membrane channels for ions and water molecules have been seen in such detail. Even these channels are formed by proteins that arrive through the protein-conducting channel. "This changes the view drastically," said Bernhard Dobberstein of the University of Heidelberg, who is writing a commentary on the structure for an upcoming issue of Science. "What it tells us is quite novel." A Channel's Ins and OutsScientists often have marveled at the function of the protein-conducting channel. It grants passage to many different proteins while holding back millions of other proteins and smaller molecules. And it works in three dimensions. It moves water-loving proteins from one side of the greasy membrane to the other, but it also directs fat-seeking membrane proteins laterally into the lipid layers.The new structure of the channel at rest suggests how it might work. To a protein eyeballing the closed portal, the protein-conducting channel looks like a small hourglass with a plug in one funnel. The channel's main subunit wraps around the hourglass opening like cold hands clutching a warm cup of coffee, Rapoport said. The complex hinges open at the fingertips, enlarging the pore or allowing some proteins to slip sideways out into the lipids. A hydrophobic ring at the funnel's waist seems to provide a seal around the protein slithering through the narrow opening.
From left, Bert van den Berg, Tom Rapoport, and William Clemons Jr. led the effort to produce the first high-resolution images of a protein-conducting channel. (Photo of van der Berg and Rapoport by Phil Farnsworth; photo of Clemons by Jeff Cleary) Proteins find the channel by a kind of zip code--a short sequence of water-hating amino acids at one end. In the channel, the signaling sequence slips between the fingertips and stays there, probably propping the hinge open and loosening the plug while the rest of the protein slides through in a hairpin maneuver. Proteins need something to push them through the channel. Most often, the ribosome docks there and extrudes limp nascent protein chains directly into the channel. Once on the other side, the protein folds into its active shape. Becoming Crystal ClearProtein-conducting channels in completely different species are uncommonly similar, probably because of the channel's importance to life. "The system is extremely ancient," Rapoport said. "In a way, it's kind of primitive. Once you form a membrane of a cell or its compartments, you have cut off the interior from the outside world, and it doesn't work. It needs energy, nutrients, and the ability to respond to its environment. Every single-celled and multicellular organism needs a system to incorporate membrane proteins and secrete other proteins."This project began five years ago with postdoctoral fellow and co-author Ian Collinson. More than a year later, co-first author and postdoc Bert van den Berg joined Collinson as he attempted to crystallize the protein-conducting channels of E. coli and then of the Methanococcus jannaschii archaebacterium. Collinson moved on to a faculty position at the Max Planck Institute of Biophysics in Germany. As van den Berg persisted in the daily trial and error of purifying and crystallizing the membrane channel complex, he experimented with nine different bacteria and even one yeast species. Eventually, he coaxed the archaebacterium channel into good crystals using a detergent to mimic the environment of the membrane. By this time, co-first author William Clemons Jr. had joined the lab as a postdoctoral fellow. Working with van den Berg, Clemons spent two years analyzing the X-ray diffraction patterns. His part of the project fell into place more than a year ago when the 3-D electron density map on his computer monitor suddenly showed the familiar spiral helices of membrane proteins. All along, the researchers had been drawing on the structural expertise of Hughes investigator Stephen Harrison, HMS professor of biological chemistry and molecular pharmacology, and postdoc Yorgo Modis, both co-authors on the new paper. Harrison calls this collaboration a stellar example of how structural biology expertise can be integrated into other research efforts to solve important structures, a model he hopes to expand with the new Center for Molecular and Cellular Dynamics at HMS. For Rapoport, this structure feels like the culmination of 25 years of work. In other ways, it is just the beginning. "The new structure allows us to postulate new hypotheses that can be tested," he said. "The next big goal is to crystallize the channel in action. In the meantime, we can do biochemical experiments to test some of the more provocative aspects." --Carol Cruzan Morton |
