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MICROBIOLOGY Electron Swapping Keeps Proteins in ShapeRegimen May Overcome Hurdle to Using Bacteria as Protein-Making Factories Bacteria like E. coli, with their ability to grow rapidly, seem like an ideal vehicle for mass producing human and genetically engineered proteins such as insulin. Slip in a foreign gene, and the new protein is churned out. But the reality is more complicated. The protein must not only be expressed but must be folded correctly, and in a foreign environment, even a difference of an electron can result in a protein that does not function. A research team led by Jonathan Beckwith, the American Cancer Society professor of microbiology and molecular genetics at HMS, has found a system for protein folding in E. coli that may be exploited to help correct some of these problems. The system, detailed in the Nov. 22 Cell, contains a novel mechanism for shuttling electrons across a membrane, raising the possibility of similar electron transfer systems in eukaryotic cells.
Jonathan Beckwith (left) and Federico Katzen have discovered a mechanism that may help correct problems in using E. coli to express foreign proteins. Photo by Graham Ramsay The system involves a series of proteins that help form disulfide bonds, covalent links between pairs of cysteines, that are necessary for keeping many extracellular and secreted proteins in their correct conformation. These bonds are not found in proteins within the cell cytoplasm, but are formed in the lumen of the endoplasmic reticulum of eukaryotic cells and in the periplasmic space between the two membranes of E. coli bacteria. Studies have shown that E. coli have difficulty producing certain foreign proteins, often because incorrect disulfide bonds are formed. The current paper focuses on the membrane protein DsbD, part of a family of proteins involved in forming and shuffling E. coli's disulfide bonds. Scientists had previously assumed the bonds formed spontaneously, without needing an enzyme as a catalyst. But about 10 years ago, Beckwith's group discovered DsbA, an enzyme in bacteria that can promote the formation of disulfide bonds in other proteins by accepting their electrons and donating its bond. Being an imperfect craftsman, however, DsbA needs a whole crew behind it. The enzyme sometimes forms incorrect disulfide bonds that must be corrected by its coworker in the periplasm, DsbC, whose task is cleaving misformed bonds. DsbC needs to maintain a supply of free electrons so it can keep adding electrons to other proteins and, for these, it relies on DsbD. Spanning the bacterial inner membrane, DsbD acquires the electrons by interacting with the cytoplasmic protein thioredoxin. So DsbD must move electrons from the cytoplasm through the membrane into the periplasm to pass them off to DsbC. "The DsbD mechanism is the first known example of how a set of sulfhydryl groups can transfer electrons across a membrane," said Federico Katzen, a research fellow in Beckwith's lab. The most familiar mechanism of transmembrane electron transfer is the respiratory chain, which uses electron transfer to fuel a proton gradient to synthesize ATP. "In contrast," said Katzen, "in the case of DsbD, the electrons themselves constitute the ultimate purpose of the system." All of these proteins have a rather simple system for shuttling electrons, which involves a little dance of disulfide exchanges. Each reaction begins with two proteins, one of which has two free cysteines with electrons to donate, and the other two oxidized cysteines that are partnered in a disulfide bond. One of the free cysteines steps in and woos one of the coupled cysteines away from its partner, joining it in a shared disulfide bond between the two proteins. But the other free cysteine, as if suddenly jealous, interrupts the couple and reclaims its partner cysteine with a disulfide bond. The formerly free cysteines have now paired up, and in doing so they have lost their electrons to the other protein, whose newly free cysteines can pass their electrons to another protein in a similar dance.
The DsbD system uses a novel mechanism to transfer electrons across the membrane. A series of disulfide exchanges—interactions between disulfide bonds (S–S) and sulfhydryl groups (–SH)—shuttles electrons from one protein to another. DsbD, shown separated into its three domains, receives electrons from the cytoplasm protein thioredoxin (TrxA). The electrons travel through DsbD across the membrane to the periplasm, where they are transferred to DsbC. Adapted from original drawing by Federico Katzen Divide and ConquerThe team wanted to figure out how DsbD was moving electrons from the cytoplasm to the periplasm, but it was difficult to isolate in which order its six cysteines were interacting. Katzen came up with a unique approach to track the path of electrons through the protein. He split the protein into its three domains, each containing a pair of cysteines, and expressed each in three separate plasmids. "It was Federico's initiative and insight to consider the possibility that we could divide the protein into three domains and express each one separately, and get them to function together," said Beckwith.With a more complete view of how this family of proteins shuffles electrons across cysteines, the team hopes to be able to enhance the process and improve the production of foreign and genetically engineered proteins. "These pathways are all limited by how rapidly you can transfer electrons in one way or another," said Beckwith. "The deeper the understanding you get of these systems, the more able people are to use them to express their own protein." Beckwith's lab is currently working with a group from the University of Texas on an NIH program to enhance the production of foreign proteins in E. coli. The studies also raise the possibility that eukaryotic cells may have an analogous system for shuffling electrons across a membrane. "This whole field of transferring electrons around using disulfide bond chemistry is something that really hasn't been explored until these systems were discovered," said Beckwith. DsbD can supply electrons to multiple proteins in the periplasm, and it may be involved in several pathways that require a constant supply of electrons. Humans have more than one hundred genes that are members of the thioredoxin superfamily to which DsbD belongs, so its system of shuttling electrons may be more than an isolated example. —Courtney Humphries |
