The discovery of the exact structure of the tail and its role in cell replication holds promise that researchers can develop new types of drugs targeting the tail or the workshop itself, turning off the unrestrained multiplication of cancer cells.
The study also shows that a slight change in a structure common among many enzymes performing similar jobs can create a new enzyme with a unique activity.
The St. Jude researchers report that the stabilizing influence of Ubc12's protein tail is based on its ability to nestle within a groove of the larger molecule called APPBP1-UBA3. These two proteins, also known by the shorthand designation of E2 (Ubc12) and E1 (APPBP1-UBA3), cooperate with each other to form a workshop where the "on switch" that accelerates cell replication is cobbled together.
The workshop makes the switch by linking a tag called NEDD8 with its "target" a molecule called Cul1. NEDD8 then modifies Cul1, causing it to set off a cascade of biochemical reactions that eliminates the molecular brake controlling cell replication. In the absence of this brake, cell replication accelerates, and if left unchecked, could cause cancer.
Until now, the function of the E2 tail was unknown, according to Brenda Schulman, Ph.D., an assistant member of the St. Jude Genetics and Tumor Cell Biology and Structural Biology departments. Schulman is senior author of the NSMB report.
The current study shows that the tail of E2 plays a crucial role in a series of hand-offs in which NEDD8 passes from one enzyme to another. First, E2's tail helps E2 clamp onto E1. That allows E1 to activate NEDD8 through a process called adenylation (putting a molecule called an "adenyl" group onto the end of NEDD8). Immediately after activating NEDD8, E1 forms a chemical bond with this protein. E1 then transfers NEDD8 to E2. Finally, E2 transfers NEDD8 to the E3 enzyme, which completes the workshop's job by binding NEDD8 to Cul1.
"This step-by-step process requires that all the molecules be brought together in a specific manner so the reactions can take place quickly," Schulman said. "That's where the E2 tail comes into play. The shape and location of E2's tail make it perfectly suited for drawing E1 close to NEDD8 so those two proteins can rapidly bind to each other and begin the process of assembling the on switch." The workshop's ability to quickly assemble the on switch is important because it reflects the need for cells to be able to react swiftly to a changing environment, according to Martine Roussel, Ph.D., member of St. Jude Genetics and Tumor Cell Biology and an author of the paper.
"Cells must be able to respond quickly to the changing demands and cues of their internal and external environments," Roussel said. "Often, the best way to respond rapidly is for the cell to modify a particular protein that already exists, rather than take the time to make a new one. Once modified, the existing protein becomes active and can quickly set off a specific cascade of biochemical reactions that performs a specific function in the cell."
The E1, E2 and E3 enzymes that make up the workshop that prepares NEDD8 to modify Cul1 are variations of a larger family of similar enzymes, according to the researchers. These other enzymes are part of the so-called ubiquitin pathway that puts molecular tags on proteins to signal that they should be destroyed. The ubiquitin pathway has dozens of different types of E2 enzymes, hundreds of different types of E3 enzymes and thousands of different targets. The NEDD8 workshop, however, has only one E2 and a few E3 enzymes, and NEDD8 itself has only a few target molecules.
"The limited number of targets NEDD8 has despite the fact that its E1, E2 and E3 enzymes look very much like those of the ubiquitin pathway makes the discovery of the E2 tail intriguing," said Danny T. Huang, Ph.D., a St. Jude postdoctoral fellow and first author of the paper. "Although most of the E2 that binds with E1 and NEDD8 looks like any other E2, its tail makes this enzyme unique."
The tail of the E2 that binds to NEDD8 and the way the tail holds E2 onto E1 give this workshop a different shape than the ones in the ubiquitin pathway. The slight modification in shape limits this workshop to working only with NEDD8 and NEDD8's few targets.
Knowing the exact shape and function of the E2 tail and the groove along E1 the tail fits into make these structures potential targets for new drugs.
"Novel drugs that are designed to disrupt the tail, the groove or both might block the ability of the NEDD8 pathway to accelerate replication of cancer cells."
The extensive study required a variety of techniques to tease apart the structure formed by the bonding of the E2 tail to E1. For example, the St. Jude team showed that deleting the tail from E2 significantly hinders the ability of E2 to transfer NEDD8 to Cul1, and blocks its ability to drive cell proliferation. This demonstrated the important role played by this unique protein.
In addition, using X-ray crystallography techniques, Schulman crystallized samples of the bonded proteins and bombarded them with a beam of X-rays. She then used the patterns formed by the diffraction of the beams off the crystals to create computer-generated, three-dimensional images of the shape of the bonded proteins.
However, images of the structure created using X-ray crystallography failed to provide detailed views of half of the individual amino acid building blocks making up the protein tail. This problem was overcome using a technique specially developed by Robert Cassell, a macromolecular specialist III in the St. Jude Hartwell Center for Biotechnology and Bioinformatics. By substituting a molecule called selenomethionine in place of certain amino acids in the E2 tail, he modified the E1-E2 combination structure in such a way that the X-ray crystallography studies were able to produce images of the entire tail structure--a crucial breakthrough that permitted the full understanding of the role this part of E2 plays in the NEDD8 pathway.
Other authors of the paper are David Miller, Rose Mathew (St. Jude) and James M. Holton (Lawrence Berkeley National Laboratory, University of California, Berkeley).
This work was supported in part by the National Institutes of Health, a National Cancer Institute Cancer Center Core Grant, the Department of Defense, a grant from Philip and Elizabeth Gross, a Pew Scholar in Biomedical Sciences Award (Brenda Schulman), a St. Jude special Fellowship (David Miller) and ALSAC.
St. Jude Children's Research Hospital
St. Jude Children's Research Hospital is internationally recognized for its pioneering work in finding cures and saving children with cancer and other catastrophic diseases. Founded by late entertainer Danny Thomas and based in Memphis, Tennessee, St. Jude freely shares its discoveries with scientific and medical communities around the world. No family ever pays for treatments not covered by insurance, and families without insurance are never asked to pay. St. Jude is financially supported by ALSAC, its fundraising organization. For more information, please visit www.stjude.org.
Journal
Nature Structural & Molecular Biology