Seeing the World in 5-D
MUN research team develops new way to visualize proteins and discovers key information on the inner workings of cancer-causing enzymes
February 4, 2019
A research team at Memorial University of Newfoundland has developed a new approach to visualizing cancer-causing proteins. This approach provides new information on how proteins work in real time.
The team, led by Memorial professor Mani Larijani, has been studying an enzyme called AID, which plays an important role in the body's immune response. AID mutates the DNA sequence of white blood cells which enables them to respond more effectively to the invasion of foreign proteins.
AID can mistakenly mutate non-immune genes or become active in non-immune cells and thus cause genetic changes that transform healthy cells into cancers. In addition to causing cancer, AID activity in cancer cells increases the aggressiveness of the cancer and makes them more resistant to treatment.
AID is a protein composed of about 200 amino acids. This molecular chain folds itself in specific ways depending on the chemical state of the cell and surrounding molecules. By understanding the structure of the protein, researchers may design drugs to block or inhibit its disease-causing activity.
Researchers traditionally use techniques such as x-ray crystallography and nuclear magnetic resonance (NMR) to visualize enzymes and other proteins to determine their molecular structure. The structures are then reproduced as 3-D models or 2-D visual illustrations in textbooks or scientific articles.
"The challenge with AID is that it's a highly insoluble protein," explains Dr. Larijani. This property makes the enzyme unsuitable for x-ray or NMR and that's why the structure of AID had remained largely unknown.
In his quest to solve the structure of AID, Dr. Larijani was not alone. His team was competing with dozens of internationally-leading structural biology teams at larger universities with much more resources. The difference was that instead of relying on the traditional techniques, Dr. Larijani and his team decided to come up with a new way of solving the problem.
Working with his PhD student Justin King, Dr. Larijani sought to identify the structure using an innovative process that they pioneered. The process combines computer modelling, evolutionary biology and functional biochemical testing of enzyme activity.
"In order for this combined and unconventional approach to work, one needs to develop a good understanding of the underlying math, physics and chemistry, and to integrate them with the biology of the system," says Dr. Larijani.
Using supercomputers, they developed hundreds of thousands of predictive models of what AID might look like. They then compared them to known variants in humans and those in more distant animals such as zebrafish. At every stage and with every new insight, they carried out extensive tests in the lab to verify aspects of the enzyme's predicted structure.
It took six years, but the tenacity of Dr. Larijani and his team was rewarded. They not only succeeded in determining the 3-D structure of AID, but using their approach they also revealed how the enzyme changes structure in real time and how it carries out its functions to mutate DNA.
One of their most important findings was that 70% of the time the active site of AID is closed and not active. In other words, enzyme has a "safe" state and a "dangerous" state. This was also a landmark discovery, because it is the first time this type of shape-shifting has been observed at the heart of a cancer-causing enzyme.
"It makes sense that an enzyme with such mutating power would have a way to regulate itself and safeguard against excess activity," says Dr. Larijani.
"This is why we consider our approach capable of providing 5-D structures," says Dr. Larijani. "We were able to reveal more about the workings of AID, in that we also understand how it moves in real time, and how much time it spends in each of those moving states, and we think of this as the 4th dimension, which is time. In addition, we understand the relevance of those moving states to the function of the enzyme in immunity and cancer, and one can consider function as the 5th dimension".
"We were very happy to have gained this insight into the structure of AID ahead of competing teams," says Dr. Larijani. "With our methodology, we have added whole new dimensions to the story. We gathered data that relates the movement of structure to function, showing how an individual molecule can move in ways that either do or do not cause cancer."
Following publication of their work, Dr. Larijani was satisfied to see other research teams follow up on their findings using x-ray and NMR on enzymes that are related to AID. "Others went on to directly observe the closed inactive state of these enzymes, as we had described it".
Based on their work, Dr. Larijani and his team have already designed a new drug to target AID in leukemia and lymphoma. "Much more work remains to be done to test and improve this drug candidate, but the results so far are exciting and promising," says Dr. Larijani.
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