Collaboration Leads to Great Achievement: Amie Boal and Squire Booker
20 September 2016
Understanding the sequence of chemical events that an enzyme employs to convert one molecule to another can provide crucial insight for drug design and the development of new synthetic strategies for clinically important natural products. However, teasing out the intricate details of these complex reaction pathways can be difficult. Protein X-ray crystallography is a powerful tool for providing detailed insight into these chemical reactions, especially if pictures can be obtained of proteins in the act of catalyzing their reactions. A fruitful collaboration between the labs of Professors Squire J. Booker and Amie Boal demonstrates the complementarity of mechanistic biochemical investigation and X-ray crystallography to provide unprecedented details of the mechanisms of some of Nature’s most compelling enzymatic reactions.
The collaboration began in 2010 while Amie was a post-doc in Amy Rosenzweig’s lab at Northwestern University. Data from biochemical studies in the Booker Lab suggested a novel mechanism for RlmN and Cfr, radical S-adenosylmethionine (SAM) methylases responsible for the methylation of RNA. Bacteria that contain Cfr are resistant to many clinically relevant antibiotics, making it an intriguing target for the development of new therapeutic compounds. The evidence for a unique chemical mechanism was substantial, but according to Booker Lab alum Tyler Grove (Ph.D., 2013), they wanted additional confirmation of the mechanism they were about to propose. “Dr. Booker and myself were sitting in his office and commiserating over my failed attempt to solve the RlmN+SAM structure. We were in the process of writing the first RlmN/Cfr paper and really wanted/needed to know if we were right or wrong about what we believed was happening, which, at the time, was a pretty wild idea about how RlmN and Cfr were functioning. So, Dr. Booker called Amy Rosenzweig (a crystallographer at Northwestern) and asked if she had someone who could help us out. Amy said ‘No problem, I have an excellent post-doc in my lab who does not mind difficult problems. Her name is Amie Boal.’ Grove explained.” Within a few months, Amie had solved the first structure of RlmN using protein purified in the Booker Lab. The structure confirmed the presence of a unique methylcysteine residue, an essential component of the proposed mechanism. The initial mechanistic characterization and the RlmN structure were published as two separate papers in Science in 2011, solidifying a place at the forefront of research on radical SAM enzymes.
When Amie came to Penn State to start her independent career in 2012, the collaboration continued, with the added benefit of adjacent lab space. The initial structure provided helpful insight into the starting steps of the mechanism, but it lacked the RNA substrate, crucial to a complete understanding of these enzymes. Protein-RNA complexes are notoriously difficult to crystallize due to their large size, heterogeneity, and highly charged nature. Using a protein variant that the Booker Lab showed was able to catalyze the first few steps but not the entire reaction, the Booker/Boal team was able to crystallize and solve two structures of RlmN cross-linked to a tRNA substrate. The first structure determined was a cross-link pulled directly from E. coli cells used to overexpress the protein, a long-shot experiment that paid off and revealed the surprising presence of the tRNA substrate. The cross-link was recapitulated using purified protein and tRNA in order to improve the resolution and clarify the finer details. Having all members of the team in the same building made it simple to share ideas, resources, and information on a day-to-day basis. In April of this year, these elaborate and elegant structures were published in Science, adding even more information to the RlmN story.
This work is only possible through the careful cooperation and hard work of these Penn State researchers. “Personally speaking, I don’t know if I have ever met a more dedicated and tireless scientist,” Grove said of Boal. The work is elevated to a whole new level, and students benefit from learning different techniques from two talented scientists. The highly collaborative environment of Penn State enables the sharing of resources and knowledge between the Booker and Boal labs to understand these complex enzymes and the strategies nature employs to catalyze essential biochemical transformations.