Revealing Elusive Protein Structures via Cryo-EM

In 2019, Ben Orlando’s research presentation at Michigan State University’s Department of Biochemistry and Molecular Biology sparked a significant collaboration focused on the elusive protein SpoIVFB. Present at the talk was Professor Emeritus Lee Kroos, who recently concluded a distinguished 30-year career at MSU. A specialist in the molecular and genetic mechanisms of bacteria, Kroos had been striving to elucidate the structure of this important protein found in the bacterium Bacillus subtilis.

SpoIVFB is classified as an intramembrane protease, a type of enzyme essential for regulating various cellular functions. It is particularly crucial for the process of sporulation, which allows bacteria to endure extreme environments. Recognizing Orlando's expertise in cryo-electron microscopy (cryo-EM), Kroos reached out to collaborate after Orlando joined the department in 2020. Their partnership has culminated in the publication of the first high-resolution structures of SpoIVFB in Nature Communications.

This research provided insights into how SpoIVFB interacts with its substrate, similar to a key fitting into a lock, facilitating vital biochemical reactions. The discovery enhances our understanding of cellular regulation across different life forms, with potential implications for microbiology, structural biology, and human health. Orlando noted that advancements in cryo-EM technology at MSU have transformed the study of membrane proteins, enabling scientists to investigate areas previously hidden from view.

By capturing the structure of SpoIVFB alongside its substrate, the researchers have significantly broadened the knowledge of critical biological processes. Typically, proteases break down proteins through proteolysis, a reaction that necessitates water to sever peptide bonds. However, for intramembrane proteases like SpoIVFB, gaining access to water within the hydrophobic lipid bilayer of the membrane presents unique challenges.

Through their research, Kroos and Orlando discovered that SpoIVFB utilizes beta-sheet augmentation to interact with its substrate, confirming a shared mechanism across all four classes of intramembrane proteases. This finding is vital for understanding various diseases linked to the malfunction of these proteases, such as neurodegenerative diseases and cancers.

The results of this study also serve as a fitting capstone to Kroos’s career, during which he has celebrated fruitful collaborations. He expressed great satisfaction in working with Orlando, stating that their discussions have been incredibly rewarding as he enjoys his well-deserved retirement.

Initially, Kroos and his colleagues attempted to study SpoIVFB using X-ray crystallography but faced difficulties related to the quality and quantity of crystals. Their collaborative efforts with cryo-EM allowed them to visualize the protein in unprecedented detail, effectively overcoming past challenges. By rapidly freezing samples, they captured high-resolution images that reveal essential details about the structure and function of SpoIVFB, paving the way for future breakthroughs in the field of biology.