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Albany 2019: 20th Conversation - Abstracts

category image Albany 2019
Conversation 20
June 11-15 2019
Adenine Press (2019)

The Conformational Landscape of the Human 26S Proteasome by Cryo-EM Structural Analysis

The 26S proteasome is a macromolecular machine (2.5-MDa) that mediates ubiquitin-dependent protein degradation in all eukaryotic species. The proteasome consists of a barrel-shaped 20S core complex flanked by two hexameric rings of AAA+ ATPases that harvest the energy from ATP hydrolysis to unfold and translocate substrates through the central pore into the degradation chamber. In a previous study, we determined how the ubiquitin configurations on substrates dictated the degradation rate by the proteasome. Currently, we aim to understand how the 26S proteasome recognizes these ubiquitin configurations and commits the substrate to a processive degradation process. In a series of studies, we used modern cryo-EM technology and classification algorithms to identify multiple conformations of the proteasomes, which provide important insights into these questions. To complement the lack of kinetic information in structural analysis, we developed a fluorescent reporter with sub-nanomolar sensitivity to measure proteasomal activities under different conditions, and established single-molecule assays to detect the transient interactions between nucleotides and the proteasome using a TIRF microscope. To synergize these results, we created a rule-based model, inspired by comparative analysis of the proteasome structures, which simulates the dynamic transitions of a translocating proteasome on a complete conformational landscape with unknown parameters that can be obtained from kinetic studies. Suggested by the results, a pathway of sequential loading/hydrolysis emerges with the highest probability, and minor pathways with lower probabilities also exist which may become the major one and consequently lower the degradation rates as in different proteasomal mutants. In summary, our study promotes a deeper understanding of the functional dynamics of the proteasome driven by ATP hydrolysis, with the goal to obtain mechanistic insights by bridging the recent structural advances and biochemical observations.

Rui Fang1
Robin Chen2
Jason Hon1
Jiayi Wu2
Yuanchen Dong2
Yanan Zhu2
Youdong Mao2
Qi Ouyang2
Daniel Finley3
Ying Lu1

1Department of Systems Biology
Harvard Medical School
Boston, MA 02115

2State Key Laboratory for Artificial Microstructures and
Mesoscopic Physics
School of Physics
Peking University
Beijing, China

3Department of Cell Biology
Harvard Medical School
Boston, MA 02115

Phone: (617)432-5143
Email: ying_lu@hms.harvard.edu