Albany 2019: 20th Conversation - Abstracts

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

Understanding the Functional Dynamics of the 26S Proteasome by Nucleotides-proteasome Interaction Study

The 26S proteasome, consisting of a barrel-shaped proteolytic 20S core complex and one or two 19S regulatory complexes, is a macromolecular machine (2.5 MDa) responsible for regulatory protein degradation in eukaryotic cells. The proteasome has a hexameric ring of AAA+ ATPases at the bottom of the 19S proteasome which converts ATP’s chemical energy to mechanical force to unfold protein substrates and translocate the denatured polypeptide through the central pore into the 20S for degradation. Though genetic, biochemical and structural studies have revealed great details about the proteasome, how the six ATPases in the 19S particle coordinate their ATP cycles to power substrate translocation and conformational transitions of other parts of the proteasome remains elusive. To understand this question, we developed a fluorescent reporter with sub-nanomolar sensitivity to measure different aspects of proteasomal activities. In a competition assay, we found that ATP-γS inhibited substrate degradation much more strongly compared to ADP, and primarily affected the rate of substrate translocation. We then developed a single-molecule assay to study the kinetics of the nucleotides-proteasome interaction. By labeling nucleotides with fluorophores, we are able to detect single nucleotide binding and disassociation events under a TIRF microscope. The result shows that proteasomal ATPases are characterized by distinct nucleotide binding kinetics. Interestingly, the overall binding kinetics of ATP is similar to that of ADP and ATP-γS, while AMP binds rather weakly. To explain the different inhibitory effects of nucleotides in the competition assay, we devised a Markov-state model representing all possible transition of the proteasomal ATPases. Simulation of this model suggests an entropy-driven mechanism underlying the inhibition by nucleotide inhibitors, which may be adaptive under low ATP conditions. Our study provides novel understanding of the working principles of proteasomal ATPases, with the goal to obtain mechanistic insights by bridging the recent structural advances and biochemical observations.

Rui Fang1
Ying Lu2


Rui Fang, a doctoral student at Harvard Univ with Prof. Ying Lu will provide a short oral from the platform.

1 Department of Molecular and Cellular Biology
Harvard University
Cambridge, MA 02138

2Department of Systems Biology
Harvard Medical School
Boston, MA 02115

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