Biophysical Society Thematic Meeting| Lima 2019

Revisiting the Central Dogma of Molecular Biology at the Single-Molecule Level

Saturday Speaker Abstracts

A FINELY TUNED MOLECULAR MOTOR: MECHANOCHEMISTRY AND POWER EFFICIENCY IN THE AAA+ PROTEASE MACHINE CLPXP Piere Rodriguez-Aliaga 1,2 ; Luis Ramirez 2 , Frank Kim 2 , Carlos Bustamante 2 ; Andreas Martin 2 ; 1 Stanford University, Biology, stanford, CA, USA 2 University of California at Berkeley, Berkeley, CA, USA ATP-dependent proteases of the AAA+ family, such as ClpX from Escherichia coli and the eukaryotic 26S proteasome, play a central role in protein degradation and protein homeostasis. Given its extensive biochemical and structural characterization, ClpX is a paradigm for the study of the operating principles of eukaryotic and prokaryotic protease machines of the AAA+ family. ClpX is a hexameric ring-shaped motor that transforms the energy from ATP into mechanical force to unfold proteins and translocate the unfolded polypeptide through its narrow axial pore into an associated protease (ClpP). Previously we showed that ClpX translocates its substrate in cycles composed of a dwell phase, during which the substrate does not move, and a burst phase, where ClpXP translocates the polypeptide by increments of 1–4 nm. However, the molecular mechanism by which ClpXP couples the energy from ATP hydrolysis into mechanical work is still incomplete. Here we used biochemical and single-molecule assays with optical tweezers to dissect i) the complete mechanochemical cycle of ClpXP, and ii) its mechanism of power generation. We show that ADP release and ATP binding happen non-sequentially during the dwell, while ATP hydrolysis and phosphate release occur during the burst. ADP release is the rate-limiting transition of the ATP cycle, and therefore it determines the duration of the dwell phase. Next, using ClpX mutants, we show that the size of the amino acids that form the highly- conserved translocating loops—which contact the protein substrate and propel its translocation along the narrow ClpX pore—has been evolutionarily optimized to maximize ClpX power generation, the coupling between chemical and mechanical cycles, and the efficiency of protein unfolding and translocation. Finally, we show that the conformational resetting of these loops between consecutive bursts determine ADP release from individual ATPase subunits, and therefore the overall duration of the motor's cycle.

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