Abstract
The primary functions of the proteasome are driven by a highly allosteric ATPase complex. ATP binding to only two subunits in this hexameric complex triggers substrate binding, ATPase–20S association and 20S gate opening. However, it is unclear how ATP binding and hydrolysis spatially and temporally coordinates these allosteric effects to drive substrate translocation into the 20S. Here, we use FRET to show that the proteasomal ATPases from eukaryotes (RPTs) and archaea (PAN) bind ATP with high affinity at neighbouring subunits, which complements the well-established spiral-staircase topology of the 26S ATPases. We further show that two conserved arginine fingers in PAN located at the subunit interface work together as a single allosteric unit to mediate the allosteric effects of ATP binding, without altering the nucleotide-binding pattern. Rapid kinetics analysis also shows that ring resetting of a sequential hydrolysis mechanism can be explained by thermodynamic equilibrium binding of ATP. These data support a model whereby these two functionally distinct allosteric networks cooperate to translocate polypeptides into the 20S for degradation.
Highlights
The primary functions of the proteasome are driven by a highly allosteric ATPase complex
Because adenosine triphosphate (ATP) binding and hydrolysis to ADP are both essential for proteasome function, understanding how these events are coordinated both spatially and temporally is critical to understanding how work is done on substrates
These findings support an allosteric model, describing how ATP binding and hydrolysis are coordinated by separate allosteric systems that control the conformational changes that drive substrate unfolding and translocation into the proteasome for degradation. Position of these high-affinity subunits within the hexameric complex, we monitored Forster resonance energy transfer (FRET) between fluorescent nucleotides bound to the high-affinity sites using a mant-ATP donor (m-ATP; Ex: 360, Em: 450) and TNPATP acceptor (t-ATP; Ex: 470, Em: 570; Forster critical distance (R0): 40 Å) (Supplementary Fig. 1)
Summary
The primary functions of the proteasome are driven by a highly allosteric ATPase complex. ATP binding to only two subunits in this hexameric complex triggers substrate binding, ATPase–20S association and 20S gate opening It is unclear how ATP binding and hydrolysis spatially and temporally coordinates these allosteric effects to drive substrate translocation into the 20S. Its C-terminal side associates with the 20S proteasome via its C-terminal HbYX motif, which induces 20S gate opening to promote substrate entry This architecture places the ATPase ring in a position where it can accept protein substrates on its N-terminal side, in an ATP-dependent manner, translocate them through its central pore and into the 20S for their degradation[1,2,3,4]. Strictly imposed allosteries indicated a patterned ATP-binding and hydrolysis mode is likely, which fits well with a sequential mechanism for ATP hydrolysis[26]
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