Conformational switching in the coiled-coil domains of a proteasomal ATPase regulates substrate processing

Protein degradation in all domains of life requires ATPases that unfold and inject proteins into compartmentalized proteolytic chambers. Proteasomal ATPases in eukaryotes and archaea contain poorly understood N-terminally conserved coiled-coil domains. In this study, we engineer disulfide crosslinks in the coiled-coils of the archaeal proteasomal ATPase (PAN) and report that its three identical coiled-coil domains can adopt three different conforma- tions: (1) in-register and zipped, (2) in-register and partially unzipped, and (3) out-of-register. This conformational heterogeneity conflicts with PAN’s symmetrical OB-coiled-coil crystal structure but resembles the conformational heterogeneity of the 26S proteasomal ATPases’ coiled-coils. Furthermore, we find that one coiled-coil can be conformationally constrained even while unfolding substrates, and conformational changes in two of the coiled-coils reg- ulate PAN switching between resting and active states. This switching functionally mimics similar states proposed for the 26S proteasome from cryo-EM. These findings thus build a mechanistic framework to understand regulation of proteasome activity

Proteasomal ATPases Hard at Work: The Inner Workings of a Protein Destruction Machine.

Across all domains of life, the proteasome is responsible for the majority of targeted protein degradation in the cell. Often, the proteasome is thought of as the molecular “garbageman” of the cell. While it is true that the proteasome degrades and eliminates misfolded proteins, the proteasome is also capable of degrading fully folded, functional proteins whose presence is no longer required (e.g. during embryonic development, cell cycle changes, etc.). Despite its crucial role in virtually every cellular process, our understanding of how the proteasome operates from a mechanistic perspective is still highly limited. In order to prevent unregulated degradation the protease sites of the proteasome are sequestered inside its hollow interior. While loosely folded proteins can enter the degradation chamber without the requirement of energy, proteins with secondary structure can only be degraded when they are properly recognized (e.g. by ubiquitin tags), unfolded, and injected into the protease chamber for degradation. Protein recognition, unfolding, and injection into the protease chamber all depend on ATP. However, very little is known about how such chemical energy is converted to mechanical work. In this dissertation we sought to understand the logistics of nucleotide binding and hydrolysis, and also to determine the conformational changes that can regulate protein entry and degradation by the proteasome. To this end, we focused on one of the most common regulators from eukaryotes-- the heterohexameric 19S ATPases, as well as its homohexameric archaeal homolog-- “PAN” (proteasome activating nucleotidase). Based on our extensive analysis, our data support a neighbor-binding sequential hydrolysis mechanism for the proteasomal ATPases. Furthermore, we show that these ATPases are highly processive, even when they reach more tightly folded domains of a protein, which is unlike what had been proposed previously based on studies of other ATP-dependent proteases (e.g. ClpX, which often “slips” and “stalls” at these more tightly folded domains). This tight binding of the proteasomal ATPases appears to be due to its crucial trans-arginine fingers that “sense” bound nucleotides in its neighboring subunit (which ClpX lacks), and we propose that this processivity arose due to the diverse client proteins the proteasome must encounter (e.g. it must engage and unfold many types of proteins, even ones that are fully folded and functional). Lastly, we have developed a disulfide engineering approach to show that PAN’s N-terminal domains adopt distinct conformations that set the rate of ATP hydrolysis. This novel approach has allowed us to isolate specific subunits from a homohexamer that are identical in their amino acid sequence, but that adopt different conformations when they form a hexameric ring. This disulfide engineering approach we’ve developed is a powerful method to analyze structural asymmetries in homomeric protein complexes with minimal structural perturbations, which has not been accomplished before and opens the door to an entire new approach to studying the function of the molecular motors. We started this work with the goal of understanding the logistics of the mechano-chemical cycle of the proteasomal ATPases. Indeed, we have developed novel methods to better understand the inner workings of this complex multimeric machine, and the groundwork we lay here has contributed greatly to our knowledge of the proteasomal ATPases, and will also push forward our understanding of other AAA+ ATPases and molecular motors in general. Ultimately, a better understanding of these complex machines will aid in the development of new therapies to combat diseases where these machines are dysregulated

ATP binding to neighbouring subunits and intersubunit allosteric coupling underlie proteasomal ATPase function

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

Endocytic myosin-1 is a force-insensitive, power-generating motor.

Myosins are required for clathrin-mediated endocytosis, but their precise molecular roles in this process are not known. This is, in part, because the biophysical properties of the relevant motors have not been investigated. Myosins have diverse mechanochemical activities, ranging from powerful contractility against mechanical loads to force-sensitive anchoring. To better understand the essential molecular contribution of myosin to endocytosis, we studied the in vitro force-dependent kinetics of the Saccharomyces cerevisiae endocytic type I myosin called Myo5, a motor whose role in clathrin-mediated endocytosis has been meticulously studied in vivo. We report that Myo5 is a low-duty-ratio motor that is activated ∼10-fold by phosphorylation and that its working stroke and actin-detachment kinetics are relatively force-insensitive. Strikingly, the in vitro mechanochemistry of Myo5 is more like that of cardiac myosin than that of slow anchoring myosin-1s found on endosomal membranes. We, therefore, propose that Myo5 generates power to augment actin assembly-based forces during endocytosis in cells