Significance: ClpB/Hsp100 chaperones protect cells from the devastating effects of protein inactivation and aggregation arising from extreme stress. This function is accomplished first by binding to the aggregates and then forcibly unraveling individual proteins by passing them through the central channel in the hexameric chaperones. Here, we investigate the role of the ClpB/Hsp100 N-terminal domain (NTD) in protein disaggregation. Our results demonstrate that ClpB recognizes exposed hydrophobic stretches in unfolded or aggregated client proteins via a substrate-binding groove in its NTD. We further show that the NTD has regulatory roles that include blocking the translocation channel in the absence of substrate and destabilizing client proteins upon binding, thus priming them for subsequent unfolding and disaggregation.

Significance: This paper uses NMR methods to compare the dynamics of a protein kinase in its active and inactive states. The results show that domain movements in the MAP kinase ERK2 are inherently constrained until the enzyme is activated by phosphorylation, with the constraint located at the hinge region. This represents an important mode for dynamical regulation in ERK2, not anticipated from previous X-ray structural analyses.

The 20S core particle proteasome is a molecular machine playing an important role in cellular function by degrading protein substrates that no longer are required or that have become damaged. Regulation of proteasome activity occurs, in part, through a gating mechanism controlling the sizes of pores at the top and bottom ends of the symmetric proteasome barrel and restricting access to catalytic sites sequestered in the lumen of the structure. Although atomic resolution models of both open and closed states of the proteasome have been elucidated, the mechanism by which gates exchange between these states remains to be understood. Here, this is investigated by using magnetization transfer NMR spectroscopy focusing on the 20S proteasome core particle from Thermoplasma acidophilum. We show from viscosity-dependent proteasome gating kinetics that frictional forces originating from random solvent motions are critical for driving the gating process. Notably, a small effective hydrodynamic radius (EHR; <4Å) is obtained, providing a picture in which gate exchange proceeds through many steps involving only very small segment sizes. A small EHR further suggests that the kinetics of gate interconversion will not be affected appreciably by large viscogens, such as macromolecules found in the cell, so long as they are inert. Indeed, measurements in cell lysate reveal that the gate interconversion rate decreases only slightly, demonstrating that controlled studies in vitro provide an excellent starting point for understanding regulation of 20S core particle function in complex, biologically relevant environments. Significance The proteasome plays a critical role in regulating cellular homeostasis by degrading target protein substrates. Gating residues that are part of the barrel-like proteasome structure provide a barrier to prevent inadvertent proteolysis. With NMR spectroscopy, the mechanism by which the gates interconvert between open and closed states is studied. We show that collisions with water molecules provide a driving force for gate interconversion and that the process takes place via many small steps that involve protein segments smaller than 4 Å. The effect of cellular lysate on proteasome gating is explored, establishing that gating equilibria and kinetics are not perturbed relative to buffer solutions under conditions of similar viscosity.