Small Peptide Binding Stiffens the Ubiquitin-like Protein SUMO1

AbstractPosttranslational modification by small ubiquitin-like modifiers (SUMOs), known as SUMOylation, is a key regulatory event in many eukaryotic cellular processes in which SUMOs interact with a large number of target proteins. SUMO binding motifs (SBMs) are small peptides derived from these target proteins that interact noncovalently with SUMOs and induce conformational changes. To determine the effect of SBMs on the mechanical properties of SUMO1 (the first member of the human SUMO family), we performed single-molecule force spectroscopy experiments on SUMO1/SBM complexes. The unfolding force of SUMO1 (at a pulling speed of 400 nm/s) increased from ∼130 pN to ∼170 pN upon binding to SBMs, indicating mechanical stabilization upon complexation. Pulling-speed-dependent experiments and Monte Carlo simulations measured a large decrease in distance to the unfolding transition state for SUMO1 upon SBM binding, which is by far the largest change measured for any ligand binding protein. The stiffness of SUMO1 (measured as a spring constant for the deformation response along the line joining the N- and C-termini) increased upon SBM binding from ∼1 N/m to ∼3.5 N/m. The relatively higher flexibility of ligand-free SUMO1 might play a role in accessing various conformations before binding to a target

Effect of directional pulling on mechanical protein degradation by ATP-dependent proteolytic machines

AAA+ proteases and remodeling machines couple hydrolysis of ATP to mechanical unfolding and translocation of proteins following recognition of sequence tags called degrons. Here, we use single-molecule optical trapping to determine the mechanochemistry of two AAA+ proteases, Escherichia coli ClpXP and ClpAP, as they unfold and translocate substrates containing multiple copies of the titin[superscript I27] domain during degradation initiated from the N terminus. Previous studies characterized degradation of related substrates with C-terminal degrons. We find that ClpXP and ClpAP unfold the wild-type titin I27 domain and a destabilized variant far more rapidly when pulling from the N terminus, whereas translocation speed is reduced only modestly in the N-to-C direction. These measurements establish the role of directionality in mechanical protein degradation, show that degron placement can change whether unfolding or translocation is rate limiting, and establish that one or a few power strokes are sufficient to unfold some protein domains. Keywords:protein degradation; AAA+ proteases; directional unfolding; AAA+ motorsNational Institutes of Health (U.S.) (Grant GM-101988)National Institutes of Health (U.S.) (Grant AI-15706

The Non-dominant AAA+ Ring in the ClpAP Protease Functions as an Anti-stalling Motor to Accelerate Protein Unfolding and Translocation

© 2020 The Authors ATP-powered unfoldases containing D1 and D2 AAA+ rings play important roles in protein homeostasis, but uncertainty about the function of each ring remains. Here we use single-molecule optical tweezers to assay mechanical unfolding and translocation by a variant of the ClpAP protease containing an ATPase-inactive D1 ring. This variant displays substantial mechanical defects in both unfolding and translocation of protein substrates. Notably, when D1 is hydrolytically inactive, ClpAP often stalls for times as long as minutes, and the substrate can back-slip through the enzyme when ATP concentrations are low. The inactive D1 variant also has more difficulty traveling in the N-to-C direction on a polypeptide track than it does moving in a C-to-N direction. These results indicate that D1 normally functions as an auxiliary/regulatory motor to promote uninterrupted enzyme advancement that is fueled largely by the D2 ring

The Intrinsically Disordered N-terminal Extension of the ClpS Adaptor Reprograms Its Partner AAA + ClpAP Protease

© 2020 Adaptor proteins modulate substrate selection by AAA + proteases. The ClpS adaptor delivers N-degron substrates to ClpAP but inhibits degradation of substrates bearing ssrA tags or other related degrons. How ClpS inhibits degradation of such substrates is poorly understood. Here, we demonstrate that ClpS impedes recognition of ssrA-tagged substrates by a non-competitive mechanism and also slows subsequent unfolding/translocation of these substrates as well as of N-degron substrates. This suppression of mechanical activity is largely a consequence of the ability of ClpS to repress ATP hydrolysis by ClpA, but several lines of evidence show that ClpS's inhibition of substrate binding and its ATPase repression are separable activities. Using ClpS mutants and ClpS-ClpA chimeras, we establish that engagement of the intrinsically disordered N-terminal extension of ClpS by ClpA is both necessary and sufficient to inhibit multiple steps of ClpAP-catalyzed degradation. These observations reveal how an adaptor can simultaneously modulate the catalytic activity of a AAA + enzyme, efficiently promote recognition of some substrates, suppress recognition of other substrates, and thereby affect degradation of its menu of substrates in a specific manner. We propose that similar mechanisms are likely to be used by other adaptors to regulate substrate choice and the catalytic activity of molecular machines