The activated ClpP peptidase forcefully grips a protein substrate.

ATPases associated with diverse cellular activities (AAA+) proteases power the maintenance of protein homeostasis by coupling ATP hydrolysis to mechanical protein unfolding, translocation, and ultimately degradation. Although ATPase activity drives a large portion of the mechanical work these molecular machines perform, how the peptidase contributes to the forceful denaturation and processive threading of substrates remains unknown. Here, using single-molecule optical trapping, we examine the mechanical activity of the caseinolytic peptidase P (ClpP) from Escherichia coli in the absence of a partner ATPase and in the presence of an activating small-molecule acyldepsipeptide.

Mechanical Protein Unfolding and Degradation.

AAA+ proteolytic machines use energy from ATP hydrolysis to degrade damaged, misfolded, or unneeded proteins. Protein degradation occurs within a barrel-shaped self-compartmentalized peptidase. Before protein substrates can enter this peptidase, they must be unfolded and then translocated through the axial pore of an AAA+ ring hexamer.

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, ClpXP and ClpAP, as they unfold and translocate substrates containing multiple copies of the titin domain during degradation initiated from the N terminus. Previous studies characterized degradation of related substrates with C-terminal degrons.

Mechanically Watching the ClpXP Proteolytic Machinery.

Energy-dependent protein degradation is studied through the dual bead ClpXP motility assay. Processing of folded proteins involves recognition, unfolding, translocation, and degradation stages. A dual optical trap, in a passive force-clamp geometry, exhibits bead-to-bead displacements that directly follow subprocesses underlying protein degradation.

Mechanistic insights into bacterial AAA+ proteases and protein-remodelling machines.

To maintain protein homeostasis, AAA+ proteolytic machines degrade damaged and unneeded proteins in bacteria, archaea and eukaryotes. This process involves the ATP-dependent unfolding of a target protein and its subsequent translocation into a self-compartmentalized proteolytic chamber. Related AAA+ enzymes also disaggregate and remodel proteins.

Dissection of Axial-Pore Loop Function during Unfolding and Translocation by a AAA+ Proteolytic Machine.

In the axial channels of ClpX and related hexameric AAA+ protein-remodeling rings, the pore-1 loops are thought to play important roles in engaging, mechanically unfolding, and translocating protein substrates. How these loops perform these functions and whether they also prevent substrate dissociation to ensure processive degradation by AAA+ proteases are open questions. Using ClpX pore-1-loop variants, single-molecule force spectroscopy, and ensemble assays, we find that the six pore-1 loops function synchronously to grip and unfold protein substrates during a power stroke but are not important in preventing substrate slipping between power strokes.

Mechanochemical basis of protein degradation by a double-ring AAA+ machine.

Molecular machines containing double or single AAA+ rings power energy-dependent protein degradation and other critical cellular processes, including disaggregation and remodeling of macromolecular complexes. How the mechanical activities of double-ring and single-ring AAA+ enzymes differ is unknown. Using single-molecule optical trapping, we determine how the double-ring ClpA enzyme from Escherichia coli, in complex with the ClpP peptidase, mechanically degrades proteins.

Stochastic but highly coordinated protein unfolding and translocation by the ClpXP proteolytic machine.

ClpXP and other AAA+ proteases recognize, mechanically unfold, and translocate target proteins into a chamber for proteolysis. It is not known whether these remarkable molecular machines operate by a stochastic or sequential mechanism or how power strokes relate to the ATP-hydrolysis cycle. Single-molecule optical trapping allows ClpXP unfolding to be directly visualized and reveals translocation steps of ∼1-4 nm in length, but how these activities relate to solution degradation and the physical properties of substrate proteins remains unclear.

Single-molecule protein unfolding and translocation by an ATP-fueled proteolytic machine.

All cells employ ATP-powered proteases for protein-quality control and regulation. In the ClpXP protease, ClpX is a AAA+ machine that recognizes specific protein substrates, unfolds these molecules, and then translocates the denatured polypeptide through a central pore and into ClpP for degradation. Here, we use optical-trapping nanometry to probe the mechanics of enzymatic unfolding and translocation of single molecules of a multidomain substrate.

Robust processivity of myosin V under off-axis loads.

The dimeric motor myosin V transports organelles along actin filament tracks over long distances in cells. Myosin V is a smart 'walker' that is able to swiftly adjust to variable 'road conditions' to continue its processive movement across dense cellular environments. Coordination between the two heads via intramolecular load modulates biochemical kinetics and ensures highly efficient unidirectional motion.

Myosin isoform determines the conformational dynamics and cooperativity of actin filaments in the strongly bound actomyosin complex.

We used transient phosphorescence anisotropy to detect the microsecond rotational dynamics of erythrosin-iodoacetamide-labeled actin strongly bound to single-headed fragments of muscle myosin subfragment 1 (S1) and non-muscle myosin V (MV). The conformational dynamics of actin filaments in solution are markedly influenced by the isoform of bound myosin. Both myosins increase the final anisotropy of actin at substoichiometric binding densities, indicating long-range, non-nearest neighbor cooperative restriction of filament rotational dynamics amplitude, but the cooperative unit is larger with MV than with muscle S1.

Watching the walk: observing chemo-mechanical coupling in a processive myosin motor.

Molecular motors are cellular nanomachines that convert the energy from nucleotide binding, hydrolysis, and product release into mechanical work. Because molecular motors contribute to fundamental processes in all living organisms, including genome replication, gene transcription, protein synthesis, organelle transport, and cell division, understanding how the chemical (ATP utilization) and mechanical (motility) cycles are linked is of fundamental importance. A recent study reports the direct visualization of simultaneous nucleotide binding and mechanical displacement of a single myosin 5a molecule, a processive molecular motor protein that takes successive approximately 36-nm steps along actin filaments of the cytoskeleton.

Structural and energetic analysis of activation by a cyclic nucleotide binding domain.

MlotiK1 is a prokaryotic homolog of cyclic-nucleotide-dependent ion channels that contains an intracellular C-terminal cyclic nucleotide binding (CNB) domain. X-ray structures of the CNB domain have been solved in the absence of ligand and bound to cAMP. Both the full-length channel and CNB domain fragment are easily expressed and purified, making MlotiK1 a useful model system for dissecting activation by ligand binding.

Load-dependent ADP binding to myosins V and VI: implications for subunit coordination and function.

Dimeric myosins V and VI travel long distances in opposite directions along actin filaments in cells, taking multiple steps in a "hand-over-hand" fashion. The catalytic cycles of both myosins are limited by ADP dissociation, which is considered a key step in the walking mechanism of these motors. Here, we demonstrate that external loads applied to individual actomyosin V or VI bonds asymmetrically affect ADP affinity, such that ADP binds weaker under loads assisting motility.

Widely distributed residues in thymosin beta4 are critical for actin binding.

We have investigated the contributions of hydrophobic residues, the conserved and variable proline residues, and the conserved lysine residues to the affinity and kinetics of thymosin beta4 (Tbeta4) binding to MgATP-actin monomers. Pro4, Lys18, Lys19, Pro27, Leu28, Pro29, and Ile34 were substituted with alanine residues. Mutagenesis of Pro4 or Pro27 has little effect ( View Article and Find Full Text PDF

How the Load and the Nucleotide State Affect the Actin Filament Binding Mode of the Molecular Motor Myosin V.

The interaction between actin and myosin V has been probed by measuring the unbinding force of individual actomyosin complexes using optical tweezers. Surprisingly, we found that in both the nucleotide-free and ADP-bound states single- and double-headed binding occurs with approximately the same probability. Estimation of the spring constant of individual actomyosin complexes confirmed that in each of the nucleotide states two distinct populations exist.

The tail domain of myosin Va modulates actin binding to one head.

Calcium activates full-length myosin Va steady-state enzymatic activity and favors the transition from a compact, folded "off" state to an extended "on" state. However, little is known of how a head-tail interaction alters the individual actin and nucleotide binding rate and equilibrium constants of the ATPase cycle. We measured the effect of calcium on nucleotide and actin filament binding to full-length myosin Va purified from chick brains.

Magnesium, ADP, and actin binding linkage of myosin V: evidence for multiple myosin V-ADP and actomyosin V-ADP states.

The [Mg(2+)] dependence of ADP binding to myosin V and actomyosin V was measured from the fluorescence of mantADP. Time courses of MgmantADP dissociation from myosin V and actomyosin V are biphasic with fast observed rate constants that depend on the [Mg(2+)] and slow observed rate constants that are [Mg(2+)]-independent. Two myosin V-MgADP states that are in reversible equilibrium, one that exchanges nucleotide and cation slowly (strong binding) and one that exchanges nucleotide and cation rapidly (weak binding), account for the data.

Mechanochemical coupling of two substeps in a single myosin V motor.

Myosin V is a double-headed processive molecular motor that moves along an actin filament by taking 36-nm steps. Using optical trapping nanometry with high spatiotemporal resolution, we discovered that there are two possible pathways for the 36-nm steps, one with 12- and 24-nm substeps, in this order, and the other without substeps. Based on the analyses of effects of ATP, ADP and 2,3-butanedione 2-monoxime (a reagent shown here to slow ADP release from actomyosin V) on the dwell time and the occurrence frequency of the main and the intermediate states, we propose that the 12-nm substep occurs after ATP binding to the bound trailing head and the 24-nm substep results from a mechanical step following the isomerization of an actomyosin-ADP state on the bound leading head.

Mechanism of nucleotide binding to actomyosin VI: evidence for allosteric head-head communication.

We have examined the kinetics of nucleotide binding to actomyosin VI by monitoring the fluorescence of pyrene-labeled actin filaments. ATP binds single-headed myosin VI following a two-step reaction mechanism with formation of a low affinity collision complex (1/K(1)' = 5.6 mm) followed by isomerization (k(+2)' = 176 s-1) to a state with weak actin affinity.

Synthesis, in vitro, and in vivo evaluation of phosphate ester derivatives of combretastatin A-4.

Combretastatin A-4 disodiumphosphate (CA4P), a prodrug formulation of the natural product combretastatin A-4 (CA4), is currently in clinical investigation for the treatment of cancer. In vivo, CA4P is rapidly enzymatically converted to CA4, a potent inhibitor of tubulin polymerization (IC(50)=1-2 microM), and rapidly causes bloodflow shutdown in tumor tissues. A variety of alkyl and aryl di- and triesters of CA4P have been synthesized and evaluated as potential CA4 prodrugs and/or stable CA4P analogues.