Search

Grp94, an ER-localized molecular chaperone, is required for the folding and activation of many membrane and secretory proteins. Client activation by Grp94 is mediated by nucleotide and conformational changes. In this work, we aim to understand how microscopic changes from nucleotide hydrolysis can potentiate large-scale conformational changes of Grp94. We performed all-atom molecular dynamics simulations on the ATP-hydrolysis competent state of the Grp94 dimer in four different nucleotide bound states. We found that Grp94 was the most rigid when ATP was bound. ATP hydrolysis or nucleotide removal enhanced mobility of the N-terminal domain and ATP lid, resulting in suppression of interdomain communication. In an asymmetric conformation with one hydrolyzed nucleotide, we identified a more compact state, similar to experimental observations. We also identified a potential regulatory role of the flexible linker, as it formed electrostatic interactions with the Grp94 M-domain helix near the region where BiP is known to bind. These studies were complemented with normal-mode analysis of an elastic network model to investigate Grp94's large-scale conformational changes. SPM analysis identified residues that are important in signaling conformational change, many of which have known functional relevance in ATP coordination and catalysis, client binding, and BiP binding. Our findings suggest that ATP hydrolysis in Grp94 alters allosteric wiring and facilitates conformational changes.

Proteolysis is essential for the control of metabolic pathways and the cell cycle. Bacterial caseinolytic proteases (Clp) use peptidase components, such as ClpP, to degrade defective substrate proteins and to regulate cellular levels of stress-response proteins. To ensure selective degradation, access to the proteolytic chamber of the double-ring ClpP tetradecamer is controlled by a critical gating mechanism of the two axial pores. The binding of conserved loops of the Clp ATPase component of the protease or small molecules, such as acyldepsipeptide (ADEP), at peripheral ClpP ring sites, triggers axial pore opening through dramatic conformational transitions of flexible N-terminal loops between disordered conformations in the "closed" pore state and ordered hairpins in the "open" pore state. In this study, we probe the allosteric communication underlying these conformational changes by comparing residue-residue couplings in molecular dynamics simulations of each configuration. Both principal component and normal mode analyses highlight large-scale conformational changes in the N-terminal loop regions and smaller amplitude motions of the peptidase core. Community network analysis reveals a switch between intra- and inter-protomer coupling in the open-closed pore transition. Allosteric pathways that connect the ADEP binding sites to N-terminal loops are rewired in this transition, with shorter network paths in the open pore configuration supporting stronger intra- and inter-ring coupling. Structural perturbations, either through the removal of ADEP molecules or point mutations, alter the allosteric network to weaken the coupling.

Although it is known that majority of kinesin motors walk predominantly toward the plus end of microtubules (MTs) in a hand-over-hand manner, the structural origin of the stepping directionality is not understood. To resolve this issue, we modeled the structures of kinesin-1 (Kin1), MT, and the Kin1-MT complex using the elastic network model and calculated the residue-dependent responses to a local perturbation in the constructs. Kin1 binding elicits an asymmetric response that is pronounced in α/β-tubulin dimers in the plus end of the MT. Kin1 opens the clefts of multiple plus end α/β-tubulin dimers, creating binding-competent conformations, which is required for processivity. Reciprocally, MT induces correlations between switches I and II in the motor and enhances fluctuations in adenosine 5'-diphosphate and the residues in the binding pocket. Our findings explain both the directionality of stepping and MT effects on a key step in the catalytic cycle of kinesin.

The 70 kDa and 90 kDa heat shock proteins Hsp70 and Hsp90 are two abundant and highly conserved ATP-dependent molecular chaperones that participate in the maintenance of cellular homeostasis. In Escherichia coli , Hsp90 (Hsp90Ec) and Hsp70 (DnaK) directly interact and collaborate in protein remodeling. Previous work has produced a model of the direct interaction of both chaperones. The locations of the residues involved have been confirmed and the model has been validated. In this study, we investigate the allosteric communication between Hsp90Ec and DnaK and how the chaperones couple their conformational cycles. Using elastic network models (ENM), normal mode analysis (NMA), and a structural perturbation method (SPM) of asymmetric and symmetric DnaK-Hsp90Ec, we extract biologically relevant vibrations and identify residues involved in allosteric signaling. When one DnaK is bound, the dominant normal modes favor biological motions that orient a substrate protein bound to DnaK within the substrate/client binding site of Hsp90Ec and release the substrate from the DnaK substrate binding domain. The presence of one DnaK molecule stabilizes the entire Hsp90Ec protomer to which it is bound. Conversely, the symmetric model of DnaK binding results in steric clashes of DnaK molecules and suggests that the Hsp90Ec and DnaK chaperone cycles operate independently. Together, this data supports an asymmetric binding of DnaK to Hsp90Ec.

The molecular motor myosin V transports cargo by stepping on actin filaments, executing a random diffusive search for actin binding sites at each step. A recent experiment suggests that the joint between the myosin lever arms may not rotate freely, as assumed in earlier studies, but instead has a preferred angle giving rise to structurally constrained diffusion. We address this controversy through comprehensive analytical and numerical modeling of myosin V diffusion and stepping. When the joint is constrained, our model reproduces the experimentally observed diffusion, allowing us to estimate bounds on the constraint energy. We also test the consistency between the constrained diffusion model and previous measurements of step size distributions and the load dependence of various observable quantities. The theory lets us address the biological significance of the constrained joint and provides testable predictions of new myosin behaviors, including the stomp distribution and the run length under off-axis force., Competing Interests: DH, RT, MH, DT No competing interests declared, (© 2020, Hathcock et al.)

The molecular motor myosin V (MyoV) exhibits a wide repertoire of pathways during the stepping process, which is intimately connected to its biological function. The best understood of these is the hand-over-hand stepping by a swinging lever arm movement toward the plus end of actin filaments. Single-molecule experiments have also shown that the motor "foot stomps," with one hand detaching and rebinding to the same site, and back-steps under sufficient load. The complete taxonomy of MyoV's load-dependent stepping pathways, and the extent to which these are constrained by motor structure and mechanochemistry, are not understood. Using a polymer model, we develop an analytical theory to describe the minimal physical properties that govern motor dynamics. We solve the first-passage problem of the head reaching the target-binding site, investigating the competing effects of backward load, strain in the leading head biasing the diffusion in the direction of the target, and the possibility of preferential binding to the forward site due to the recovery stroke. The theory reproduces a variety of experimental data, including the power stroke and slow diffusive search regimes in the mean trajectory of the detached head, and the force dependence of the forward-to-backward step ratio, run length, and velocity. We derive a stall force formula, determined by lever arm compliance and chemical cycle rates. By exploring the MyoV design space, we predict that it is a robust motor whose dynamical behavior is not compromised by reasonable perturbations to the reaction cycle and changes in the architecture of the lever arm.

Chaperonins are molecular machines that use ATP-driven cycles to assist misfolded substrate proteins to reach the native state. During the functional cycle, these machines adopt distinct nucleotide-dependent conformational states, which reflect large-scale allosteric changes in individual subunits. Distinct allosteric kinetics has been described for the two chaperonin classes. Bacterial (group I) chaperonins, such as GroEL, undergo concerted subunit motions within each ring, whereas archaeal and eukaryotic chaperonins (group II) undergo sequential subunit motions. We study these distinct mechanisms through a comparative normal mode analysis of monomer and double-ring structures of the archaeal chaperonin thermosome and GroEL. We find that thermosome monomers of each type exhibit common low-frequency behavior of normal modes. The observed distinct higher-frequency modes are attributed to functional specialization of these subunit types. The thermosome double-ring structure has larger contribution from higher-frequency modes, as it is found in the GroEL case. We find that long-range intersubunit correlation of amino-acid pairs is weaker in the thermosome ring than in GroEL. Overall, our results indicate that distinct allosteric behavior of the two chaperonin classes originates from different wiring of individual subunits as well as of the intersubunit communications., (Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The detachment kinetics from actin upon ATP binding is a key step in the reaction cycle of myosin V. We show that a network of residues, constituting the allostery wiring diagram (AWD), that trigger the rigor (R) to post-rigor (PR) transition, span key structural elements from the ATP and actin-binding regions. Several of the residues are in the 33 residue helix (H18), P loop, and switch I. Brownian dynamics simulations show that a hierarchy of kinetically controlled local structural changes leads to the opening of the "cleft" region, resulting in the detachment of the motor domain from actin. Movements in switch I and P loop facilitate changes in the rest of the motor domain, in particular the rotation of H18, whose stiffness within the motor domain is crucial in the R --> PR transition. The finding that residues in the AWD also drive the kinetics of the R --> PR transition shows how the myosin architecture regulates the allosteric movements during the reaction cycle., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Determining the network of residues that transmit allosteric signals is crucial to understanding the function of biological nanomachines. During the course of a reaction cycle, biological machines in general, and Escherichia coli chaperonin GroEL in particular, undergo large-scale conformational changes in response to ligand binding. Normal mode analyses, based on structure-based coarse-grained models where each residue is represented by an alpha carbon atom, have been widely used to describe the motions encoded in the structures of proteins. Here, we propose a new Calpha-side chain elastic network model of proteins that includes information about the physical identity of each residue and accurately accounts for the side-chain topology and packing within the structure. Using the Calpha-side chain elastic network model and the structural perturbation method, which probes the response of a local perturbation at a given site at all other sites in the structure, we determine the network of key residues (allostery wiring diagram) responsible for the T-->R and R''-->T transitions in GroEL. A number of residues, both within a subunit and at the interface of two adjacent subunits, are found to be at the origin of the positive cooperativity in the ATP-driven T-->R transition. Of particular note are residues G244, R58, D83, E209, and K327. Of these, R38, D83, and K327 are highly conserved. G244 is located in the apical domain at the interface between two subunits; E209 and K327 are located in the apical domain, toward the center of a subunit; R58 and D83 are equatorial domain residues. The allostery wiring diagram shows that the network of residues are interspersed throughout the structure. Residues D83, V174, E191, and D359 play a critical role in the R''-->T transition, which implies that mutations of these residues would compromise the ATPase activity. D83 and E191 are also highly conserved; D359 is moderately conserved. The negative cooperativity between the rings in the R''-->T transition is orchestrated through several interface residues within a single ring, including N10, E434, D435, and E451. Signal from the trans ring that is transmitted across the interface between the equatorial domains is responsible for the R''-->T transition. The cochaperonin GroES plays a passive role in the R''-->T transition. Remarkably, the binding affinity of GroES for GroEL is allosterically linked to GroEL residues 350-365 that span helices K and L. The movements of helices K and L alter the polarity of the cavity throughout the GroEL functional cycle and undergo large-scale motions that are anticorrelated with the other apical domain residues. The allostery wiring diagrams for the T-->R and R''-->T transitions of GroEL provide a microscopic foundation for the cooperativity (anticooperativity) within (between) the ring (rings). Using statistical coupling analysis, we extract evolutionarily linked clusters of residues in GroEL and GroES. We find that several substrate protein binding residues as well as sites related to ATPase activity belong to a single functional network in GroEL. For GroES, the mobile loop residues and GroES/GroES interface residues are linked.

The bacterial chaperonin GroEL and the co-chaperonin GroES assist in the folding of a number of structurally unrelated substrate proteins (SPs). In the absence of chaperonins, SP folds by the kinetic partitioning mechanism (KPM), according to which a fraction of unfolded molecules reaches the native state directly, while the remaining fraction gets trapped in a potentially aggregation-prone misfolded state. During the catalytic reaction cycle, GroEL undergoes a series of allosteric transitions (T<-->R-->R"-->T) triggered by SP capture, ATP binding and hydrolysis, and GroES binding. We developed a general kinetic model that takes into account the coupling between the rates of the allosteric transitions and the folding and aggregation of the SP. Our model, in which the GroEL allosteric rates and SP-dependent folding and aggregation rates are independently varied without prior assumption, quantitatively fits the GroEL concentration-dependent data on the yield of native ribulose bisphosphate carboxylase/oxygenase (Rubisco) as a function of time. The extracted kinetic parameters for the GroEL reaction cycle are consistent with the available values from independent experiments. In addition, we also obtained physically reasonable parameters for the kinetic steps in the reaction cycle that are difficult to measure. If experimental values for GroEL allosteric rates are used, the time-dependent changes in native-state yield at eight GroEL concentrations can be quantitatively fit using only three SP-dependent parameters. The model predicts that the differences in the efficiencies (as measured by yields of the native state) of GroEL, single-ring mutant (SR1), and variants of SR1, in the rescue of mitochondrial malate dehydrogenase, citrate synthase, and Rubisco, are related to the large variations in the allosteric transition rates. We also show that GroEL/S mutants that efficiently fold one SP at the expense of all others are due to a decrease in the rate of a key step in the reaction cycle, which implies that wild-type GroEL has evolved as a compromise between generality and specificity. We predict that, under maximum loading conditions and saturating ATP concentration, the efficiency of GroEL (using parameters for Rubisco) depends predominantly on the rate of R-->R" transition, while the equilibrium constant of the T<-->R has a small effect only. Both under sub- and superstoichiometric GroEL concentrations, enhanced efficiency is achieved by rapid turnover of the reaction cycle, which is in accord with the predictions of the iterative annealing mechanism. The effects are most dramatic at substoichiometric conditions (most relevant for in vivo situations) when SP aggregation can outcompete capture of SP by chaperonins.