Hydrolysis Events Research Articles

MINFLUX reveals dynein stepping in live neurons

Dynein is the primary molecular motor responsible for retrograde intracellular transport of a variety of cargoes, performing successive nanometer-sized steps within milliseconds. Due to the limited spatiotemporal precision of established methods for molecular tracking, current knowledge of dynein stepping is essentially limited to slowed-down measurements in vitro. Here, we use MINFLUX fluorophore localization to directly track CRISPR/Cas9-tagged endogenous dynein with nanometer/millisecond precision in living primary neurons. We show that endogenous dynein primarily takes 8 nm steps, including frequent sideways steps but few backward steps. Strikingly, the majority of direction reversals between retrograde and anterograde movement occurred on the time scale of single steps (16 ms), suggesting a rapid regulatory reversal mechanism. Tug-of-war-like behavior during pauses or reversals was unexpectedly rare. By analyzing the dwell time between steps, we concluded that a single rate-limiting process underlies the dynein stepping mechanism, likely arising from just one adenosine 5'-triphosphate hydrolysis event being required during each step. Our study underscores the power of MINFLUX localization to elucidate the spatiotemporal changes underlying protein function in living cells.

Mechanistic behavior and subtle key events during DNA clamp opening and closing in T4 bacteriophage

Clamp loaders ensure processive DNA replication by loading the toroidal shaped sliding clamps onto the DNA. The sliding clamps serve as a platform for the attachment of polymerases and several other proteins associated with the regulation of various cellular processes. Clamp loaders are fascinating as nanomachines that engage in protein-protein and protein-DNA interactions. The loading mechanism of the clamp around dsDNA at the atomic level has not yet been fully explored. We performed microsecond timescale molecular dynamics simulations to reveal the dynamics of two different intermediate complexes involved in loading of the clamps around DNA. We conducted various time-dependent MD-driven analyses including the highly robust Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) calculations to observe changes in the structural elements of the clamp loader-clamp-DNA complexes in open and closed states. Our studies revealed the structural consequences of ATP hydrolysis events at different subunits of the clamp loader. This study would help in a better understanding of the clamp loading mechanism and would allow tackling various complications that might arise due to irregularities in this process.

A multivariate insight into the organoleptic properties of porcine muscle by ultrasound-assisted brining: Protein oxidation, water state and microstructure

Ultrasound could accelerate mass transfer of brine into postmortem muscle, but the biochemical consequences and architecture of cured products require more validations. Herein, we comparatively evaluated the water holding capacity (WHC), instrumental color, textural parameters, protein oxidation and hydrolysis events, and microstructure of porcine biceps femoris (BF) after static brining (SB) and ultrasound-assisted brining (UAB, 350 W for 1 h). Results showed that brining significantly increased oxidative susceptibility of muscle proteins, improved WHC and color (L*, internal a* and surface b* values) as well as textural properties (hardness, springiness and chewiness) compared to the control samples (P < 0.05). SDS-PAGE pattern suggested that UAB led to a great loss of major myofilament components with generation of 15–20 kDa extracellular protein fragments. Additionally, microscopic observation proved that UAB samples rendered more compact and enhanced microfissures in tissue fibers than SB samples, implying an increasing course of salt diffusion. Multivariant analysis and chemometrics revealed that water-protein interactions should be the most important variables used to explain the textural changes of BF muscles upon ultrasound-assisted brining.

Inflammatory endothelium-targeted and cathepsin responsive nanoparticles are effective against atherosclerosis.

Rationale: Atherosclerosis is characterized by lipid accumulation, plaque formation, and artery stenosis. The pharmacological treatment is a promising therapy for atherosclerosis, but this approach faces major challenges such as targeted drug delivery, controlled release, and non-specific clearance.Methods: Based on the finding that the cathepsin k (CTSK) enzyme is enriched in atherosclerotic lesions, we constructed an integrin αvβ3 targeted and CTSK-responsive nanoparticle to control the release of rapamycin (RAP) locally. The targeted and responsive nanoparticles (T/R NPs) were engineered by the self-assembly of a targeting polymer PLGA-PEG-c(RGDfC) and a CTSK-sensitive polymer PLGA-Pep-PEG. PLGA-Pep-PEG was also modified with a pair of FRET probe to monitor the hydrolysis events.Results: Our results indicated that RAP@T/R NPs accelerated the release of RAP in response to CTSK stimulation in vitro, which significantly inhibited the phagocytosis of OxLDL and the release of cytokines by inflammatory macrophages. Additionally, T/R NPs had prolonged blood retention time and increased accumulation in the early and late stage of atherosclerosis lesions. RAP@T/R NPs significantly blocked the development of atherosclerosis and suppressed the systemic and local inflammation in ApoE-/- mice.Conclusions: RAP@T/R NPs hold a great promise as a drug delivery system for safer and more efficient therapy of atherosclerosis.

Batch Electrogenic Systems Resolve Hydrolysis Events in Digestion of Complex Organic Compounds with High-Temporal Resolution

Batch Electrogenic Systems Resolve Hydrolysis Events in Digestion of Complex Organic Compounds with High-Temporal Resolution

A STUDY ON KINETIC CHARACTERIZATION OF CHANNEL-BLOCKING MUTANTS IN BRADYRHIZOBIUM JAPONICUM UTILIZATION A

Proline utilization A (PutA) from Bradyrhizobium japonicum (BjPutA) is a bifunctional avoenzyme that catalyzes the oxidation of proline to glutamate using fused proline dehydrogenase (PRODH) and ∆1-pyrroline-5-carboxylate dehydrogenase (P5CDH) domains. Recent crystal structures and kinetic data suggest an intramolecular channel connects the two active sites, promoting substrate channeling of the intermediate P5C from the PRODH domain to the P5CDH domain. In this work several mutations were made along the channel in an effort to block passage of P5C to the second active site. Analysis of several site-specic mutants in the substrate channel of BjPutA revealed an important role for D779 in the channeling path. BjPutA mutants D779Y and D779W signicantly decreased the overall PRODH-P5CDH channeling reaction indicating that bulky mutations at residue D779 impede travel of P5C through the channel. Interestingly, D779Y and D779W also exhibited lower P5CDH activity, suggesting that exogenous P5C must enter the channel upstream of D779. Replacing D779 with a smaller residue (D779A) had no effect on the catalytic and channeling properties of BjPutA showing that the carboxylate group of D779 is not essential for channeling. An identical mutation at D778 (D778Y) did not impact BjPutA channeling activity. Thus, D779 is optimally orientated so that replacement with the larger side chains of Tyr/Trp blocks P5C movment through the channel. The kinetic data reveal not only that bulky mutations at residue D779 hinder passage of P5C to the second active site, but also P5C must use the channel to efciently access the P5CDH domain. Moreover, these mutants may be used to learn more about the hydrolysis event that is thought to take place within the channel

Atomistic basis of force generation, translocation, and coordination in a viral genome packaging motor.

Double-stranded DNA viruses package their genomes into pre-assembled capsids using virally-encoded ASCE ATPase ring motors. We present the first atomic-resolution crystal structure of a multimeric ring form of a viral dsDNA packaging motor, the ATPase of the asccφ28 phage, and characterize its atomic-level dynamics via long timescale molecular dynamics simulations. Based on these results, and previous single-molecule data and cryo-EM reconstruction of the homologous φ29 motor, we propose an overall packaging model that is driven by helical-to-planar transitions of the ring motor. These transitions are coordinated by inter-subunit interactions that regulate catalytic and force-generating events. Stepwise ATP binding to individual subunits increase their affinity for the helical DNA phosphate backbone, resulting in distortion away from the planar ring towards a helical configuration, inducing mechanical strain. Subsequent sequential hydrolysis events alleviate the accumulated mechanical strain, allowing a stepwise return of the motor to the planar conformation, translocating DNA in the process. This type of helical-to-planar mechanism could serve as a general framework for ring ATPases.

Combining Single-Molecule Fluorescence and MD-Simulations to Delineate the Kinetics and Regulation of Proteins

Protein conformational kinetics and its regulation occur on many time- and length-scales. While for example post-translational modifications act very locally, specific protein-protein interaction typically affect larger domains, and global changes (like crowding) affect the whole protein (complex) non-specifically. In a first part we use a combination of single-molecule FRET, FRET-FCS, ns-FCS and MD simulations to delineate the timescales for information transfer from the hydrolysis event in the nucleotide binding site of the heat shock protein 90 (Hsp90) to large conformational changes in the Hsp90 dimer[1]. This allosteric process occurs via a hierarchical mechanism that involves timescales from pico- to microseconds. In a second part we show protein regulation under different degrees of localization, and present the effects on the Hsp90 chaperone system. Interestingly using single-molecule FRET, we find that similar functional and conformational steady states are caused by completely different underlying kinetics. We will introduce a new mechanistic concept, namely functional stimulation through conformational confinement[2]. Finally, we use a network of distance distributions obtained with single molecule FRET to investigate a Hsp90 - model client interaction[3]. We find negligible effects of the client on the open-close equilibrium of Hsp90, but strong effects on Hsp90's conformational fluctuations. This hints towards an additional (orthogonal) regulation mechanism. Altogether, these applications demonstrate that a combination of (single-molecule) methods is ideal to investigate the biophysics of multi-component (protein) systems. [1] S. Wolf et al., Allosteric action of nucleotides on Hsp90 across several time- and length scales. BioRxiv, 2020.02.15.950725 (2020); https://doi.org/10.1101/2020.02.15.950725 [2] S. Schmid and T. Hugel, Controlling protein function by fine-tuning conformational flexibility. eLife, 9, 1013 (2020); https://doi.org/10.7554/eLife.57180 [3] Hellenkamp et al., Multidomain structure and correlated dynamics determined by self-consistent FRET networks. Nat Methods, 14 (2), 174 (2017). https://doi.org/10.1038/nmeth.4081

Sisyphus observed: Unraveling the high ATP usage of an RNA chaperone

DEAD-box proteins are nonprocessive RNA helicases that can function as RNA chaperones by coupling ATP binding and hydrolysis to structural reorganization of RNA. Here, Jarmoskaite et al. quantify the ATP utilization of an RNA chaperone during refolding of a misfolded ribozyme substrate. Strikingly, 100 ATP hydrolysis events are needed per successfully refolded ribozyme, suggesting that each round of unfolding requires ten ATP molecules, since 90% of substrate unfolding cycles only lead back to the kinetically favored misfolded state. This near-Sisyphean effort reveals a potentially conserved model for RNA reorganization by RNA chaperones.

Refined measurement of SecA-driven protein secretion reveals that translocation is indirectly coupled to ATP turnover

The universally conserved Sec system is the primary method cells utilize to transport proteins across membranes. Until recently, measuring the activity-a prerequisite for understanding how biological systems work-has been limited to discontinuous protein transport assays with poor time resolution or reported by large, nonnatural tags that perturb the process. The development of an assay based on a split superbright luciferase (NanoLuc) changed this. Here, we exploit this technology to unpick the steps that constitute posttranslational protein transport in bacteria. Under the conditions deployed, the transport of a model preprotein substrate (proSpy) occurs at 200 amino acids (aa) per minute, with SecA able to dissociate and rebind during transport. Prior to that, there is no evidence for a distinct, rate-limiting initiation event. Kinetic modeling suggests that SecA-driven transport activity is best described by a series of large (∼30 aa) steps, each coupled to hundreds of ATP hydrolysis events. The features we describe are consistent with a nondeterministic motor mechanism, such as a Brownian ratchet.

Spatio-temporal correlations between catastrophe events in a microtubule bundle: a computational study.

We explore correlations between dynamics of different microtubules in a bundle, via numerical simulations, using a one-dimensional stochastic model of a microtubule. The guanosine triphosphate (GTP)-bound tubulins undergo diffusion-limited binding to the tip. Random hydrolysis events take place along the microtubule and converts the bound GTP in tubulin to guanosine diphosphate (GDP). The microtubule starts depolymerising when the monomer at the tip becomes GDP-bound; in this case, detachment of GDP-tubulin ensues and continues until either GTP-bound tubulin is exposed or complete depolymerisation is achieved. In the latter case, the microtubule is defined to have undergone a "catastrophe". Our results show that, in general, the dynamics of growth and catastrophe in different microtubules are coupled to each other; the closer the microtubules are, the stronger the coupling. In particular, all microtubules grow slower, on average, when brought closer together. The reduction in growth velocity also leads to more frequent catastrophes. More dramatically, catastrophe events in the different microtubules forming a bundle are found to be correlated; a catastrophe event in one microtubule is more likely to be followed by a similar event in the same microtubule. This propensity of bunching disappears when the microtubules move farther apart.

Single-molecule analysis reveals rotational substeps and chemo-mechanical coupling scheme of Enterococcus hirae V1-ATPase

V1-ATPase (V1), the catalytic domain of an ion-pumping V-ATPase, is a molecular motor that converts ATP hydrolysis–derived chemical energy into rotation. Here, using a gold nanoparticle probe, we directly observed rotation of V1 from the pathogen Enterococcus hirae (EhV1). We found that 120° steps in each ATP hydrolysis event are divided into 40 and 80° substeps. In the main pause before the 40° substep and at low ATP concentration ([ATP]), the time constant was inversely proportional to [ATP], indicating that ATP binds during the main pause with a rate constant of 1.0 × 107 m−1 s−1. At high [ATP], we observed two [ATP]-independent time constants (0.5 and 0.7 ms). One of two time constants was prolonged (144 ms) in a rotation driven by slowly hydrolyzable ATPγS, indicating that ATP is cleaved during the main pause. In another subpause before the 80° substep, we noted an [ATP]-independent time constant (2.5 ms). Furthermore, in an ATP-driven rotation of an arginine-finger mutant in the presence of ADP, −80 and −40° backward steps were observed. The time constants of the pauses before −80° backward and +40° recovery steps were inversely proportional to [ADP] and [ATP], respectively, indicating that ADP- and ATP-binding events trigger these steps. Assuming that backward steps are reverse reactions, we conclude that 40 and 80° substeps are triggered by ATP binding and ADP release, respectively, and that the remaining time constant in the main pause represents phosphate release. We propose a chemo-mechanical coupling scheme of EhV1, including substeps largely different from those of F1-ATPases.

Discovery of processive catalysis by an exo-hydrolase with a pocket-shaped active site

Substrates associate and products dissociate from enzyme catalytic sites rapidly, which hampers investigations of their trajectories. The high-resolution structure of the native Hordeum exo-hydrolase HvExoI isolated from seedlings reveals that non-covalently trapped glucose forms a stable enzyme-product complex. Here, we report that the alkyl β-d-glucoside and methyl 6-thio-β-gentiobioside substrate analogues perfused in crystalline HvExoI bind across the catalytic site after they displace glucose, while methyl 2-thio-β-sophoroside attaches nearby. Structural analyses and multi-scale molecular modelling of nanoscale reactant movements in HvExoI reveal that upon productive binding of incoming substrates, the glucose product modifies its binding patterns and evokes the formation of a transient lateral cavity, which serves as a conduit for glucose departure to allow for the next catalytic round. This path enables substrate-product assisted processive catalysis through multiple hydrolytic events without HvExoI losing contact with oligo- or polymeric substrates. We anticipate that such enzyme plasticity could be prevalent among exo-hydrolases.

Proteolysis mediated by the membrane-integrated ATP-dependent protease FtsH has a unique nonlinear dependence on ATP hydrolysis rates.

ATPases associated with diverse cellular activities (AAA+) proteases utilize ATP hydrolysis to actively unfold native or misfolded proteins and translocate them into a protease chamber for degradation. This basic mechanism yields diverse cellular consequences, including the removal of misfolded proteins, control of regulatory circuits, and remodeling of protein conformation. Among various bacterial AAA+ proteases, FtsH is only membrane-integrated and plays a key role in membrane protein quality control. Previously, we have shown that FtsH has substantial unfoldase activity for degrading membrane proteins overcoming a dual energetic burden of substrate unfolding and membrane dislocation. Here, we asked how efficiently FtsH utilizes ATP hydrolysis to degrade membrane proteins. To answer this question, we measured degradation rates of the model membrane substrate GlpG at various ATP hydrolysis rates in the lipid bilayers. We find that the dependence of degradation rates on ATP hydrolysis rates is highly nonlinear: (i) FtsH cannot degrade GlpG until it reaches a threshold ATP hydrolysis rate; (ii) after exceeding the threshold, the degradation rates steeply increase and saturate at the ATP hydrolysis rates far below the maxima. During the steep increase, FtsH efficiently utilizes ATP hydrolysis for degradation, consuming only 40-60% of the total ATP cost measured at the maximal ATP hydrolysis rates. This behavior does not fundamentally change against water-soluble substrates as well as upon addition of the macromolecular crowding agent Ficoll 70. The Hill analysis shows that the nonlinearity stems from coupling of three to five ATP hydrolysis events to degradation, which represents unique cooperativity compared to other AAA+ proteases including ClpXP, HslUV, Lon, and proteasomes.

Multi-scale structure and pasting/digestion features of yam bean tuber starches

This work discloses the multi-scale structure and pasting/digestion behaviors of yam bean tuber starches (YB-GD and YB-SC). Compared to potato starch, YB starches exhibited varied pasting/digestion features that were understood from a multi-scale structural view. Especially, YB starches showed relatively polydisperse lamellae, less perfect crystallites, and fewer amylose molecules. These features should reduce the bulk density of starch chain packing, and could facilitate the water or enzyme diffusion in starch matrixes. Consistently, not only was starch’s resistance to hydrothermal effects weakened (shown by reduced pasting temperature), but also the enzyme absorption to starch chains and the subsequent hydrolysis events were accelerated. Furthermore, YB starch molecules had more short chains, which played roles in reducing the paste viscosity along with the reduced granule size and in enhancing the paste stabilities during heating and cooling. Also, those molecular features tended to speed up the enzyme diffusion and digestion events.

Cryo-EM structures and dynamics of substrate-engaged human 26S proteasome.

The proteasome is an ATP-dependent, 2.5-megadalton machine responsible for selective protein degradation in eukaryotic cells. Here we present cryo-EM structures of the substrate-engaged human proteasome in seven conformational states at 2.8-3.6 Å resolution, captured during breakdown of a polyubiquitylated protein. These structures visualize a continuum of dynamic substrate-proteasome interactions from ubiquitin recognition to substrate translocation, during which ATP hydrolysis sequentially navigates through all six ATPases. Three principal modes of coordinated hydrolysis are observed, featuring hydrolytic events in two oppositely positioned ATPases, in two adjacent ATPases, and in one ATPase at a time. These hydrolytic modes regulate deubiquitylation, translocation initiation and processive unfolding of substrates, respectively. ATP hydrolysis powers a hinge-like motion in each ATPase that regulates its substrate interaction. Synchronization of ATP binding, ADP release and ATP hydrolysis in three adjacent ATPases drives rigid-body rotations of substrate-bound ATPases that are propagated unidirectionally in the ATPase ring and unfold the substrate.

Substrate-engaged 26S proteasome structures reveal mechanisms for ATP-hydrolysis-driven translocation.

The 26S proteasome is the primary eukaryotic degradation machine and thus is critically involved in numerous cellular processes. The heterohexameric adenosine triphosphatase (ATPase) motor of the proteasome unfolds and translocates targeted protein substrates into the open gate of a proteolytic core while a proteasomal deubiquitinase concomitantly removes substrate-attached ubiquitin chains. However, the mechanisms by which ATP hydrolysis drives the conformational changes responsible for these processes have remained elusive. Here we present the cryo-electron microscopy structures of four distinct conformational states of the actively ATP-hydrolyzing, substrate-engaged 26S proteasome. These structures reveal how mechanical substrate translocation accelerates deubiquitination and how ATP-binding, -hydrolysis, and phosphate-release events are coordinated within the AAA+ (ATPases associated with diverse cellular activities) motor to induce conformational changes and propel the substrate through the central pore.

The Hydrazine–O2 Redox Couple as a Platform for Organocatalytic Oxidation: Benzo[c]cinnoline‐Catalyzed Oxidation of Alkyl Halides to Aldehydes

AbstractAn organocatalytic oxidation platform that capitalizes on the capacity of hydrazines to undergo rapid autoxidation to diazenes is described. Commercially available benzo[c]cinnoline is shown to catalyze the oxidation of alkyl halides to aldehydes in a novel mechanistic paradigm involving nucleophilic attack, prototropic shift, and hydrolysis. The hydrolysis and reoxidation events occur readily with only adventitious oxygen and water. A survey of the scope of viable substrates is shown along with mechanistic and computational studies that give insight into this mode of catalysis.

The Hydrazine-O2 Redox Couple as a Platform for Organocatalytic Oxidation: Benzo[c]cinnoline-Catalyzed Oxidation of Alkyl Halides to Aldehydes.

An organocatalytic oxidation platform that capitalizes on the capacity of hydrazines to undergo rapid autoxidation to diazenes is described. Commercially available benzo[c]cinnoline is shown to catalyze the oxidation of alkyl halides to aldehydes in a novel mechanistic paradigm involving nucleophilic attack, prototropic shift, and hydrolysis. The hydrolysis and reoxidation events occur readily with only adventitious oxygen and water. A survey of the scope of viable substrates is shown along with mechanistic and computational studies that give insight into this mode of catalysis.

Ribosome recycling is coordinated by processive events in two asymmetric ATP sites of ABCE1.

Ribosome recycling orchestrated by ABCE1 is a fundamental process in protein translation and mRNA surveillance, connecting termination with initiation. Beyond the plenitude of well-studied translational GTPases, ABCE1 is the only essential factor energized by ATP, delivering the energy for ribosome splitting via two nucleotide-binding sites by a yet unknown mechanism. Here, we define how allosterically coupled ATP binding and hydrolysis events in ABCE1 empower ribosome recycling. ATP occlusion in the low-turnover control site II promotes formation of the pre-splitting complex and facilitates ATP engagement in the high-turnover site I, which in turn drives the structural re-organization required for ribosome splitting. ATP hydrolysis and ensuing release of ABCE1 from the small subunit terminate the post-splitting complex. Thus, ABCE1 runs through an allosterically coupled cycle of closure and opening at both sites consistent with a processive clamp model. This study delineates the inner mechanics of ABCE1 and reveals why various ABCE1 mutants lead to defects in cell homeostasis, growth, and differentiation.