Conformational Gating Research Articles

Gating residues govern ligand unbinding kinetics from the buried cavity in HIF-2α PAS-B.

While transcription factors have been generally perceived as "undruggable," an exception is the HIF-2 hypoxia-inducible transcription factor, which contains an internal cavity that is sufficiently large to accommodate a range of small-molecules, including the therapeutically used inhibitor belzutifan. Given the relatively long ligand residence times of these small molecules and the lack of any experimentally observed pathway connecting the cavity to solvent, there has been great interest in understanding how these drug ligands exit the buried receptor cavity. Here, we focus on the relevant PAS-B domain of hypoxia-inducible factor 2α (HIF-2α) and examine how one such small molecule (THS-017) exits from the buried cavity within this domain on the seconds-timescale using atomistic simulations and ZZ-exchange NMR. To enable the simulations, we applied the weighted ensemble path sampling strategy, which generates continuous pathways for a rare-event process [e.g., ligand (un)binding] with rigorous kinetics in orders of magnitude less computing time compared to conventional simulations. Results reveal the formation of an encounter complex intermediate and two distinct classes of pathways for ligand exit. Based on these pathways, we identified two pairs of conformational gating residues in the receptor: one for the major class (N288 and S304) and another for the minor class (L272 and M309). ZZ-exchange NMR validated the kinetic importance of N288 for ligand unbinding. Our results provide an ideal simulation dataset for rational manipulation of ligand unbinding kinetics.

Reducing Off-State and Leakage Currents by Dielectric Permittivity-Graded Stacked Gate Oxides on Trigate FinFETs: A TCAD Study.

Since its invention in the 1960s, one of the most significant evolutions of metal-oxide semiconductor field effect transistors (MOSFETs) would be the 3D version that makes the semiconducting channel vertically wrapped by conformal gate electrodes, also recognized as FinFET. During recent decades, the width of fin (Wfin) and the neighboring gate oxide width (tox) in FinFETs has shrunk from about 150 nm to a few nanometers. However, both widths seem to have been leveling off in recent years, owing to the limitation of lithography precision. Here, we show that by adapting the Penn model and Maxwell-Garnett mixing formula for a dielectric constant (κ) calculation for nanolaminate structures, FinFETs with two- and three-stage κ-graded stacked combinations of gate dielectrics with SiO2, Si3N4, Al2O3, HfO2, La2O3, and TiO2 perform better against the same structures with their single-layer dielectrics counterparts. Based on this, FinFETs simulated with κ-graded gate oxides achieved an off-state drain current (IOFF) reduced down to 6.45 × 10-15 A for the Al2O3: TiO2 combination and a gate leakage current (IG) reaching down to 2.04 × 10-11 A for the Al2O3: HfO2: La2O3 combination. While our findings push the individual dielectric laminates to the sub 1 nm limit, the effects of dielectric permittivity matching and κ-grading for gate oxides remain to have the potential to shed light on the next generation of nanoelectronics for higher integration and lower power consumption opportunities.

High-performance monolayer MoS2 nanosheet GAA transistor

In this article, a 0.7 nm thick monolayer MoS2 nanosheet gate-all-around field effect transistors (NS-GAAFETs) with conformal high-κ metal gate deposition are demonstrated. The device with 40 nm channel length exhibits a high on-state current density of ~410 μA μm−1 with a large on/off ratio of 6 × 108 at drain voltage = 1 V. The extracted contact resistance is 0.48 ± 0.1 kΩ μm in monolayer MoS2 NS-GAAFETs, thereby showing the channel-dominated performance with the channel length scaling from 80 to 40 nm. The successful demonstration of device performance in this work verifies the integration potential of transition metal dichalcogenides for future logic transistor applications.

Excited State Dynamics of a Conformationally Fluxional Copper Coordination Complex.

The conversion of solar energy into chemical fuel represents a capstone goal of the 21st century and has the potential to supply terawatts of power in a globally distributed manner. However, the disparate time scales of photodriven charge separation (∼fs) and steps in chemical reactions (∼μs) represent an inherent bottleneck in solar-to-fuels technology. To address this discrepancy, we are developing earth-abundant coordination complexes that undergo light-induced conformational rearrangements such that charge separation (CS) is hastened, while charge recombination (CR) is slowed. To these ends, we report the preparation and characterization of a new series of conformationally fluxional copper coordination complexes that contain a twisted intramolecular charge transfer (TICT) fluorophore as part of their ligand scaffold. Structural and spectroscopic characterization of the Cu(I) and Cu(II) complexes formed with these ligands in their ground states establish oxidation state-dependent conformational dynamicity, while time-resolved emission and transient absorption spectroscopies define the photophysical parameters of photo-induced excited states. Building on initial reports with a related set of molecules, the improved ligand design presented here greatly simplifies the observed photophysics, effectively shutting down unwanted ligand-centered excited states previously observed. Time-dependent density functional theory (TDDFT) analyses reveal an unusual metal-to-TICT electronic transition only reported once before, and though the formation of a CS state is not observed directly through experiments, TDDFT geometry optimizations in the excited states support the formation of transient Cu(II) CS species, lending credence to the potential success of our approach. These studies establish a clear model for the excited state dynamics at play in proof-of-concept systems and clarify key design parameters for future optimizations toward achieving long-lived CS via photoinduced conformational gating.

Regulation Mechanism of Electron Transfer Reaction from Cytochrome C to Cytochrome C Oxidase

The respiratory chain in mitochondria is quite essential for the energy generation in cells, effectively producing “the energy currency in living organisms”, ATP. The driving force for this system is a series of electron transfers form Complex I (NADH:quinone oxidoreductase) to Complex IV (Cytochrome c Oxidase; CcO) in inner mitochondria membrane, and these electron transfer reactions promote the proton pumping from the matrix to the intermembrane space in mitochondria, and, by use of the resulting proton concentration gradient across the membrane, Complex V (ATP synthase) produces ATP. These series of electron transfer reactions terminate at CcO to reduce molecular oxygen to water molecules. The electrons required for this four-electron reduction of molecular oxygen in CcO are donated from a small soluble electron transfer hemoprotein, Cytochrome c (Cyt c), and this electron transfer is also associated with the proton pumping for the ATP generation in the respiratory chain. To examine the molecular regulation mechanism of the electron transfer reaction to terminate the electron transport chain for four-electron reduction of molecular oxygen and proton pumping for the ATP generation, we kinetically followed the electron transfer reaction and analyzed the electron transfer reaction by the Michaelis-Menten equation. By utilizing the Cyt c mutants, we identified the amino acid residues to regulate the electron transfer reactions (Biochem. J., 2020, 477,1565). The kinetic analysis of the electron transfer reaction also revealed that the apparent rate for the electron transfer reaction determined by the steady-state kinetics is much slower than the intramolecular electron transfer rate in the Cyt c – CcO complex. On the other hand, we successfully determined the interaction site between Cyt c and CcO by using NMR (Proc. Natl. Acad. Sci., 2011, 108, 12271) and protein-protein docking simulation (J. Biol. Chem., 2016, 291, 15320). The Cyt c – CcO complex structure estimated from the NMR and protein-protein docking simulation clearly showed that the two redox centers, heme iron in Cyt c and CuA in subunit II of CcO, were located more than 20 Å apart, and the complexes exhibiting shorter redox center distances were energetically unstable. Combined with the results from kinetic and structural studies, we proposed a “conformationally gated electron transfer mechanism”, where the thermal fluctuation of the protein structure controls the electron transfer rate. However, direct experimental evidence that protein conformational fluctuations regulate electron transfer reactions has not been obtained. In this study, we focused on the viscosity of the protein solution as a factor to regulate the conformational fluctuation of the proteins, based on the assumption that high viscosity of the solution suppresses the conformational fluctuation. The electron transfer reaction from Cyt c to CcO was examined in the high viscosity solution and NMR relaxation measurements under high viscosity were also performed to identify the local structural fluctuation responsible for the regulation of the electron transfer rate. Together with the transient complex structures between Cyt c and CcO estimated by the MD simulation, the regulation mechanism of the electron transfer from Cyt c to CcO based on the “conformational gating” will be discussed.

Inverse effects of APOC2 and ANGPTL4 on the conformational dynamics of lid-anchoring structures in lipoprotein lipase

The lipolytic processing of triglyceride-rich lipoproteins (TRLs) by lipoprotein lipase (LPL) is crucial for the delivery of dietary lipids to the heart, skeletal muscle, and adipose tissue. The processing of TRLs by LPL is regulated in a tissue-specific manner by a complex interplay between activators and inhibitors. Angiopoietin-like protein 4 (ANGPTL4) inhibits LPL by reducing its thermal stability and catalyzing the irreversible unfolding of LPL's α/β-hydrolase domain. We previously mapped the ANGPTL4 binding site on LPL and defined the downstream unfolding events resulting in LPL inactivation. The binding of LPL to glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 protects against LPL unfolding. The binding site on LPL for an activating cofactor, apolipoprotein C2 (APOC2), and the mechanisms by which APOC2 activates LPL have been unclear and controversial. Using hydrogen-deuterium exchange/mass spectrometry, we now show that APOC2's C-terminal α-helix binds to regions of LPL surrounding the catalytic pocket. Remarkably, APOC2's binding site on LPL overlaps with that for ANGPTL4, but their effects on LPL conformation are distinct. In contrast to ANGPTL4, APOC2 increases the thermal stability of LPL and protects it from unfolding. Also, the regions of LPL that anchor the lid are stabilized by APOC2 but destabilized by ANGPTL4, providing a plausible explanation for why APOC2 is an activator of LPL, while ANGPTL4 is an inhibitor. Our studies provide fresh insights into the molecular mechanisms by which APOC2 binds and stabilizes LPL-and properties that we suspect are relevant to the conformational gating of LPL's active site.

Mapping the determinants of substrate selectivity in a bacterial metal transporter with model-guided deep mutagenesis.

Membrane transporter proteins control what molecules are able to enter and exit the cell based on their ability to discriminate between substrates. The model bacterial metal transporter Deinococcus radiodurans (Dra)Nramp is responsible for importing rare divalent transition metals such as manganese and iron while excluding common alkaline earth metals such as magnesium and calcium. While recent high-resolution structures of DraNramp have mapped the conformational cycle underlying transport and identified key residues involved in metal binding and conformational gating, exactly which features of DraNramp control selectivity remains unclear, especially given the recent discovery of homologous proteins with altered substrate selectivity. We are simultaneously profiling the effect of thousands of DraNramp variants on import of manganese and magnesium, with the hypothesis that specificity may involve epistatic interactions between key active site positions and residues distal to the active site. To probe beyond single mutants, we have designed a strategy to bias our variant library using evolutionary sequence modeling to test a set of single- to octuple-mutants of DraNramp at positions likely to be involved in controlling substrate selectivity and unlikely to dramatically destabilize the protein. We have also designed novel assays to couple manganese and magnesium import to fluorescence in E. coli, and are testing our biased library in high-throughput with these assays to map the mutational landscape of substrate selectivity in DraNramp. This study will advance our understanding of how proteins bind metals as well as broadly what features of membrane transporter proteins are key to selecting between substrates. The methods for developing and assessing model-guided deep mutagenesis strategies also provide a framework bridging sequencing modeling and experimental biochemistry for future researchers.

Carbon nanotubes field-effect transistor pressure sensor based on three-dimensional conformal force-sensitive gate modulation

Pressure sensors based on field-effect transistor mechanism has received a lot of attention from researchers, which can effectively suppress low contrast and crosstalk effects. In this paper, we demonstrate a pressure sensor based on carbon nanotube field-effect transistor (CNTs-FET) and three-dimensional conformal force-sensitive top electrode with high sensitivity and easy integration. The high mobility field-effect transistor sensors were prepared using CNTs films with 99.9999% semiconductor concentration as the channel material. Conformal graphene nanowalls (GNWs) film on pyramidal micro-structure arrays worked as force-sensitive top electrode, the field-effect transistor sensor shows a sensitivity of 946.23 kPa−1 and 43.79 kPa−1 in the linear range of 0–30 Pa and 0.03–10 kPa, much higher than piezoresistive sensor (the sensitivity of about 377.73 kPa−1) and capacitive sensor (the sensitivity of 115.78 kPa−1). And the sensitivity and range of the sensor can be adjusted by the force-sensitive layer orientation design. Additionally, the sensor's response time is less than 30 ms, the recovery time is about 30 ms. The field-effect pressure sensor could detect ultra-low pressure (about 1.3 mg) of the chrysanthemum petals, and also could record the dynamic crawling process of the insect.

Molecular mechanism underlying ABC exporter gating: a computational study

ATP-binding cassette (ABC) exporters are a class of molecular machines that transport substrates out of biological membranes by gating movements leading to transitions between outward-facing (OF) and inward-facing (IF) conformational states. Despite significant advances in structural and functional studies, the molecular mechanism underlying conformational gating in ABC exporters is not completely understood. A complete elucidation of the state transitions during the transport cycle is beyond the capability of the all-atom molecular dynamics (MD) method because of the limited time scale of MD. In the present work, a coarse-grained molecular dynamics (CG-MD) method with an improved sampling strategy is performed for the bacterial ABC exporter MsbA. The resultant potential of the mean force (PMF) along the center-of-mass (COM) distances, <i>d</i><sub>1</sub> and <i>d</i><sub>2</sub>, between the two opposing subunits of the internal and external gates, respectively, are obtained, delicately showing the details of the <inline-formula><tex-math id="M1">\begin{document}$ {\rm{OF}}\to {\rm{IF}} $\end{document}</tex-math><alternatives><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="JUSTC-2022-0134_M1.jpg"/><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="JUSTC-2022-0134_M1.png"/></alternatives></inline-formula> transition occurring via an occluded (OC) state, in which the internal and external gates are both closed. The OC state has an important role in the unidirectionality of the transport function of ABC exporters. Our CG-MD simulations dynamically show that upon NBD dissociation, the opening of the internal gate occurs in a highly cooperative manner with the closure of the external gate. Based on our PMF calculations and CG-MD simulations in this paper, we proposed a mechanistic model that is significantly different from those recently published in the literature, shedding light on the molecular mechanism by which the ABC exporter executes conformational gating for substrate translocation.

Functional Water Networks in Fully Hydrated Photosystem II.

Water channels and networks within photosystem II (PSII) of oxygenic photosynthesis are critical for enzyme structure and function. They control substrate delivery to the oxygen-evolving center and mediate proton transfer at both the oxidative and reductive endpoints. Current views on PSII hydration are derived from protein crystallography, but structural information may be compromised by sample dehydration and technical limitations. Here, we simulate the physiological hydration structure of a cyanobacterial PSII model following a thorough hydration procedure and large-scale unconstrained all-atom molecular dynamics enabled by massively parallel simulations. We show that crystallographic models of PSII are moderately to severely dehydrated and that this problem is particularly acute for models derived from X-ray free electron laser (XFEL) serial femtosecond crystallography. We present a fully hydrated representation of cyanobacterial PSII and map all water channels, both static and dynamic, associated with the electron donor and acceptor sides. Among them, we describe a series of transient channels and the attendant conformational gating role of protein components. On the acceptor side, we characterize a channel system that is absent from existing crystallographic models but is likely functionally important for the reduction of the terminal electron acceptor plastoquinone QB. The results of the present work build a foundation for properly (re)evaluating crystallographic models and for eliciting new insights into PSII structure and function.

Photoactivation and conformational gating for manganese binding and oxidation in bacterial reaction centers

The influence of illumination history of native bacterial reaction centers (BRCs) on the ability of binding and photo-induced oxidation of manganous ions was investigated in the pH range between 8.0 and 9.4. Binding of manganous ions to a buried site required 6 to 11-fold longer incubation periods, depending on the pH, in dark-adapted BRCs than in BRCs that were previously illuminated prior to manganese binding. The intrinsic electron transfer from the bound manganese ion to the photo-oxidized primary electron donor was found to be limited by a 2 to 5-fold slower precursor conformational step in the dark-adapted samples for the same pH range. The conformational gating could be eliminated by photoactivation, namely if the BRCs were illuminated prior to binding. Unlike in Photosystem II, photoactivation in BRCs did not involve cluster assembly. Photoactivation with manganese already bound was only possible at elevated detergent concentration. In addition, also exclusively in dark-adapted BRCs, a marked breaking point in the Arrhenius-plot was discovered around 15 °C at pH 9.4 indicating a change in the reaction mechanism, most likely caused by the change of orientation of the 2-acetyl group of the inactive bacteriochlorophyll monomer located near the manganese binding site.

Structural and Functional Characterization of Electron Transfer Complex between Cytochrome C and Cytochrome C Oxidase

The electron transfer (ET) system in the respiratory chain is terminated by the four-electron reduction of molecular oxygen in cytochrome c oxidase, concomitant with the proton pumping from the matrix to the intermembrane space in mitochondria. The last ET reaction in the respiratory chain is mediated by a small ET protein, cytochrome c (Cyt c), and the interprotein ET reaction from Cyt c to CcO plays crucial roles in the four-electron reduction of molecular oxygen and proton pumping essential for the ATP generation in mitochondria. Although the well-defined structures of Cyt c and CcO have been already reported and the structure of the ET complex between Cyt c and CcO is indispensable to examine the regulation mechanism for the ET reaction from Cyt c to CcO, the huge molecular weight of CcO as a membrane-bound protein and large dissociation constant of CcO to Cyt c have been prevented us from the detailed analysis of the ET reaction based on molecular spectroscopies and X-ray crystallography. However, we successfully determined the structure of the ET complex between Cyt c and CcO under steady-state turnover conditions by utilizing protein-protein docking simulation combined with the experimentally determined interaction site for CcO in Cyt c and the Michaelis constants for the ET reaction from various kinds of the Cyt c mutants to CcO. The complex structure determined by the simulation revealed that the ET distance between the redox centers of two proteins, heme iron of Cyt c (Fe c ) and cupper of the CuA site of CcO (CuA), is nearly 23 Å, which is much longer than that previously suggested. To experimentally confirm the ET distance between two redox centers, the ET rate from Cyt c to CcO in the Cyt c – CcO complex was determined by temperature-dependent flow-flash measurements. In the flow-flash measurements, the solution containing carbon monoxide bound CcO was rapidly mixed with the oxygen-saturated solution, and, after the mixing, the four-electron reduction reaction of molecular oxygen in CcO was initiated by irradiating flash light to the reaction mixture to dissociate carbon monoxide from heme a 3 of CcO and to bind molecular oxygen to the heme a 3 site. Associated with the reduction of molecular oxygen, the successive ET reactions from Cyt c to CcO are induced. The ET rate from Fe c of Cyt c to CuA of CcO can be determined by the oxidation rate of Fe c of Cyt c. Based on the Marcus equation for the ET reaction, the ET distance estimated from the oxidation rate of Cyt c was less than 19 Å, which is substantially shorter than the Fe c –CuA distance in the simulated Cyt c–CcO complex. This finding allows us to speculate that the “ET-active complex” showing the ET rate as detected in the flow-flush experiments would have a different binding orientation of Cyt c on CcO from that in the complex, “energetically stable complex”, determined by the previous docking simulation. To structurally characterize the “ET-active complex”, a protein-protein docking simulation under the structural restraint of the short Fe c –CuA distance was applied, and we found a stable complex structure showing that the Fe c –CuA distance is ~ 18 Å, corresponding to the experimentally determined ET distance (< 18.6 Å). The orientation of Cyt c on CcO in the newly determined Cyt c – CcO complex was significantly different from that in the “energetically stable complex”, suggesting the “conformational gating” where the thermal motions of the protein induce the conformational changes of the protein from the most energetically stable structure to the less stable but more active structure of the proteins. In the ET reaction from Cyt c to CcO, the “ET-active complex showing fast ET rate and the short ET distance between two redox centers would not be the most energetically stable structure in the Cyt c – CcO complexes and the structural fluctuation induced by the thermal motion would be a trigger to the conformational changes from the “energetically stable complex” to the “ET-active complex”. The much slower oxidation rate of Cyt c by CcO observed for the steady-state kinetics measurements also supports the “conformational gating” mechanism in the ET reaction from Cyt c to CcO, where the rate for the conformational changes from the “energetically stable complex” to the “ET-active complex” would be the rate-determinant step for the ET reaction from Cyt c to CcO.

The bare bones of bifurcation: small flavoproteins as artificial electron bifurcases

Flavin‐based electron bifurcation (FBEB) is an essential mode of energy conservation in bacteria and archaea wherein specific flavoenzymes couple exergonic and endergonic metabolic electron flux from a single electron donor. While our understanding of the role of FBEB in metabolism continues to grow, the mechanism by which the flavin cofactor and the protein scaffold coordinate electron bifurcation remains elusive, largely due to the complexity of native bifurcases. We have leveraged the light‐oxygen‐voltage (LOV) domain flavoprotein as a robust and straight‐forward scaffold to interrogate the operant principles of electron bifurcation, including the role of the flavin binding pocket on tuning the flavin redox potential, proton‐coupled electron transfer, and potential “inversion,” as well as the role of conformational gating. Using bioinformatics and structural homology, we have remodeled the LOV flavin binding site to mimic that of native bifurcases and demonstrated key residues stabilize the anionic hydroquinone and increases potential inversion by 60 mV relative to wild‐type LOV. Furthermore, reactions with single electron donors/acceptors implicates a reactive neutral semiquinone, and lay the groundwork for kinetic electron bifurcation studies. Using cytochrome c as a model one‐electron acceptor, we can model the conformational gating mechanism by controlling donor‐acceptor concentrations, and have demonstrated the facile inter‐protein electron transfer, and photochemical crosslinking indicates a unique electron transfer interface. Kinetic modeling, including short‐circuit pathways, suggests that a bifurcation prone state should be long enough lived for direct observation, and studies are ongoing to spectroscopically identify this species. These results illustrate the utility of this reductionist approach in gaining insight into the physical principles that dictate FBEB and inform novel bifurcase design.

Conformational changes and CO2-induced channel gating in connexin26

SummaryConnexins form large-pore channels that function either as dodecameric gap junctions or hexameric hemichannels to allow the regulated movement of small molecules and ions across cell membranes. Opening or closing of the channels is controlled by a variety of stimuli, and dysregulation leads to multiple diseases. An increase in the partial pressure of carbon dioxide (PCO2) has been shown to cause connexin26 (Cx26) gap junctions to close. Here, we use cryoelectron microscopy (cryo-EM) to determine the structure of human Cx26 gap junctions under increasing levels of PCO2. We show a correlation between the level of PCO2 and the size of the aperture of the pore, governed by the N-terminal helices that line the pore. This indicates that CO2 alone is sufficient to cause conformational changes in the protein. Analysis of the conformational states shows that movements at the N terminus are linked to both subunit rotation and flexing of the transmembrane helices.

Determination of binding affinity and efficacy for NMDA receptor agonists and GluN2A selective allosteric modulators

NMDA-type ionotropic glutamate receptors are composed of two glycine-binding GluN1 and two glutamate-binding GluN2 subunits. Simultaneous occupation of all 4 agonist binding domains allows the receptor to undergo all the conformational changes that lead to opening of the ion channel. Agonist-induced conformational changes in turn alter agonist affinity. Thus, the efficacy by which agonists induce conformational changes and channel gating influences the apparent affinity. Allosteric modulators influence the agonist-induced conformational changes and can therefore alter agonist binding affinity and efficacy.

Multiscale Approach for Computing Gated Ligand Binding from Molecular Dynamics and Brownian Dynamics Simulations.

We develop an approach to characterize the effects of gating by a multiconformation protein consisting of macrostate conformations that are either accessible or inaccessible to ligand binding. We first construct a Markov state model of the apo-protein from atomistic molecular dynamics simulations from which we identify macrostates and their conformations, compute their relative macrostate populations and interchange kinetics, and structurally characterize them in terms of ligand accessibility. We insert the calculated first-order rate constants for conformational transitions into a multistate gating theory from which we derive a gating factor γ that quantifies the degree of conformational gating. Applied to HIV-1 protease, our approach yields a kinetic network of three accessible (semi-open, open, and wide-open) and two inaccessible (closed and a newly identified, "parted") macrostate conformations. The parted conformation sterically partitions the active site, suggesting a possible role in product release. We find that the binding kinetics of drugs and drug-like inhibitors to HIV-1 protease falls in the slow gating regime. However, because γ = 0.75, conformational gating only modestly slows ligand binding. Brownian dynamics simulations of the diffusional association of eight inhibitors to the protease─having a wide range of experimental association constants (∼104-1010 M-1 s-1)─yields gated rate constants in the range of ∼0.5-5.7 × 108 M-1 s-1. This indicates that, whereas the association rate of some inhibitors could be described by the model, for many inhibitors either subsequent conformational transitions or alternate binding mechanisms may be rate-limiting. For systems known to be modulated by conformational gating, the approach could be scaled computationally efficiently to screen association kinetics for a large number of ligands.

Detection of Catalytically Linked Conformational Changes in Wild-Type Class Ia Ribonucleotide Reductase Using Reaction-Induced FTIR Spectroscopy

The enzyme, ribonucleotide reductase (RNR), is essential for DNA synthesis in all cells. The class Ia Escherichia coli RNR consists of two dimeric subunits, α2 and β2, which form an active but unstable heterodimer of dimers, α2β2. The structure of the wild-type form of the enzyme has been challenging to study due to the instability of the catalytic complex. A long-range proton-coupled electron-transfer (PCET) pathway facilitates radical migration from the Y122 radical-diiron cofactor in the β subunit to an active site cysteine, C439, in the α subunit to initiate the RNR chemistry. The PCET reactions and active site chemistry are spectroscopically masked by a rate-limiting, conformational gate. Here, we present a reaction-induced Fourier transform infrared (RIFTIR) spectroscopic method to monitor the mechanism of the active, wild-type RNR α2β2 complex. This method is employed to obtain new information about conformational changes accompanying RNR catalysis, including the role of carboxylate interactions, deprotonation, and oxidation of active site cysteines, and a detailed description of reversible secondary structural changes. Labeling of tyrosine revealed a conformationally active tyrosine in the β subunit, assigned to Y356β, which is part of the intersubunit PCET pathway. New insights into the roles of the inhibitors, azidoUDP and dATP, and the sensitivity of RIFTIR spectroscopy to detect subtle conformational motions arising from protein allostery are also presented.

Conformational gating, dynamics and allostery in human monoacylglycerol lipase

Inhibition of human Monoacylglycerol Lipase (hMGL) offers a novel approach for treating neurological diseases. The design of inhibitors, targeting active-inactive conformational transitions of the enzyme, can be aided by understanding the interplay between structure and dynamics. Here, we report the effects of mutations within the catalytic triad on structure, conformational gating and dynamics of hMGL by combining kinetics, NMR, and HDX-MS data with metadynamics simulations. We found that point mutations alter delicate conformational equilibria between active and inactive states. HDX-MS reveals regions of the hMGL that become substantially more dynamic upon substitution of catalytic acid Asp-239 by alanine. These regions, located far from the catalytic triad, include not only loops but also rigid α-helixes and β-strands, suggesting their involvement in allosteric regulation as channels for long-range signal transmission. The results identify the existence of a preorganized global communication network comprising of tertiary (residue-residue contacts) and quaternary (rigid-body contacts) networks that mediate robust, rapid intraprotein signal transmission. Catalytic Asp-239 controls hMGL allosteric communications and may be considered as an essential residue for the integration and transmission of information to enzymes’ remote regions, in addition to its well-known role to facilitate Ser-122 activation. Our findings may assist in the identification of new druggable sites in hMGL.

A “Drug Sweeping” State of the TriABC Triclosan Efflux Pump from Pseudomonas aeruginosa

The structure of the TriABC inner membrane component of the triclosan/SDS-specific efflux pump from Pseudomonas aeruginosa was determined by cryoelectron microscopy to 4.5Å resolution. The complete structure of the inner membrane transporter TriC of the resistance-nodulation-division (RND) superfamily was solved, including a partial structure of the fused periplasmic membrane fusion subunits, TriA and TriB. The substrate-free conformation of TriABC represents an intermediate step in efflux complex assembly before the engagement of the outer membrane channel. Structural analysis identified a tunnel network whose constriction impedes substrate efflux, indicating inhibition of TriABC in the unengaged state. Blind docking studies revealed binding to TriC at the same loci by substrates and bulkier non-substrates. Together with functional analyses, we propose that selective substrate translocation involves conformational gating at the tunnel narrowing that, together with conformational ordering of TriA and TriB, creates an engaged state capable of mediating substrate efflux.

Conformational gating in ammonia lyases

BackgroundAmmonia lyases are enzymes of industrial and biomedical interest. Knowledge of structure-dynamics-function relationship in ammonia lyases is instrumental for exploiting the potential of these enzymes in industrial or biomedical applications. MethodsWe investigated the conformational changes in the proximity of the catalytic pocket of a 3-methylaspartate ammonia lyase (MAL) as a model system. At this scope, we used microsecond all-atom molecular dynamics simulations, analyzed with dimensionality reduction techniques, as well as in terms of contact networks and correlated motions. ResultsWe identify two regulatory elements in the MAL structure, i.e., the β5-α2 loop and the helix-hairpin-loop subdomain. These regulatory elements undergo conformational changes switching from ‘occluded’ to ‘open’ states. The rearrangements are coupled to changes in the accessibility of the active site. The β5-α2 loop and the helix-hairpin-loop subdomain modulate the formation of tunnels from the protein surface to the catalytic site, making the active site more accessible to the substrate when they are in an open state. ConclusionsOur work pinpoints a sequential mechanism, in which the helix-hairpin-loop subdomain of MAL needs to break a subset of intramolecular interactions first to favor the displacement of the β5-α2 loop. The coupled conformational changes of these two elements contribute to modulate the accessibility of the catalytic site. General significanceSimilar molecular mechanisms can have broad relevance in other ammonia lyases with similar regulatory loops. Our results also imply that it is important to account for protein dynamics in the design of variants of ammonia lyases for industrial and biomedical applications.