Concerted Conformational Changes Research Articles

Influence of Wobbling Tryptophan and Mutations on PET Degradation Explored by QM/MM Free Energy Calculations.

Plastic-degrading enzymes, particularly poly(ethylene terephthalate) (PET) hydrolases, have garnered significant attention in recent years as potential eco-friendly solutions for recycling plastic waste. However, understanding of their PET-degrading activity and influencing factors remains incomplete, impeding the development of uniform approaches for enhancing PET hydrolases for industrial applications. A key aspect of PET hydrolase engineering is optimizing the PET-hydrolysis reaction by lowering the associated free energy barrier. However, inconsistent findings have complicated these efforts. Therefore, our goal is to elucidate various aspects of enzymatic PET degradation by means of quantum mechanics/molecular mechanics (QM/MM) reaction simulations and analysis, focusing on the initial reaction step, acylation, in two thermophilic PET hydrolases, LCC and PES-H1, along with their highly active variants, LCCIG and PES-H1FY. Our findings highlight the impact of semiempirical QM methods on proton transfer energies, affecting the distinction between a two-step reaction involving a metastable tetrahedral intermediate and a one-step reaction. Moreover, we uncovered a concerted conformational change involving the orientation of the PET benzene ring, altering its interaction with the side-chain of the "wobbling" tryptophan from T-stacking to parallel π-π interactions, a phenomenon overlooked in prior research. Our study thus enhances the understanding of the acylation mechanism of PET hydrolases, in particular by characterizing it for the first time for the promising PES-H1FY using QM/MM simulations. It also provides insights into selecting a suitable QM method and a reaction coordinate, valuable for future studies on PET degradation processes.

Illuminating the mechanism and allosteric behavior of NanoLuc luciferase

NanoLuc, a superior β-barrel fold luciferase, was engineered 10 years ago but the nature of its catalysis remains puzzling. Here experimental and computational techniques are combined, revealing that imidazopyrazinone luciferins bind to an intra-barrel catalytic site but also to an allosteric site shaped on the enzyme surface. Structurally, binding to the allosteric site prevents simultaneous binding to the catalytic site, and vice versa, through concerted conformational changes. We demonstrate that restructuration of the allosteric site can boost the luminescent reaction in the remote active site. Mechanistically, an intra-barrel arginine coordinates the imidazopyrazinone component of luciferin, which reacts with O2 via a radical charge-transfer mechanism, and then it also protonates the resulting excited amide product to form a light-emitting neutral species. Concomitantly, an aspartate, supported by two tyrosines, fine-tunes the blue color emitter to secure a high emission intensity. This information is critical to engineering the next-generation of ultrasensitive bioluminescent reporters.

Outer membrane utilisomes mediate glycan uptake in gut Bacteroidetes.

Bacteroidetes are abundant members of the human microbiota, utilizing a myriad of diet- and host-derived glycans in the distal gut1. Glycan uptake across the bacterial outer membrane of these bacteria is mediated by SusCD protein complexes, comprising a membrane-embedded barrel and a lipoprotein lid, which is thought to open and close to facilitate substrate binding and transport. However, surface-exposed glycan-binding proteins and glycoside hydrolases also play critical roles in the capture, processing and transport of large glycan chains. The interactions between these components in the outer membrane are poorly understood, despite being crucial for nutrient acquisition by our colonic microbiota. Here we show that for both the levan and dextran utilization systems of Bacteroides thetaiotaomicron, the additional outer membrane components assemble on the core SusCD transporter, forming stable glycan-utilizing machines that we term utilisomes. Single-particle cryogenic electron microscopy structures in the absence and presence of substrate reveal concerted conformational changes that demonstrate the mechanism of substrate capture, and rationalize the role of each component in the utilisome.

Concerted conformational changes control metabotropic glutamate receptor activity.

Allosteric modulators bear great potential to fine-tune neurotransmitter action. Promising targets are metabotropic glutamate (mGlu) receptors, which are associated with numerous brain diseases. Orthosteric and allosteric ligands act in synergy to control the activity of these multidomain dimeric GPCRs. Here, we analyzed the effect of such molecules on the concerted conformational changes of full-length mGlu2 at the single-molecule level. We first established FRET sensors through genetic code expansion combined with click chemistry to monitor conformational changes on live cells. We then used single-molecule FRET and show that orthosteric agonist binding leads to the stabilization of most of the glutamate binding domains in their closed state, while the reorientation of the dimer into the active state remains partial. Allosteric modulators, interacting with the transmembrane domain, are required to stabilize the fully reoriented active dimer. These results illustrate how concerted conformational changes within multidomain proteins control their activity, and how these are modulated by allosteric ligands.

Molecular mechanism and energetics of coupling between substrate binding and product release in the F1-ATPase catalytic cycle

F1-ATPase is a motor protein that couples the rotation of its rotary [Formula: see text] subunit with ATP synthesis or hydrolysis. Single-molecule experiments indicate that nucleotide binding and release events occur almost simultaneously during the synthesis cycle, allowing the energy gain due to spontaneous binding of ADP to one catalytic [Formula: see text] subunit to be directly harnessed for driving the release of ATP from another rather than being dissipated as heat. Here, we examine the unknown mechanism of this coupling that is critical for an exceptionally high mechanochemical efficiency of F1-ATPase by means of all-atom free-energy simulations. We find that nondissipative and kinetically fast progression of the motor in the synthesis direction requires a concerted conformational change involving the closure of the ADP-binding [Formula: see text] subunit followed by the gradual opening of the ATP-releasing [Formula: see text] subunit over the course of the 30 to 40° rotary substep of the [Formula: see text] subunit. This rotary substep, preceding the ATP-dependent metastable state, allows for the recovery of a large portion of the ADP binding energy in the conformation of ATP-bound [Formula: see text] that gradually adopts the low-affinity conformation, captured also by the recent cryo-EM structure of this elusive state. The release of ATP from this nearly open conformation leads to its further opening, which enables the progression of the motor to the next catalytic metastable state. Our simulations explain this energy conversion mechanism in terms of intersubunit and ligand-protein interactions.

Competitive Hydrogen Bonding and Unprecedented Polymorphism in Selected Chiral Phosphorylated Thioureas

New racemic and enantiopure N-phosphorylated thioureas bearing 1-phenylethyl or tetrahydronaphthalenyl fragments were synthesized. According to NMR data assisted by DFT calculations, the preferred conformation is stabilized by an intramolecular hydrogen bond. This form in solution is in equilibrium with dimeric N–H···S hydrogen-bonded associates, the population depending on the concentration. In the crystalline phase the low-energy conformation with an intramolecular H-bond is realized only in the racemic tetrahydronaphthalenyl derivative. In most crystals various types of intermolecular hydrogen bonding are observed, accompanied by the formation of infinite linear chains or helical structures. Due to the conformational lability of compounds and competitive intermolecular H-bonding, multiple polymorphic modifications are formed. Therefore, crystallization of enantiopure 1-phenylethyl derivatives from various solvents results in concomitant polymorphs at room temperature. One of them undergoes reversible two-step phase transitions from the high-symmetry I41 space group (Z′ = 1, no disorder) via the P41 space group (Z′ = 6) to the monoclinic P21 space group (Z′ = 16) accompanied by drastic concerted conformational changes. Notably, the optimization of the crystal packing is observed upon phase transitions with a gradual reduction of the void space in the unit cell from 4.5% to 0.8%. This is a rare case of several high-Z′ polymorphs for one compound, with chirality playing an important role.

Mobility of Lower MA-Helices for Ion Conduction through Lateral Portals in 5-HT3A Receptors

The intracellular domain of the serotonin type 3A receptor, a pentameric ligand-gated ion channel, is crucial for regulating conductance. Ion permeation through the extracellular vestibule and the transmembrane channel is well understood, whereas the specific ion conduction pathway through the intracellular domain is less clear. The intracellular domain starts with a short loop after the third transmembrane segment, followed by a short α-helical segment, a large unstructured loop, and finally, the membrane-associated MA-helix that continues into the last transmembrane segment. The MA-helices from all five subunits form the extension of the transmembrane ion channel and shape what has been described as a “closed vestibule,” with their lateral portals obstructed by loops and their cytosolic ends forming a tight hydrophobic constriction. The question remains whether the lateral portals or cytosolic constriction conduct ions upon channel opening. In our study, we used disulfide bond formation between pairs of engineered cysteines to probe the proximity and mobility of segments of the MA-helices most distal to the membrane bilayer. Our results indicate that the proximity and orientation for cysteine pairs at I409C/R410C, in close proximity to the lateral windows, and L402C/L403C, at the cytosolic ends of the MA-helices, are conducive for disulfide bond formation. Although conformational changes associated with gating promote cross-linking for I409C/R410C, which in turn decreases channel currents, cross-linking of L402C/L403C is functionally silent in macroscopic currents. These results support the hypothesis that concerted conformational changes open the lateral portals for ion conduction, rendering ion conduction through the vertical portal unlikely.

Water-mediated interactions enable smooth substrate transport in a bacterial efflux pump

BackgroundEfflux pumps of the Resistance-Nodulation-cell Division superfamily confer multi-drug resistance to Gram-negative bacteria. The most-studied polyspecific transporter belonging to this class is the inner-membrane trimeric antiporter AcrB of Escherichia coli. In previous studies, a functional rotation mechanism was proposed for its functioning, according to which the three monomers undergo concerted conformational changes facilitating the extrusion of substrates. However, the molecular determinants and the energetics of this mechanism still remain unknown, so its feasibility must be proven mechanistically. MethodsA computational protocol able to mimic the functional rotation mechanism in AcrB was developed. By using multi-bias molecular dynamics simulations we characterized the translocation of the substrate doxorubicin driven by conformational changes of the protein. In addition, we estimated for the first time the free energy profile associated to this process. ResultsWe provided a molecular view of the process in agreement with experimental data. Moreover, we showed that the conformational changes occurring in AcrB enable the formation of a layer of structured waters on the internal surface of the transport channel. This water layer, in turn, allows for a fairly constant hydration of the substrate, facilitating its diffusion over a smooth free energy profile. ConclusionsOur findings reveal a new molecular mechanism of polyspecific transport whereby water contributes by screening potentially strong substrate-protein interactions. General significanceWe provided a mechanistic understanding of a fundamental process related to multi-drug transport. Our results can help rationalizing the behavior of other polyspecific transporters and designing compounds avoiding extrusion or inhibitors of efflux pumps.

Coiled-Coil Formation Conveys a STIM1 Signal from ER Lumen to Cytoplasm

STIM1 and STIM2 are endoplasmic reticulum (ER) membrane proteins that sense decreases in ER-luminal free Ca2+ and, through a conformational change in the STIM cytoplasmic domain, control gating of the plasma membrane Ca2+ channel ORAI1. To determine how STIM1 conveys a signal from the ER lumen to the cytoplasm, we studied the Ca2+-dependent conformational change of engineered STIM1 proteins in isolated ER membranes and, in parallel, physiological activation of these proteins in cells. We find that conserved "sentinel" features of the CC1 region help to prevent activation while Ca2+ is bound to STIM ER-luminal domains. Reduced ER-luminal Ca2+ drives a concerted conformational change, in which STIM luminal domains rearrange and the STIM transmembrane helices and initial parts of the CC1 regions pair in an extended coiled coil. This intradimer rearrangement overcomes the relatively weak CC1-SOAR/CAD interactions that hold STIM in an inactive conformation, releasing the SOAR/CAD domain to activate ORAI channels.

Retrospective on Cholesterol Homeostasis: The Central Role of Scap.

Scap is a polytopic membrane protein that functions as a molecular machine to control the cholesterol content of membranes in mammalian cells. In the 21 years since our laboratory discovered Scap, we have learned how it binds sterol regulatory element-binding proteins (SREBPs) and transports them from the endoplasmic reticulum (ER) to the Golgi for proteolytic processing. Proteolysis releases the SREBP transcription factor domains, which enter the nucleus to promote cholesterol synthesis and uptake. When cholesterol in ER membranes exceeds a threshold, the sterol binds to Scap, triggering several conformational changes that prevent the Scap-SREBP complex from leaving the ER. As a result, SREBPs are no longer processed, cholesterol synthesis and uptake are repressed, and cholesterol homeostasis is restored. This review focuses on the four domains of Scap that undergo concerted conformational changes in response to cholesterol binding. The data provide a molecular mechanism for the control of lipids in cell membranes.

Crystal structures of an atypical aldehyde dehydrogenase having bidirectional oxidizing and reducing activities

Aldehyde dehydrogenases (ALDHs) are NAD(P)+-dependent oxidoreductases that catalyze the oxidation of a variety of aldehydes to their acid forms. In this study, we determined the crystal structures of ALDH from Bacillus cereus (BcALDH), alone, and in complex with NAD+ and NADP+. This enzyme can oxidize all-trans-retinal to all-trans-retinoic acid using either NAD+ or NADP+ with equal efficiency, and atypically, as a minor activity, can reduce all-trans-retinal to all-trans-retinol using NADPH. BcALDH accommodated the additional 2′-phosphate of NADP+ by expanding the cofactor-binding pocket and upshifting the AMP moiety in NADP+. The nicotinamide moiety in NAD+ and NADP+ had direct interactions with the conserved catalytic residues (Cys300 and Glu266) and caused concerted conformational changes. We superimposed the structure of retinoic acid bound to human ALDH1A3 onto the BcALDH structure and speculated a model of the substrate all-trans-retinal bound to BcALDH. We also proposed a plausible mechanism for the minor reducing activity of BcALDH. These BcALDH structures will be useful in understanding cofactor specificity and the catalytic mechanism of an atypical bacterial BcALDH and should help the development of a new biocatalyst to produce retinoic acid and related high-end products.

Structural Basis of Eco1-Mediated Cohesin Acetylation

Sister-chromatid cohesion is established by Eco1-mediated acetylation on two conserved tandem lysines in the cohesin Smc3 subunit. However, the molecular basis of Eco1 substrate recognition and acetylation in cohesion is not fully understood. Here, we discover and rationalize the substrate specificity of Eco1 using mass spectrometry coupled with in-vitro acetylation assays and crystallography. Our structures of the X. laevis Eco2 (xEco2) bound to its primary and secondary Smc3 substrates demonstrate the plasticity of the substrate-binding site, which confers substrate specificity by concerted conformational changes of the central β hairpin and the C-terminal extension.

Transport Mechanism in the RND Transporter AcrD of E. coli

The RND efflux pump AcrAB-TolC is involved in antibiotic resistance in E. coli [1-2]. The core of this machinery is the proton-gradient-driven antiporter AcrB, a homotrimeric protein with an unreached ability to recognize antibiotics belonging to many different families. On the basis of available experimental data, a functional rotation mechanism has been hypothesized for this transporter, in which recognition and expulsion of substrates are coupled to concerted conformational changes occurring in each monomer of AcrB.

Emergence of ion channel modal gating from independent subunit kinetics

Many ion channels exhibit a slow stochastic switching between distinct modes of gating activity. This feature of channel behavior has pronounced implications for the dynamics of ionic currents and the signaling pathways that they regulate. A canonical example is the inositol 1,4,5-trisphosphate receptor (IP3R) channel, whose regulation of intracellular Ca(2+) concentration is essential for numerous cellular processes. However, the underlying biophysical mechanisms that give rise to modal gating in this and most other channels remain unknown. Although ion channels are composed of protein subunits, previous mathematical models of modal gating are coarse grained at the level of whole-channel states, limiting further dialogue between theory and experiment. Here we propose an origin for modal gating, by modeling the kinetics of ligand binding and conformational change in the IP3R at the subunit level. We find good agreement with experimental data over a wide range of ligand concentrations, accounting for equilibrium channel properties, transient responses to changing ligand conditions, and modal gating statistics. We show how this can be understood within a simple analytical framework and confirm our results with stochastic simulations. The model assumes that channel subunits are independent, demonstrating that cooperative binding or concerted conformational changes are not required for modal gating. Moreover, the model embodies a generally applicable principle: If a timescale separation exists in the kinetics of individual subunits, then modal gating can arise as an emergent property of channel behavior.

Probing the Secondary Active Transport Mechanism of the Bacterial Efflux Pump EmrE

Secondary active transporters are able to confer bacterial multidrug resistance to both antibiotics and antiseptics. While the energy source for the transport mechanism is derived from the electrochemical potential across the membrane, this thermodynamic view does not provide a mechanistic understanding regarding how the energy is converted into useful work. To gain insight into the ion-coupled transport process, we applied a plethora of biophysical techniques including solid-state and solution NMR spectroscopy in conjunction with resistance assays to the drug transporter EmrE from E. coli. EmrE is an anti-parallel homodimer from the small multidrug resistance (SMR) family that effluxes a wide variety of quaternary cationic compounds and has served as a model for ion-coupled transport. We carried out pH titrations using NMR spectroscopy to reveal an elevated acid dissociation constant for a conserved and membrane embedded glutamate residue that is responsible for conferring drug resistance in E. coli. Furthermore, we found that the acid/base chemistry at this anionic residue was responsible for regulating the global dynamics of the transporter based on the protonation state. The implication of this result is that an open conformation of EmrE in the presence of a pH gradient would be oriented toward the higher pH side of the membrane. Indeed, tryptophan fluorescence experiments acquired in lipid bilayers strongly validate this interpretation. Finally, protonation at E14 showed long-range chemical shift perturbations far removed from the drug-binding pocket, which is evidence of an intricate allosteric network involving both concerted conformational changes to the transmembrane helices and loops that are involved in regulating the inward-open to outward-open structural switch.

Asymmetric properties of rod cGMP Phosphodiesterase 6 (PDE6): structural and functional analysis

Photoreceptor cGMP Phsophodiesterase 6 (PDE6) is the effector molecule of visual signal transduction and mediates fast response of light signals. The rod holo-PDE6 comprises catalytic (α, β; each ~ 90 kDa) and two identical inhibitiory (γ; ~ 10 kDa) subunits. The catalytic subunits comprise N-terminal tandem GAF domains followed by C-terminal catalytic domains and isoprenylations for membrane-association. Contrary to activation of other tandem GAF comprising PDEs, PDE6 activation does not occur via cGMP-induced concerted conformational changes. Rather two copies of the α-subunit of retinal G-Protein (Gα*), transducin, activate PDE6 by partially displacing the inhibitory subunits. The activation of PDE6 has therefore been described as a “de-inhibition”. The affinity of Gα* to PDE6 and the enzymatic activity of the intermediary 1:1 complex is highly disputed, therefore a conclusive activation model is lacking so far. Our combined structural, enzymatic and computational investigations deal with the activation-mechanism of PDE6. Our cryo electron microscopy (EM) structure of PDEαβ catalytic core shows an elongated bell-shaped structure with symmetric side-by-side arrangement of the two subunits with flexible membrane-binding domains. A comparison with nearly full-length inactive PDE2A structure [1] suggests that less compaction of both subunits and higher degree of conformational freedom of the catalytic domains result in constitutive activation of PDE6αβ, which is kept inactive by the inhibitory γ subunits. Furthermore, the structure of PDE6 suggests Gα* binding-sites pointing to opposing faces. The enzymatic characterization using Gα* titration of the PDE6 however reveal striking asymmetry of the two catalytic subunits with a high and a low affinity binding site for Gα*. Occupancy of the PDE6 with one Gα* induces negligible activity, whereas occupancy with two copies of Gα* leads to full enzyme activity. Such an activation mechanism constitutes a “coincidence switch” that allows noise filtering (i.e., spontaneously produced Gα* do not activate PDE6). Our spatiotemporal simulation work indeed confirms that spontaneously generated Gα* lead to the formation of singly occupied PDE6 and only a high local concentration of Gα*, as produced by an active receptor (rhodopsin), leads to doubly Gα* occupied effector complex. Therefore the localized large concentration of Gα* combined with the asymmetric properties of PDE6 constitutes a “density switch” that allows suppression effector level noise and reliable reporting of single quantum events in rod photoreceptor cells.

Substrate binding accelerates the conformational transitions and substrate dissociation in multidrug efflux transporter AcrB.

The tripartite efflux pump assembly AcrAB-TolC is the major multidrug resistance transporter in E. coli. The inner membrane transporter AcrB is a homotrimer, energized by the proton movement down the transmembrane electrochemical gradient. The asymmetric crystal structures of AcrB with three monomers in distinct conformational states [access (A), binding (B) and extrusion (E)] support a functional rotating mechanism, in which each monomer of AcrB cycles among the three states in a concerted way. However, the relationship between the conformational changes during functional rotation and drug translocation has not been totally understood. Here, we explored the conformational changes of the AcrB homotrimer during the ABE to BEA transition in different substrate-binding states using targeted MD simulations. It was found that the dissociation of substrate from the distal binding pocket of B monomer is closely related to the concerted conformational changes in the translocation pathway, especially the side chain reorientation of Phe628 and Tyr327. A second substrate binding at the proximal binding pocket of A monomer evidently accelerates the conformational transitions as well as substrate dissociation in B monomer. The acceleration effect of the multi-substrate binding mode provides a molecular explanation for the positive cooperativity observed in the kinetic studies of substrate efflux and deepens our understanding of the functional rotating mechanism of AcrB.

Conformational Changes Underlying Glutamate Receptor Gating

The gating mechanism of glutamate receptor ion channels involves conformational changes triggered by the binding of glutamate that open the ion channel, followed within milliseconds by nearly complete desensitization. In a collaboration with the Subramaniam Lab at NCI, cryo-EM structures of full length AMPA (GluA2) receptors, stabilized in the active state by allosteric modulators, reveal concerted conformational changes in the ligand binding domain that produce a vertical contraction accompanied by a corkscrew like rotation that leads to expansion of the linkers leading to the M3 helix bundle; in the desensitized state the ATD dimer pairs adopts multiple conformations with evidence for substantial reorganization of the ligand binding domain tetramer. By contrast, for the GluK2 kainate receptor the ATD dimer assembly remains intact in the desensitized state, while the LBD rearranges into a 4-fold symmetric structure in which the linkers leading to the M3 helix bundle adopt a contracted conformation that is however different from the closed state. Measurements of ATD tetramer association by analytical ultracentrifugation reveal a 1000-fold high higher affinity for GluK2 versus GluA2 that underlies the different behavior of the ATD in the desensitized state, movements of which however are secondary to and play no major role in desensitization.

Stepwise visualization of membrane pore formation by suilysin, a bacterial cholesterol-dependent cytolysin.

Membrane attack complex/perforin/cholesterol-dependent cytolysin (MACPF/CDC) proteins constitute a major superfamily of pore-forming proteins that act as bacterial virulence factors and effectors in immune defence. Upon binding to the membrane, they convert from the soluble monomeric form to oligomeric, membrane-inserted pores. Using real-time atomic force microscopy (AFM), electron microscopy (EM), and atomic structure fitting, we have mapped the structure and assembly pathways of a bacterial CDC in unprecedented detail and accuracy, focussing on suilysin from Streptococcus suis. We show that suilysin assembly is a noncooperative process that is terminated before the protein inserts into the membrane. The resulting ring-shaped pores and kinetically trapped arc-shaped assemblies are all seen to perforate the membrane, as also visible by the ejection of its lipids. Membrane insertion requires a concerted conformational change of the monomeric subunits, with a marked expansion in pore diameter due to large changes in subunit structure and packing.

Crystal structure of EndoS, an immunomodulatory endoglycosidase specific for human IgG antibodies

In order to evade host immune mechanisms, many bacteria secrete immunomodulatory enzymes. Streptococcus pyogenes, one of the most common human pathogens, secretes a large endoglycosidase, EndoS, which removes carbohydrates in a highly specific manner from IgG antibodies. This modification renders antibodies incapable of eliciting host effector functions through either complement or Fc γ receptors, providing the bacteria with a survival advantage. On account of this antibody-specific modifying activity, EndoS is being developed as a promising injectable therapeutic for autoimmune diseases that rely on autoantibodies. Additionally, EndoS is a key enzyme used in the chemoenzymatic synthesis of homogenously glycosylated antibodies with tailored Fc γ receptor-mediated effector functions. Despite the tremendous utility of this enzyme, the molecular basis of EndoS specificity for, and processing of, IgG antibodies has remained poorly understood. Here, we report the X-ray crystal structure of EndoS and provide a model of its encounter complex with its substrate, the IgG1 Fc domain. We show that EndoS is composed of five distinct protein domains, including glycosidase, leucine-rich repeat, hybrid Ig, carbohydrate binding module, and three-helix bundle domains, arranged in a distinctive V-shaped conformation. Our data suggest that the substrate enters the concave interior of the enzyme structure, is held in place by the carbohydrate binding module, and that concerted conformational changes in both enzyme and substrate are required for subsequent antibody deglycosylation. The EndoS structure presented here provides a framework from which novel endoglycosidases could be engineered for additional clinical and biotechnological applications.