Showing papers on "Conformational change published in 2022"

Activation of STING by targeting a pocket in the transmembrane domain

Abstract: Stimulator of interferon genes (STING) is an adaptor protein in innate immunity against DNA viruses or bacteria1-5. STING-mediated immunity could be exploited in the development of vaccines or cancer immunotherapies. STING is a transmembrane dimeric protein that is located in the endoplasmic reticulum or in the Golgi apparatus. STING is activated by the binding of its cytoplasmic ligand-binding domain to cyclic dinucleotides that are produced by the DNA sensor cyclic GMP-AMP (cGAMP) synthase or by invading bacteria1,6,7. Cyclic dinucleotides induce a conformational change in the STING ligand-binding domain, which leads to a high-order oligomerization of STING that is essential for triggering the downstream signalling pathways8,9. However, the cGAMP-induced STING oligomers tend to dissociate in solution and have not been resolved to high resolution, which limits our understanding of the activation mechanism. Here we show that a small-molecule agonist, compound 53 (C53)10, promotes the oligomerization and activation of human STING through a mechanism orthogonal to that of cGAMP. We determined a cryo-electron microscopy structure of STING bound to both C53 and cGAMP, revealing a stable oligomer that is formed by side-by-side packing and has a curled overall shape. Notably, C53 binds to a cryptic pocket in the STING transmembrane domain, between the two subunits of the STING dimer. This binding triggers outward shifts of transmembrane helices in the dimer, and induces inter-dimer interactions between these helices to mediate the formation of the high-order oligomer. Our functional analyses show that cGAMP and C53 together induce stronger activation of STING than either ligand alone.

Activation of STING by targeting a pocket in the transmembrane domain

Abstract: Stimulator of interferon genes (STING) is an adaptor protein in innate immunity against DNA viruses or bacteria1-5. STING-mediated immunity could be exploited in the development of vaccines or cancer immunotherapies. STING is a transmembrane dimeric protein that is located in the endoplasmic reticulum or in the Golgi apparatus. STING is activated by the binding of its cytoplasmic ligand-binding domain to cyclic dinucleotides that are produced by the DNA sensor cyclic GMP-AMP (cGAMP) synthase or by invading bacteria1,6,7. Cyclic dinucleotides induce a conformational change in the STING ligand-binding domain, which leads to a high-order oligomerization of STING that is essential for triggering the downstream signalling pathways8,9. However, the cGAMP-induced STING oligomers tend to dissociate in solution and have not been resolved to high resolution, which limits our understanding of the activation mechanism. Here we show that a small-molecule agonist, compound 53 (C53)10, promotes the oligomerization and activation of human STING through a mechanism orthogonal to that of cGAMP. We determined a cryo-electron microscopy structure of STING bound to both C53 and cGAMP, revealing a stable oligomer that is formed by side-by-side packing and has a curled overall shape. Notably, C53 binds to a cryptic pocket in the STING transmembrane domain, between the two subunits of the STING dimer. This binding triggers outward shifts of transmembrane helices in the dimer, and induces inter-dimer interactions between these helices to mediate the formation of the high-order oligomer. Our functional analyses show that cGAMP and C53 together induce stronger activation of STING than either ligand alone.

Temperature-dependent local conformations and conformational distributions of cyanine dimer labeled single-stranded–double-stranded DNA junctions by 2D fluorescence spectroscopy

Abstract: DNA replication and the related processes of genome expression require binding, assembly, and function of protein complexes at and near single-stranded (ss)-double-stranded (ds) DNA junctions. These central protein-DNA interactions are likely influenced by thermally induced conformational fluctuations of the DNA scaffold across an unknown distribution of functionally relevant states to provide regulatory proteins access to properly conformed DNA binding sites. Thus, characterizing the nature of conformational fluctuations and the associated structural disorder at ss-dsDNA junctions is critical for understanding the molecular mechanisms of these central biological processes. Here, we describe spectroscopic studies of model ss-dsDNA fork constructs that contain dimers of "internally labeled" cyanine (iCy3) chromophore probes that have been rigidly inserted within the sugar-phosphate backbones of the DNA strands. Our combined analyses of absorbance, circular dichroism, and two-dimensional fluorescence spectroscopy permit us to characterize the local conformational parameters and conformational distributions. We find that the DNA sugar-phosphate backbones undergo abrupt successive changes in their local conformations-initially from a right-handed and ordered DNA state to a disordered splayed-open structure and then to a disordered left-handed conformation-as the dimer probes are moved across the ss-dsDNA junction. Our results suggest that the sugar-phosphate backbones at and near ss-dsDNA junctions adopt specific position-dependent local conformations and exhibit varying extents of conformational disorder that deviate widely from the Watson-Crick structure. We suggest that some of these conformations can function as secondary-structure motifs for interaction with protein complexes that bind to and assemble at these sites.

Lipoprotein-associated phospholipase A2: A paradigm for allosteric regulation by membranes

Abstract: Significance Lp-PLA2 is a physiologically important human enzyme and an inflammatory biomarker for assessing risk factors associated with cardiovascular diseases. It is associated with low- and high-density lipoproteins in human plasma and acts on the outside of the phospholipid monolayer that coats these particles, in stark contrast to traditional PLA2 enzymes that act on bilayer membranes. This study addresses the allosteric activation of Lp-PLA2 by phospholipid monolayers and membranes, its precise selectivity and specificity for particular oxidized and short acyl-chain phospholipid substrates not previously possible. Of particular importance, this work identifies and confirms by site-directed mutagenesis a phospholipid head-group binding pocket distinct from known drug inhibitor binding pockets that informs us about Lp-PLA2’s mechanism of action and creates opportunities for additional therapeutic approaches. Lipoprotein-associated phospholipase A2 (Lp-PLA2) associates with low- and high-density lipoproteins in human plasma and specifically hydrolyzes circulating oxidized phospholipids involved in oxidative stress. The association of this enzyme with the lipoprotein’s phospholipid monolayer to access its substrate is the most crucial first step in its catalytic cycle. The current study demonstrates unequivocally that a significant movement of a major helical peptide region occurs upon membrane binding, resulting in a large conformational change upon Lp-PLA2 binding to a phospholipid surface. This allosteric regulation of an enzyme’s activity by a large membrane-like interface inducing a conformational change in the catalytic site defines a unique dimension of allosterism. The mechanism by which this enzyme associates with phospholipid interfaces to select and extract a single phospholipid substrate molecule and carry out catalysis is key to understanding its physiological functioning. A lipidomics platform was employed to determine the precise substrate specificity of human recombinant Lp-PLA2 and mutants. This study uniquely elucidates the association mechanism of this enzyme with membranes and its resulting conformational change as well as the extraction and binding of specific oxidized and short acyl-chain phospholipid substrates. Deuterium exchange mass spectrometry coupled with molecular dynamics simulations was used to define the precise specificity of the subsite for the oxidized fatty acid at the sn-2 position of the phospholipid backbone. Despite the existence of several crystal structures of this enzyme cocrystallized with inhibitors, little was understood about Lp-PLA2‘s specificity toward oxidized phospholipids.

Enabling Role of Ligand-Driven Conformational Changes in Enzyme Evolution

Abstract: Many enzymes that show a large specificity in binding the enzymatic transition state with a higher affinity than the substrate utilize substrate binding energy to drive protein conformational changes to form caged substrate complexes. These protein cages provide strong stabilization of enzymatic transition states. Using part of the substrate binding energy to drive the protein conformational change avoids a similar strong stabilization of the Michaelis complex and irreversible ligand binding. A seminal step in the development of modern enzyme catalysts was the evolution of enzymes that couple substrate binding to a conformational change. These include enzymes that function in glycolysis (triosephosphate isomerase), the biosynthesis of lipids (glycerol phosphate dehydrogenase), the hexose monophosphate shunt (6-phosphogluconate dehydrogenase), and the mevalonate pathway (isopentenyl diphosphate isomerase), catalyze the final step in the biosynthesis of pyrimidine nucleotides (orotidine monophosphate decarboxylase), and regulate the cellular levels of adenine nucleotides (adenylate kinase). The evolution of enzymes that undergo ligand-driven conformational changes to form active protein–substrate cages is proposed to proceed by selection of variants, in which the selected side chain substitutions destabilize a second protein conformer that shows compensating enhanced binding interactions with the substrate. The advantages inherent to enzymes that incorporate a conformational change into the catalytic cycle provide a strong driving force for the evolution of flexible protein folds such as the TIM barrel. The appearance of these folds represented a watershed event in enzyme evolution that enabled the rapid propagation of enzyme activities within enzyme superfamilies.

Molecular mechanism of a large conformational change of the quinone cofactor in the semiquinone intermediate of bacterial copper amine oxidase

Abstract: Copper amine oxidase from Arthrobacter globiformis (AGAO) catalyses the oxidative deamination of primary amines via a large conformational change of a topaquinone (TPQ) cofactor during the semiquinone formation step. This conformational change of TPQ occurs in the presence of strong hydrogen bonds and neighboring bulky amino acids, especially the conserved Asn381, which restricts TPQ conformational changes over the catalytic cycle. Whether such a semiquinone intermediate is catalytically active or inert has been a matter of debate in copper amine oxidases. Here, we show that the reaction rate of the Asn381Ala mutant decreases 160-fold, and the X-ray crystal structures of the mutant reveals a TPQ-flipped conformation in both the oxidized and reduced states, preceding semiquinone formation. Our hybrid quantum mechanics/molecular mechanics (QM/MM) simulations show that the TPQ conformational change is realized through the sequential steps of the TPQ ring-rotation and slide. We determine that the bulky side chain of Asn381 hinders the undesired TPQ ring-rotation in the oxidized form, favoring the TPQ ring-rotation in reduced TPQ by a further stabilization leading to the TPQ semiquinone form. The acquired conformational flexibility of TPQ semiquinone promotes a high reactivity of Cu(i) to O2, suggesting that the semiquinone form is catalytically active for the subsequent oxidative half-reaction in AGAO. The ingenious molecular mechanism exerted by TPQ to achieve the “state-specific” reaction sheds new light on a drastic environmental transformation around the catalytic center.

Integrated AlphaFold2 and DEER investigation of the conformational dynamics of a pH-dependent APC antiporter

Abstract: The Amino Acid-Polyamine-Organocation transporter GadC contributes to the survival of pathogenic bacteria under extreme acid stress by exchanging extracellular glutamate for intracellular GABA. Its structure, determined exclusively in an inward-facing conformation at alkaline pH, consists of the canonical LeuT-fold of a conserved five-helix inverted repeat, thereby resembling functionally divergent transporters such as the serotonin reuptake transporter SERT and the glucose-sodium symporter transporter SGLT1. However, despite this structural similarity, it is unclear if the conformational dynamics of antiporters such as GadC follows the blueprint of these or other well-studied LeuT-fold transporters. Here, we used double electron-electron resonance (DEER) spectroscopy to monitor the conformational dynamics of GadC in lipid bilayers in response to acidification and substrate binding. To guide experimental design and facilitate the interpretation of the DEER data, we generated an ensemble of structural models in multiple conformations using a recently introduced AlphaFold2 methodology. Our experimental results reveal acid-induced conformational changes that dislodge the C-terminus from the permeation pathway coupled with rearrangement of helices that enable isomerization between both inward- and outward-facing states. The substrate glutamate, but not GABA, modulates the dynamics of an extracellular thin gate without shifting the equilibrium between inward- and outward-facing conformations. In addition to introducing an integrated methodology for probing transporter conformational dynamics, the congruence of the DEER data with patterns of structural rearrangements deduced from ensembles of AlphaFold2 models illuminate the conformational cycle of GadC underpinning transport and exposes yet another example of the divergence between the dynamics of different functional families in the LeuT-fold. SIGNIFICANCE STATEMENT The transporter GadC contributes to acid resistance in bacterial pathogens by exchanging two substrates, glutamate and GABA, using a mechanism termed alternating access. In this study, the conformational dynamics underlying alternating access was studied using a combination of spectroscopy and computational modeling. A conformationally diverse ensemble of models, generated using AlphaFold2, guided the design and interpretation of double electron-electron resonance spectroscopy experiments. We found that whereas GadC was inactive and conformationally homogeneous at neutral pH, low pH induced isomerization between two conformations. From our integrated computational/experimental investigation emerges a transport model that may be relevant to eukaryotic homologs that are involved in other cellular processes.

Role of Repeated Conformational Transitions in Substrate Binding of Adenylate Kinase

Abstract: The catalytic cycle of the enzyme adenylate kinase involves large conformational motions between open and closed states. A previous single-molecule experiment showed that substrate binding tends to accelerate both the opening and the closing rates and that a single turnover event often involves multiple rounds of conformational switching. In this work, we showed that the repeated conformational transitions of adenylate kinase are essential for the relaxation of incorrectly bound substrates into the catalytically competent conformation by combining all-atom and coarse-grained molecular simulations. In addition, free energy calculations based on all-atom and coarse-grained models demonstrated that the enzyme with incorrectly bound substrates has much a lower free energy barrier for domain opening compared to that with the correct substrate conformation, which may explain the the acceleration of the domain opening rate by substrate binding. The results of this work provide mechanistic understanding to previous experimental observations and shed light onto the interplay between conformational dynamics and enzyme catalysis.

Engineered disulfide reveals structural dynamics of locked SARS-CoV-2 spike

Abstract: The spike (S) protein of SARS-CoV-2 has been observed in three distinct pre-fusion conformations: locked, closed and open. Of these, the function of the locked conformation remains poorly understood. Here we engineered a SARS-CoV-2 S protein construct “S-R/x3” to arrest SARS-CoV-2 spikes in the locked conformation by a disulfide bond. Using this construct we determined high-resolution structures confirming that the x3 disulfide bond has the ability to stabilize the otherwise transient locked conformations. Structural analyses reveal that wild-type SARS-CoV-2 spike can adopt two distinct locked-1 and locked-2 conformations. For the D614G spike, based on which all variants of concern were evolved, only the locked-2 conformation was observed. Analysis of the structures suggests that rigidified domain D in the locked conformations interacts with the hinge to domain C and thereby restrains RBD movement. Structural change in domain D correlates with spike conformational change. We propose that the locked-1 and locked-2 conformations of S are present in the acidic high-lipid cellular compartments during virus assembly and egress. In this model, release of the virion into the neutral pH extracellular space would favour transition to the closed or open conformations. The dynamics of this transition can be altered by mutations that modulate domain D structure, as is the case for the D614G mutation, leading to changes in viral fitness. The S-R/x3 construct provides a tool for the further structural and functional characterization of the locked conformations of S, as well as how sequence changes might alter S assembly and regulation of receptor binding domain dynamics.

Conformational plasticity of DNA secondary structures: probing the conversion between i-motif and hairpin species by circular dichroism and ultraviolet resonance Raman spectroscopies.

Abstract: The promoter regions of important oncogenes such as BCL2 and KRAS contain GC-rich sequences that can form distinctive noncanonical DNA structures involved in the regulation of transcription: G-quadruplexes on the G-rich strand and i-motifs on the C-rich strand. Interestingly, BCL2 and KRAS promoter i-motifs are highly dynamic in nature and exist in a pH-dependent equilibrium with hairpin and even with hybrid i-motif/hairpin species. Herein, the effects of pH and presence of cell-mimicking molecular crowding conditions on conformational equilibria of the BCL2 and KRAS i-motif-forming sequences were investigated by ultraviolet resonance Raman (UVRR) and circular dichroism (CD) spectroscopies. Multivariate analysis of CD data was essential to model the presence and identity of the species involved. Analysis of UVRR spectra measured as a function of pH, performed also by the two-dimensional correlation spectroscopy (2D-COS) technique, showed the role of several functional groups in the DNA conformational transitions, and provided structural and dynamic information. Thus, the UVRR investigation of intramolecular interactions and of local and environmental dynamics in promoting the different species induced by the solution conditions provided valuable insights into i-motif conformational transitions. The combined use of the two spectroscopic tools is emphasized by the relevant possibility of working in the same DNA concentration range and by the heterospectral UVRR/CD 2D-COS analysis. The results of this study shed light on the factors that can influence at the molecular level the equilibrium between the different conformational species putatively involved in the oncogene expression.

Structures of a large prolate virus capsid in unexpanded and expanded states generate insights into the icosahedral virus assembly

Abstract: Many icosahedral viruses assemble proteinaceous precursors called proheads or procapsids. Proheads are metastable structures that undergo a profound structural transition known as expansion that transforms an immature unexpanded head into a mature genome-packaging head. Bacteriophage T4 is a model virus, well studied genetically and biochemically, but its structure determination has been challenging because of its large size and unusually prolate-shaped, ∼1,200-Å-long and ∼860-Å-wide capsid. Here, we report the cryogenic electron microscopy (cryo-EM) structures of T4 capsid in both of its major conformational states: unexpanded at a resolution of 5.1 Å and expanded at a resolution of 3.4 Å. These are among the largest structures deposited in Protein Data Bank to date and provide insights into virus assembly, head length determination, and shell expansion. First, the structures illustrate major domain movements and ∼70% additional gain in inner capsid volume, an essential transformation to contain the entire viral genome. Second, intricate intracapsomer interactions involving a unique insertion domain dramatically change, allowing the capsid subunits to rotate and twist while the capsomers remain fastened at quasi-threefold axes. Third, high-affinity binding sites emerge for a capsid decoration protein that clamps adjacent capsomers, imparting extraordinary structural stability. Fourth, subtle conformational changes at capsomers' periphery modulate intercapsomer angles between capsomer planes that control capsid length. Finally, conformational changes were observed at the symmetry-mismatched portal vertex, which might be involved in triggering head expansion. These analyses illustrate how small changes in local capsid subunit interactions lead to profound shifts in viral capsid morphology, stability, and volume.

A two-dimensional polymer memristor based on conformational changes with tunable resistive switching behaviours

Abstract: A resistive switching memory device based on a 2DP TPAK+TAPB film with the conformational change mechanism was prepared and the memory behaviors can be adjusted by the degree of conformational changes.

Ligand-induced transmembrane conformational coupling in monomeric EGFR

Abstract: Single pass cell surface receptors regulate cellular processes by transmitting ligand-encoded signals across the plasma membrane via changes to their extracellular and intracellular conformations. This transmembrane signaling is generally initiated by ligand binding to the receptors in their monomeric form. While subsequent receptor-receptor interactions are established as key aspects of transmembrane signaling, the contribution of monomeric receptors has been challenging to isolate due to the complexity and ligand-dependence of these interactions. By combining membrane nanodiscs produced with cell-free expression, single-molecule Förster Resonance Energy Transfer measurements, and molecular dynamics simulations, we report that ligand binding induces intracellular conformational changes within monomeric, full-length epidermal growth factor receptor (EGFR). Our observations establish the existence of extracellular/intracellular conformational coupling within a single receptor molecule. We implicate a series of electrostatic interactions in the conformational coupling and find the coupling is inhibited by targeted therapeutics and mutations that also inhibit phosphorylation in cells. Collectively, these results introduce a facile mechanism to link the extracellular and intracellular regions through the single transmembrane helix of monomeric EGFR, and raise the possibility that intramolecular transmembrane conformational changes upon ligand binding are common to single-pass membrane proteins.

Regulation of integrin α5β1 conformational states and intrinsic affinities by metal ions and the ADMIDAS

Abstract: Mn 2 + activates integrins by increasing the ligand-binding affinity of all three of its conformational states ∼30-fold. Mn 2 + also increases the population of the high-affinity state, but other states still predominate. The ADMIDAS increases the affinity difference between high- and low- affinity states and the fidelity of conformational change.

An intrabody sensor to monitor conformational activation of β-arrestins.

Abstract: Agonist-induced interaction of β-arrestins with GPCRs is critically involved in downstream signaling and regulation. This interaction is associated with activation and major conformational changes in β-arrestins. Although there are some assays available to monitor the conformational changes in β-arrestins in cellular context, additional sensors to report β-arrestin activation, preferably with high-throughput capability, are likely to be useful considering the structural and functional diversity in GPCR-β-arrestin complexes. We have recently developed an intrabody-based sensor as an integrated approach to monitor GPCR-β-arrestin interaction and conformational change, and generated a luminescence-based reporter using NanoBiT complementation technology. This sensor is derived from a synthetic antibody fragment referred to as Fab30 that selectively recognizes activated and receptor-bound conformation of β-arrestin1. Here, we present a step-by-step protocol to employ this intrabody sensor to measure the interaction and conformational activation of β-arrestin1 upon agonist-stimulation of a prototypical GPCR, the complement C5a receptor (C5aR1). This protocol is potentially applicable to other GPCRs and may also be leveraged to deduce qualitative differences in β-arrestin1 conformations induced by different ligands and receptor mutants.

Redox-Regulated Conformational Change of Disulfide-Rich Assembling Peptides.

Abstract: Disulfide bond formation is a common mechanism for regulating conformational changes in proteins during oxidative folding. Despite extensive studies of the use of multiple disulfide bonds to constrain peptide conformation, few studies have explored their usage in developing self-assembling peptides. Herein, we report that a thiol-rich peptide could fold into an amphiphilic β-hairpin conformation through the formation of two hetero-disulfide bonds upon oxidation, subsequently self-assembling into a mechanically rigid hydrogel. Breaking disulfide bonds under reductive condition, the hydrogel exhibited a transition from hydrogel to solution. Molecular simulation revealed that intermolecular interaction between two tryptophan residues was indispensable for hydrogelation. This work is the first case of the use of multiple disulfide bonds to control conformational change and self-assembly, and provides a cell-compatible hydrogel material for potential biomedical application.

Structural insights into ClpP protease side exit pore‐opening by a pH drop coupled with substrate hydrolysis

Abstract: The ClpP serine peptidase is a tetradecameric degradation molecular machine involved in many physiological processes. It becomes a competent ATP‐dependent protease when coupled with Clp‐ATPases. Small chemical compounds, acyldepsipeptides (ADEPs), are known to cause the dysregulation and activation of ClpP without ATPases and have potential as novel antibiotics. Previously, structural studies of ClpP from various species revealed its structural details, conformational changes, and activation mechanism. Although product release through side exit pores has been proposed, the detailed driving force for product release remains elusive. Herein, we report crystal structures of ClpP from Bacillus subtilis (BsClpP) in unforeseen ADEP‐bound states. Cryo‐electron microscopy structures of BsClpP revealed various conformational states under different pH conditions. To understand the conformational change required for product release, we investigated the relationship between substrate hydrolysis and the pH‐lowering process. The production of hydrolyzed peptides from acidic and basic substrates by proteinase K and BsClpP lowered the pH values. Our data, together with those of previous findings, provide insight into the molecular mechanism of product release by the ClpP self‐compartmentalizing protease.

Mechanisms of long-distance allosteric couplings in proton-binding membrane transporters

Abstract: Membrane transporters that use proton binding and proton transfer for function couple local protonation change with changes in protein conformation and water dynamics. Changes of protein conformation might be required to allow transient formation of hydrogen-bond networks that bridge proton donor and acceptor pairs separated by long distances. Inter-helical hydrogen-bond networks adjust rapidly to protonation change, and ensure rapid response of the protein structure and dynamics. Membrane transporters with known three-dimensional structures and proton-binding groups inform on general principles of protonation-coupled protein conformational dynamics. Inter-helical hydrogen bond motifs between proton-binding carboxylate groups and a polar sidechain are observed in unrelated membrane transporters, suggesting common principles of coupling protonation change with protein conformational dynamics.

Photoinduced Single-Crystal to Single-Crystal Transformation via Conformational Change with Turn-On Fluorescence

Abstract: Single-crystal to single-crystal (SCSC) transformation by phototrigger is presently attracting immense research interest in many fields such as sensors, actuators, artificial muscles, soft robotics, and energy harvesting. However, the only success of rare examples has been achieved by SCSC transformation so far, involving a change in molecular composition based on photocyclization reactions. Here, we show photoinduced SCSC transformation of 3-methylene-2-(quinolin-8-yl) isoindolin-1-one (MQIO) derivatives from monoclinic to triclinic phase via conformational change, just as “small input and big output”. UV light induces a molecular conformation change of MQIO-H with rotation (11.3°) of the carbon–nitrogen single bond. The conformational changes of MQIO-H lead to the slides of molecular packing in crystals, finally resulting in the SCSC transformation from the monoclinic to the triclinic phase. In addition, the SCSC transformation was accompanied by turn-on fluorescence and shape changes of single crystal. The shape changes of single crystals can be clearly observed by UV light triggering under an optical microscope. This work provides a strategy to achieve SCSC transformation via photoinduction.

Structural and functional role of Domain I for the insecticidal activity of the Vip3Aa protein from Bacillus thuringiensis

Abstract: Vip3 proteins are produced by Bacillus thuringiensis and are toxic against lepidopterans, reason why the vip3Aa gene has been introduced into cotton and corn to control agricultural pests. Recently, the structure of Vip3 proteins has been determined and consists of a tetramer where each monomer is composed of five structural domains. The transition from protoxin to the trypsin‐activated form involves a major conformational change of the N‐terminal Domain I, which is remodelled into a tetrameric coiled‐coil structure that is thought to insert into the apical membrane of the midgut cells. To better understand the relevance of this major change in Domain I for the insecticidal activity, we have generated several mutants aimed to alter the activity and remodelling capacity of this central region to understand its function. These mutants have been characterized by proteolytic processing, negative staining electron microscopy, and toxicity bioassays against Spodoptera exigua. The results show the crucial role of helix α1 for the insecticidal activity and in restraining the Domain I in the protoxin conformation, the importance of the remodelling of helices α2 and α3, the proteolytic processing that takes place between Domains I and II, and the role of the C‐t Domains IV and V to sustain the conformational change necessary for toxicity.

Monoclonal Antibodies as Probes to Study Ligand-Induced Conformations of Troponin Subunits

Abstract: Striated muscle contraction and relaxation is regulated by Ca2+ at the myofilament level via conformational modulations of the troponin complex. To understand the structure–function relationship of troponin in normal muscle and in myopathies, it is necessary to study the functional effects of troponin isoforms and mutations at the level of allosteric conformations of troponin subunits. Traditional methodologies assessing such conformational studies are laborious and require significant amounts of purified protein, while many current methodologies require non-physiological conditions or labeling of the protein, which may affect their physiological conformation and function. To address these issues, we developed a novel approach using site-specific monoclonal antibodies (mAb) as molecular probes to detect and monitor conformational changes of proteins. Here, we present examples for its application in studies of two subunits of troponin: the Ca2+-binding subunit, TnC, and the tropomyosin-binding/thin filament-anchoring subunit, TnT. Studies using a high-throughput microplate assay are compared with that using localized surface plasmon resonance (LSPR) to demonstrate the effectiveness of using mAb probes to assess ligand-induced conformations of troponin subunits in physiological conditions. The assays utilize relatively small amounts of protein and are free of protein modification, which may bias results. Detailed methodologies using various monoclonal antibodies (mAbs) are discussed with considerations for the optimization of assay conditions and the broader application in studies of other proteins as well as in screening of therapeutic reagents that bind a specific target site with conformational and functional effects.

The cyclic octapeptide antibiotic argyrin B inhibits translation by trapping EF-G on the ribosome during translocation

Abstract: Significance The increase in multidrug-resistant bacteria highlights the urgent need for compounds with novel target sites that can be developed as antibiotics. The argyrins represent a family of naturally produced octapeptides that display promising activity against Pseudomonas aeruginosa by inhibiting protein synthesis. Our structural and kinetic analyses reveal that argyrins inhibit protein synthesis by interacting with, and trapping, the translation elongation factor G (EF-G) on the ribosome, analogous to that reported previously for the unrelated antibiotic fusidic acid. However, the binding site of argyrin on EF-G is distinct from that of fusidic acid, indicating that intramolecular movements at the domain III/V interface of EF-G are also essential for facilitating late events in the translocation mechanism.

Phosphorylation Induced Conformational Transitions in DNA Polymerase β

Abstract: DNA polymerase β (pol β) is a member of the X- family of DNA polymerases that catalyze the distributive addition of nucleoside triphosphates during base excision DNA repair. Previous studies showed that the enzyme was phosphorylated in vitro with PKC at two serines (44 and 55), causing loss of DNA polymerase activity but not DNA binding. In this work, we have investigated the phosphorylation-induced conformational changes in DNA polymerase β in the presence of Mg ions. We report a comprehensive atomic resolution study of wild type and phosphorylated DNA polymerase using molecular dynamics (MD) simulations. The results are examined via novel methods of internal dynamics and energetics analysis to reveal the underlying mechanism of conformational transitions observed in DNA pol β. The results show drastic conformational changes in the structure of DNA polymerase β due to S44 phosphorylation. Phosphorylation-induced conformational changes transform the enzyme from a closed to an open structure. The dynamic cross-correlation shows that phosphorylation enhances the correlated motions between the different domains. Centrality network analysis reveals that the S44 phosphorylation causes structural rearrangements and modulates the information pathway between the Lyase domain and base pair binding domain. Further analysis of our simulations reveals that a critical hydrogen bond (between S44 and E335) disruption and the formation of three additional salt bridges are potential drivers of these conformational changes. In addition, we found that two of these additional salt bridges form in the presence of Mg ions on the active sites of the enzyme. These results agree with our previous study of DNA pol β S44 phosphorylation without Mg ions which predicted the deactivation of DNA pol β. However, the phase space of structural transitions induced by S44 phosphorylation is much richer in the presence of Mg ions.