Conformational Changes Research Articles

Kinetics-Based State Definitions for Discrete Binding Conformations of T4 L99A in MD via Markov State Modeling.

As a model system, the binding pocket of the L99A mutant of T4 lysozyme has been the subject of numerous computational free energy studies. However, previous studies have failed to fully sample and account for the observed changes in the binding pocket of T4 L99A upon binding of a congeneric ligand series, limiting the accuracy of results. In this work, we resolve the closed, intermediate, and open states for T4 L99A previously reported in experiment in MD and establish definitions for these states based on the dynamics of the system. From this analysis, we arrive at two primary conclusions. First, assignment of simulation trajectories into discrete states should not be done simply based on RMSD to crystal structures as this can result in misassignment of states. Second, the different metastable conformations studied here need to be carefully treated, as we estimate the time scales for conformational interconversion to be on the order of 102 to 103 ns─far longer than time scales for typical binding calculations. We conclude with a discussion on the need to develop enhanced sampling methods to generally account for significant changes in protein conformation due to relatively small ligand perturbations.

An Organic Vapor-Responsive Actuator Based on a Novel Urea Macrocycle.

The mechanical actuation of smart materials has garnered considerable attention in biological and medical research due to their ability to mimic biological processes at both molecular level, such as conformational changes in individual compounds, and at the macroscopic level, where polymeric substrates respond to external stimuli. In this study, we present a polymeric composite incorporating a novel urea macrocycle as a filler, forming a soft actuator that responds to various organic solvent vapors. The underlying actuation mechanism is attributed to crystalline phase transition of urea macrocycle, driven by the host‒guest interactions with diverse guest molecules. This work provides valuable insights for advancing the design of supramolecular hosts in smart material applications.

Water and chloride as allosteric inhibitors in WNK kinase osmosensing.

Osmotic stress and chloride regulate the autophosphorylation and activity of the WNK1 and WNK3 kinase domains. The kinase domain of unphosphorylated WNK1 (uWNK1) is an asymmetric dimer possessing water molecules conserved in multiple uWNK1 crystal structures. Conserved waters are present in two networks, referred to here as conserved water networks 1 and 2 (CWN1 and CWN2). Here, we show that PEG400 applied to crystals of dimeric uWNK1 induces de-dimerization. Both the WNK1 the water networks and the chloride-binding site are disrupted by PEG400. CWN1 is surrounded by a cluster of pan-WNK-conserved charged residues. Here, we mutagenized these charges in WNK3, a highly active WNK isoform kinase domain, and WNK1, the isoform best studied crystallographically. Mutation of E314 in the Activation Loop of WNK3 (WNK3/E314Q and WNK3/E314A, and the homologous WNK1/E388A) enhanced the rate of autophosphorylation, and reduced chloride sensitivity. Other WNK3 mutants reduced the rate of autophosphorylation activity coupled with greater chloride sensitivity than wild-type. The water and chloride regulation thus appear linked. The lower activity of some mutants may reflect effects on catalysis. Crystallography showed that activating mutants introduced conformational changes in similar parts of the structure to those induced by PEG400. WNK activating mutations and crystallography support a role for CWN1 in WNK inhibition consistent with water functioning as an allosteric ligand.

Water and chloride as allosteric inhibitors in WNK kinase osmosensing

Osmotic stress and chloride regulate the autophosphorylation and activity of the WNK1 and WNK3 kinase domains. The kinase domain of unphosphorylated WNK1 (uWNK1) is an asymmetric dimer possessing water molecules conserved in multiple uWNK1 crystal structures. Conserved waters are present in two networks, referred to here as conserved water networks 1 and 2 (CWN1 and CWN2). Here, we show that PEG400 applied to crystals of dimeric uWNK1 induces de-dimerization. Both the WNK1 the water networks and the chloride-binding site are disrupted by PEG400. CWN1 is surrounded by a cluster of pan-WNK-conserved charged residues. Here, we mutagenized these charges in WNK3, a highly active WNK isoform kinase domain, and WNK1, the isoform best studied crystallographically. Mutation of E314 in the Activation Loop of WNK3 (WNK3/E314Q and WNK3/E314A, and the homologous WNK1/E388A) enhanced the rate of autophosphorylation, and reduced chloride sensitivity. Other WNK3 mutants reduced the rate of autophosphorylation activity coupled with greater chloride sensitivity than wild-type. The water and chloride regulation thus appear linked. The lower activity of some mutants may reflect effects on catalysis. Crystallography showed that activating mutants introduced conformational changes in similar parts of the structure to those induced by PEG400. WNK activating mutations and crystallography support a role for CWN1 in WNK inhibition consistent with water functioning as an allosteric ligand.

Spectral fingerprints of DOM-tungsten interactions: Linking molecular binding to conformational changes

Tungsten (W), a widely used yet understudied emerging contaminant, forms oxyanions in aqueous environments, distinguishing it from conventional heavy metals. While dissolved organic matter (DOM) demonstrates considerable potential for W binding, DOM-W interactions remain largely unexplored. Of particular significance, yet frequently overlooked, are the conformational changes in DOM during W binding processes. This study proposes a novel theoretical framework integrating superposition and charge transfer models to elucidate the complexity of these interactions. By combining spectroscopic techniques and photophysical models, we revealed that aromatic compounds containing 1–3 rings, especially monocyclic aromatic protein-like components, exhibit high affinity for W (logK=3.74–4.00). Phenolic hydroxyls served as primary binding sites for W, with aromatic rings facilitating binding through π interactions. Importantly, W binding to aromatic compounds induced conformational changes in DOM, transitioning from a loosely aggregated state to a more compact configuration. These changes facilitated W encapsulation within DOM through the synergistic effects of hydrophobic interactions, hydrogen/π-hydrogen bonding and π-stacking, potentially leading to stable trapping of W. Two-dimensional correlation spectroscopy analysis elucidated the sequential encapsulation process, involving phenolic, aromatic carboxylic/aliphatic carboxylic, polysaccharides, and aliphatics. The intricate behavior of DOM-W binding profoundly reshapes DOM's conformation, subtly yet significantly orchestrating W's binding affinity, environmental transport, and bioavailability in aquatic ecosystems.

Structural insights into the ATP-dependent activation of NOD-like receptor with pyrin 3 (NLRP3) protein by molecular dynamics simulation

The inflammasome-forming NOD-like receptor containing pyrin-3 (NLRP3) protein is a critical player in the innate immune responses to cellular danger signals. New structural data of NLRP3 provide a framework to probe the conformational impact of nucleotide binding. In this study, microsecond molecular dynamics (MD) simulations were used to detail information on the unique structural conformations adopted by NLRP3 with ATP or ADP binding. Sampling convergence reflected a high degree of confidence in the MD simulations as shown by RMSD and protein-nucleotide concordance, favourable overall MM-PBSA ligand binding energies for both nucleotides and low cosine coefficients of the principal eigenvectors obtained with essential dynamics (ED) analysis. NLRP3-ADP simulations provide relatively stable conformations with few global rearrangements as shown by decreased protein RMSD, Rg, SASA, and solvent accessibility for the ADP-bound structure. In contrast, ATP binding induced increased flexibility and resulted in substantive conformational changes to the NLRP3 structure. Binding of ATP was thermodynamically favourable as shown by the ΔGsolv and MM-PBSA calculations of complex free energies, and these NLRP3-ATP simulations resulted in similar structural transitions as observed in the activated NLRC4 empirical structure. Lastly, the active conformation of NLRP3 critically depends on hinging between the HD2 and LRR domains, whereby ATP binding drives local conformational changes that are conveyed to the global structure.

Significance of the Double Bond in the Acyl Chain of Cardiolipin Revealed by the Partial Unfolding Dynamics of Cytochrome c Using Femtosecond Transient Absorption Spectroscopy.

Cytochrome c (Cyt c) released from the mitochondrion acts as a trigger for the onset of apoptosis in which a double bond of cardiolipin (CL) is oxidized upon interaction with Cyt c. To understand the interaction dynamics of Cyt c with the double bond of CL, CLs having acyl chains with a systematic increase in the number of double bonds, 0 (18:0 CL), 1 (18:1 CL), and 2 (18,2 CL), were complexed with Cyt c, and their excited-state dynamics were studied using femtosecond time-resolved pump-probe spectroscopy. Steady-state and femtosecond transient absorption spectra revealed a systematic increase in the partial unfolding of Cyt c with an increase in double bonds in CL, as observed by the enhanced fluorescence intensity and lifetime of tryptophan due to variations in the resonance energy transfer and extended global conformational relaxation time constants. These studies reflect the significance of occurrence of global conformational changes of Cyt c by structural modification near the double bond of CL in the Cyt c-CL complex, which could be prerequisites for the apoptosis.

Large-scale Conformational Changes of Diindole Functional Groups Driven by Cation-π Interactions and the Toughening Mechanism of Thermosets.

This is described that a new concept for the design of toughening supramolecular thermosets by incorporating diindole groups, which are functionalized via cation-π interactions in covalently crosslinked networks. Large-scale conformational changes of the diindole functional groups could occur under external forces, endowing the thermosets with excellent toughening properties.

Structural dynamics of calcium and integrin-binding protein 2 (CIB2) reveal uncommon flexibility and heterogeneous calcium and magnesium loading

Calcium- and Integrin-Binding protein 2 (CIB2) is a widely expressed protein with an uncertain biological role. Two of its four EF-hand motifs bind Mg(II) and/or Ca(II), thus triggering conformational changes. Although previous studies suggested that CIB2 preferentially binds Mg(II) over Ca(II) under physiological conditions, an atomic level characterization of CIB2 in the presence of both cations was lacking. Based on a combination of solution NMR, exhaustive molecular dynamics simulations and isothermal titration and differential scanning calorimetry, we characterized the interaction of CIB2 with both Ca(II) and Mg(II) ions and elucidated the protein regions involved in the interaction with the α7B integrin target. Analysis of experimental amide nitrogen relaxation rates shows that the EF4 motif exhibits high mobility regardless of the specific bound metal ion and demonstrates that the Ca(II)- and Mg(II)-bound state of CIB2 is relatively floppy, with pico-nanosecond motions induced in a region involved in target recognition. Overall, our data indicate a preferential, thermodynamically stable but structurally flexible state for CIB2, in which an Mg(II) ion is bound to EF3 and a Ca(II) ion to EF4. These results unveil the role of metal binding events in CIB2 and offer new insights into the dynamic regulation of target recognition.

Single-molecule imaging reveals the kinetics of non-homologous end-joining in living cells

Non-homologous end joining (NHEJ) is the predominant pathway that repairs DNA double-stranded breaks (DSBs) in vertebrates. However, due to challenges in detecting DSBs in living cells, the repair capacity of the NHEJ pathway is unknown. The DNA termini of many DSBs must be processed to allow ligation while minimizing genetic changes that result from break repair. Emerging models propose that DNA termini are first synapsed ~115 Å apart in one of several long-range synaptic complexes before transitioning into a short-range synaptic complex that juxtaposes DNA ends to facilitate ligation. The transition from long-range to short-range synaptic complexes involves both conformational and compositional changes of the NHEJ factors bound to the DNA break. Importantly, it is unclear how NHEJ proceeds in vivo because of the challenges involved in analyzing recruitment of NHEJ factors to DSBs over time in living cells. Here, we develop an approach to study the temporal and compositional dynamics of NHEJ complexes using live cell single-molecule imaging. Our results provide direct evidence for stepwise maturation of the NHEJ complex, pinpoint key regulatory steps in NHEJ progression, and allowed us to estimate the overall repair capacity of the NHEJ pathway in living cells.

Allostery can convert binding free energies into concerted domain motions in enzymes

Enzymatic mechanisms are typically inferred from structural data. However, understanding enzymes require unravelling the intricate dynamic interplay between dynamics, conformational substates, and multiple protein structures. Here, we use single-molecule nanopore analysis to investigate the catalytic conformational changes of adenylate kinase (AK), an enzyme that catalyzes the interconversion of various adenosine phosphates (ATP, ADP, and AMP). Kinetic analysis validated by hidden Markov models unravels the details of domain motions during catalysis. Our findings reveal that allosteric interactions between ligands and cofactor enable converting binding energies into directional conformational changes of the two catalytic domains of AK. These coordinated motions emerged to control the exact sequence of ligand binding and the affinity for the three different substrates, thereby guiding the reactants along the reaction coordinates. Interestingly, we find that about 10% of enzymes show altered allosteric regulation and ligand affinities, indicating that a subset of enzymes folds in alternative catalytically active forms. Since molecules or proteins might be able to selectively stabilize one of the folds, this observation suggests an evolutionary path for allostery in enzymes. In AK, this complex catalytic framework has likely emerged to prevent futile ATP/ADP hydrolysis and to regulate the enzyme for different energy needs of the cell.

Structural insight into Okazaki fragment maturation mediated by PCNA-bound FEN1 and RNaseH2.

PCNA is a master coordinator of many DNA-metabolic events. During DNA replication, the maturation of Okazaki fragments involves at least four DNA enzymes, all of which contain PCNA-interacting motifs. However, the temporal relationships and functional modulations between these PCNA-binding proteins are unclear. Here, we developed a strategy to purify endogenous PCNA-containing complexes from native chromatin, and characterized their structures using cryo-EM. Two structurally resolved classes (PCNA-FEN1 and PCNA-FEN1-RNaseH2 complexes) have captured a series of 3D snapshots for the primer-removal steps of Okazaki fragment maturation. These structures show that product release from FEN1 is a rate-liming step. Furthermore, both FEN1 and RNaseH2 undergo continuous conformational changes on PCNA that result in constant fluctuations in the bending angle of substrate DNA at the nick site, implying that these enzymes could regulate each other through conformational modulation of the bound DNA. The structures of the PCNA-FEN1-RNaseH2 complex confirm the toolbelt function of PCNA and suggests a potential unrecognized role of RNaseH2, as a dsDNA binding protein, in promoting the 5'-flap cleaving activity of FEN1.

Structural basis for the reaction cycle and transport mechanism of human Na+-sulfate cotransporter NaS1 (SLC13A1).

Sulfate (SO42-) is a pivotal inorganic anion with essential roles in mammalian physiology. NaS1, a member of solute carrier 13 family and divalent anion/sodium symporter family, functions as a Na+-sulfate cotransporter, facilitating sulfate (re)absorption across renal proximal tubule and small intestine epithelia. While previous studies have linked several human disorders to mutations in the NaS1 gene, its transport mechanism remains unclear. Here, we report the cryo-electron microscopy structures of five distinct conformations of the human NaS1 at resolutions of 2.7 to 3.3 angstroms, revealing the substrates recognition mechanism and the conformational change of NaS1 during the Na+-sulfate cotransport cycle. Our studies delineate the molecular basis of the detailed dynamic transport cycle of NaS1. These findings advance the current understanding of the Na+-sulfate cotransport mechanism, human sulfate (re)absorption, and the implications of disease-associated NaS1 mutations.

Effect of Acetonitrile on the Conformation of Bovine Serum Albumin.

The use of organic solvents in drug delivery systems (DDSs) either to produce albumin nanoparticles or to manipulate the binding of target molecules to albumin, a promising nanocarrier material, presents challenges due to the conformational changes induced in the protein. In this study, we investigated the alterations in the conformation of bovine serum albumin (BSA) caused by acetonitrile (ACN) in aqueous solution by using a combination of spectroscopic analysis and molecular dynamics (MD) simulations. Ultraviolet (UV) absorption, fluorescence, and infrared (IR) absorption spectroscopy were used to analyze the BSA conformation in the solutions containing 0-60 vol % ACN. Additionally, MD simulations were conducted to elucidate the interactions between BSA and solvent components, focusing on the structural changes in the hydrophobic pocket with Trp residues of the albumin. Increasing the ACN concentration leads to significant changes in the BSA conformation, as evidenced by shifts in UV fluorescence wavelength, decreased intensity, and alterations in IR absorption bands. Furthermore, the formation of protein aggregates was observed at high ACN concentration (30 vol % ACN), shown by increased hydrodynamic diameter distribution. MD simulations further demonstrate that the presence of ACN molecules near the hydrophobic pocket with the Trp-213 residue increases the fluctuations in the positions of amino acids observed near the hydrophobic pocket with Trp-213. Moreover, the intrusion of water molecules into the hydrophobic pocket under 60% ACN conditions with highly decreased solvent polarity was correlated with the changes in the BSA secondary structure. These findings enhance our understanding of how solvent polarity affects the albumin conformation, which is crucial for optimizing albumin-based DDS applications.

Hybrid Mass Spectrometry Applied across the Production of Antibody Biotherapeutics.

Post expression from the host cells, biotherapeutics undergo downstream processing steps before final formulation. Mass spectrometry and biophysical characterization methods are valuable for examining conformational and stoichiometric changes at these stages, although typically not used in biomanufacturing, where stability is assessed via bulk property studies. Here we apply hybrid MS methods to understand how solution condition changes impact the structural integrity of a biopharmaceutical across the processing pipeline. As an exemplar product, we use the model IgG1 antibody, mAb4. Flexibility, stability, aggregation propensity, and bulk properties are evaluated in relation to perfusion media, purification stages, and formulation solutions. Comparisons with Herceptin, an extensively studied IgG1 antibody, were conducted in a mass spectrometry-compatible solution. Despite presenting similar charge state distributions (CSD) in native MS, mAb4, and Herceptin show distinct unfolding patterns in activated ion mobility mass spectrometry (aIM-MS) and differential scanning fluorimetry (DSF). Herceptin's greater structural stability and aggregation onset temperature (Tagg) are attributed to heavier glycosylation and kappa-class light chains, unlike the lambda-class light chains in mAb4. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) revealed that mAb4 undergoes substantial structural changes during purification, marked by high flexibility, low melting temperature (Tm), and prevalent repulsive protein-protein interactions but transitions to a compact and stable structure in high-salt and formulated environments. Notably, in formulation, the third constant domain (CH3) of the heavy chain retains flexibility and is a region of interest for aggregation. Future work could translate features of interest from comprehensive studies like this to targeted approaches that could be utilized early in the development stage to aid in decision-making regarding targeted mutations or to guide the design space of bioprocesses and formulation choices.

Three STEPs forward: A trio of unexpected structures of PTPN5.

Protein tyrosine phosphatases (PTPs) play pivotal roles in myriad cellular processes by counteracting protein tyrosine kinases. Striatal-enriched protein tyrosine phosphatase (STEP, PTPN5) regulates synaptic function and neuronal plasticity in the brain and is a therapeutic target for several neurological disorders. Here, we present three new crystal structures of STEP, each with unexpected features. These include high-resolution conformational heterogeneity at multiple sites, a highly coordinated citrate molecule that inhibits enzyme activity, a previously unseen conformational change at an allosteric site, an intramolecular disulfide bond that was characterized biochemically but had never been visualized structurally, and two serendipitous covalent ligand binding events at surface-exposed cysteines that are nearly or entirely unique to STEP among human PTPs. Together, our results offer new views of the conformational landscape of STEP that may inform structure-based design of allosteric small molecules to specifically inhibit this biomedically important enzyme.

Unveiling the Mechanism of Deprotonation and Proton Transfer of DNA Polymerase Catalysis via Single-Molecule Conductance.

DNA polymerases (Pols) play important roles in the transmission of genetic information. Although the function and (de)regulation of Pols are linked to many human diseases, the key mechanism of 3'-OH deprotonation and the PPi formation are not totally clear. In this work, a method is presented to detect the full catalytic cycle of human Pol (hPol β) in graphene-molecule-graphene single-molecule junctions. Real-time in situ monitoring successfully revealed the spatial and temporal properties of the open and closed conformation states of hPol β, distinguishing the reaction states in the Pols catalytic cycle and unveiling 3'-OH deprotonation and pyrophosphate (PPi) formation mechanism of hPol β. Proton inventory experiment demonstrated that the rate-limiting step of PPi formation is deprotonation, which occurs before a reverse conformational change. Additionally, by detecting the acidity (pKa), it is found that MgA-bound OH- acted as a general base and activated the nucleophile of 3'-OH, and that acidic residue D190 or D192 coordinated with MgB as a proton donor to PPi. This work provides useful insights into a fundamental chemical reaction that impacts genome synthesis efficiency and Pol fidelity, which the discovery of Pol-targeting drugs and design of artificial Pols for DNA synthetic applications are expected to accelerated.

Ultrasound-mediated catheter delivery of tissue plasminogen activator promotes thrombolysis by altering fibrin fibre thickness and clot permeability.

It has been proposed that low power, high frequency ultrasound can augment the ability of thrombolytic agents to dissolve clot in patients with venous thromboembolism. We created a bench model to examine what role and mechanism ultrasound may have in this process. Fibrin polymerisation was analysed through modified light-scattering experiments with the inclusion of catheter-mediated ultrasound application. We studied fibrin fibre diameters through scanning electron microscopy of ultrasound treated fibrin clots. Clot porosity was investigated using permeation tests, while fibrinolysis was analysed through lightscattering experiments, and by changes in porosity of lysing clots under flow. Application of ultrasound did not change initial fibrin polymerisation but did induce a reversible change in maximal turbidity of already formed fibrin clots. This change in turbidity was caused by a reduction in fibrin fibre diameter and was associated with an increase in clot porosity. These reversible structural changes were associated with a linear increase in fibrinolysis rates under static conditions, while an exponential increase in rates was observed under flow. The use of ultrasound augmentation of thrombolysis enhances clot dissolution through greater and more rapid fibrin degradation. This is due to conformational change created by the ultrasound in clot structure, a reversible phenomenon that may increase binding sites for lytic agent and could potentially allow the use of lower doses and shorter infusion times of ultrasound-assisted thrombolytic to treat venous thromboembolism in-vivo.

Enrichable Protein Footprinting for Structural Proteomics.

Protein footprinting is a useful method for studying protein higher order structure and conformational changes induced by interactions with various ligands via addition of covalent modifications onto the protein. Compared to other methods that provide single amino acid-level structural resolution, such as cryo-EM, X-ray diffraction, and NMR, mass spectrometry (MS)-based methods can be advantageous as they require lower protein amounts and purity. As with other MS-based proteomic methods, such as post-translational modification analysis, enrichment techniques have proven necessary for both optimal sensitivity and sequence coverage when analyzing highly complex proteomes. Currently used reagents for footprinting via covalent labeling, such as hydroxyl radicals and carbodiimide-based methods, do not yet have a suitable enrichment method, limiting their applicability to whole proteome analysis. Here, we report a method for enrichable covalent labeling built upon the GEE/EDC system commonly used to covalently label aspartic acid and glutamic acid residues. Novel labeling reagents containing alkynyl functionality can be "clicked" to any azido-containing molecule with copper-catalyzed azide-alkyne cycloaddition (CuAAC), allowing for enrichment or further labeling. Multiple azide- and alkyne-containing GEE-like molecules were tested, and the most efficient method was determined to be the EDC-facilitated coupling of glycine propargyl amide (GPA) to proteins. The pipeline we report includes clicking via CuAAC to a commercially available biotin-azide containing a photocleavable linker, followed by enrichment using a streptavidin resin and subsequent cleavage under ultraviolet light. The enrichment process was optimized through the screening of clickable amines, coupling reagents, and enrichment scaffolds and methods with pure model proteins and has also been applied to complex mixtures of proteins isolated from the model plant, Arabidopsis thaliana, suggesting that our method may ultimately be used to measure protein conformation on a proteomic scale.

Effect of NaCl concentration on the interfacial and foaming properties of wheat aqueous phase protein

In this study, the structure, foaming properties, and air-water interfacial behavior of wheat aqueous phase protein (WAP) with different NaCl concentrations were investigated. With the addition of NaCl, the particle size and the β-sheet content increased, indicating the formation of larger aggregates. In the FT-IR spectrum NaCl also increased the peak intensity for WAP, this peak often associated with C-O and C-N stretching vibrations, indicating that NaCl may induce conformational changes, such as protein unfolding. As NaCl concentration increased, the foam capacity of WAP increased from 106.25 ± 1.41% to 142.38 ± 15.73%, while native WAP exhibited higher foam stability. Interfacial adsorption kinetics revealed that NaCl favored WAP adsorption at the air-water interface. The interfacial viscoelasticity modulus of all samples increased over time, forming an interfacial layer primarily characterized by elastic behavior. Native WAP exhibited a stronger viscoelasticity modulus, forming a stable adsorption layer at the air-water interface, which contributed to enhanced foam stability. This study provides valuable insights into the regulation of WAP structure and foaming properties by salt ions, which may offer a new strategy for improving the interfacial properties of wheat-based food products.