Conformational Reorganization Research Articles

Tracing Allostery in the Spliceosome Ski2-like RNA Helicase Brr2.

RNA ATPases/helicases remodel substrate RNA-protein complexes in distinct ways. The different RNA ATPases/helicases, taking part in the spliceosome complex, reshape the RNA/RNA-protein contacts to enable premature-mRNA splicing. Among them, the bad response to refrigeration 2 (Brr2) helicase promotes U4/U6 small nuclear (sn)RNA unwinding via ATP-driven translocation of the U4 snRNA strand, thus playing a pivotal role during the activation, catalytic, and disassembly phases of splicing. The plastic Brr2 architecture consists of an enzymatically active N-terminal cassette (N-cassette) and a structurally similar but inactive C-terminal cassette (C-cassette). The C-cassette, along with other allosteric effectors and regulators, tightly and timely controls Brr2's function via an elusive mechanism. Here, microsecond-long molecular dynamics simulations, dynamical network theory, and community network analysis are combined to elucidate how allosteric effectors/regulators modulate the Brr2 function. We unexpectedly reveal that U4 snRNA itself acts as an allosteric regulator, amplifying the cross-talk of distal Brr2 domains and triggering a conformational reorganization of the protein. Our findings offer fundamental understanding into Brr2's mechanism of action and broaden our knowledge on the sophisticated regulatory mechanisms by which spliceosome ATPases/helicases control gene expression. This includes their allosteric regulation exerted by client RNA strands, a mechanism that may be broadly applicable to other RNA-dependent ATPases/helicases.

Structural basis for autoinhibition by the dephosphorylated regulatory domain of Ycf1

Yeast Cadmium Factor 1 (Ycf1) sequesters glutathione and glutathione-heavy metal conjugates into yeast vacuoles as a cellular detoxification mechanism. Ycf1 belongs to the C subfamily of ATP Binding Cassette (ABC) transporters characterized by long flexible linkers, notably the regulatory domain (R-domain). R-domain phosphorylation is necessary for activity, whereas dephosphorylation induces autoinhibition through an undefined mechanism. Because of its transient and dynamic nature, no structure of the dephosphorylated Ycf1 exists, limiting understanding of this R-domain regulation. Here, we capture the dephosphorylated Ycf1 using cryo-EM and show that the unphosphorylated R-domain indeed forms an ordered structure with an unexpected hairpin topology bound within the Ycf1 substrate cavity. This architecture and binding mode resemble that of a viral peptide inhibitor of an ABC transporter and the secreted bacterial WXG peptide toxins. We further reveal the subset of phosphorylation sites within the hairpin turn that drive the reorganization of the R-domain conformation, suggesting a mechanism for Ycf1 activation by phosphorylation-dependent release of R-domain mediated autoinhibition.

What to Make of Zero: Resolving the Statistical Noise from Conformational Reorganization in Alchemical Binding Free Energy Estimates with Metadynamics Sampling.

We introduce the self-relative binding free energy (self-RBFE) approach to evaluate the intrinsic statistical variance of dual-topology alchemical binding free energy estimators. The self-RBFE is the relative binding free energy between a ligand and a copy of the same ligand, and its true value is zero. Nevertheless, because the two copies of the ligand move independently, the self-RBFE value produced by a finite-length simulation fluctuates and can be used to measure the variance of the model. The results of this validation provide evidence that a significant fraction of the errors observed in benchmark studies reflect the statistical fluctuations of unconverged estimates rather than the models' accuracy. Furthermore, we find that ligand reorganization is a significant contributing factor to the statistical variance of binding free energy estimates and that metadynamics-accelerated conformational sampling of the torsional degrees of freedom of the ligand can drastically reduce the time to convergence.

Revisiting the conformational transition model for the pH dependence of BSA structure using photoluminescence, circular dichroism, and ellipsometric Raman spectroscopy

Changes in pH affect metabolic pathways, primarily by modulating enzyme conformations, which is why a detailed analysis of pH-driven conformational transitions is required to understand the underlying biochemistry of diseases and biological organisms. In this work, we examined the pH-driven conformational dynamics of Bovine Serum Albumin (BSA), within the framework of the Foster Model. Circular Dichroism and Raman Optical Activity showed the conversion of helical into β-rich structures in the acid and basic regions, while an opening of BSA tertiary structure was shown by the upsurging of accessibility of ANS-BSA binding sites and the increasing of random contributions at regions F and B. We could then revisit the Foster Model by introducing two additional intermediate conformational states and structural reorganization at extreme pH values. This expanded model opens up new possibilities concerning protein-molecule interactions, promising far-reaching implications for fields such as drug design and biomaterials.

In-situ Raman spectroscopic study of conformational reorganization during heat treatment for high-density polyethylene solid

We investigated in-situ conformational reorganization in a quenched high-density polyethylene solid during heat treatments at various temperatures using in-situ Raman spectroscopic technique. The heat-treatment cycle included three steps: heating from room temperature, maintaining at a fixed sub-melting temperature (tempering), and cooling to room temperature. The heating process caused a conformational transformation from orthorhombic crystalline trans conformers to non-crystalline long trans segments outside the crystalline lattice, which were then amorphized. During the tempering or isothermal duration region, the amorphized long trans segments were steadily subjected to crystalline rearrangement. Finally, the cooling process modified the long trans component (including the amorphized component) into crystalline trans conformers. Overall, a single heat-treatment cycle increased the crystallinity through reorganization of non-crystalline long-trans segments without changing the interfacial content where the original amorphous content remained the same.

Structures of the interleukin 11 signalling complex reveal gp130 dynamics and the inhibitory mechanism of a cytokine variant

Interleukin (IL-)11, an IL-6 family cytokine, has pivotal roles in autoimmune diseases, fibrotic complications, and solid cancers. Despite intense therapeutic targeting efforts, structural understanding of IL-11 signalling and mechanistic insights into current inhibitors are lacking. Here we present cryo-EM and crystal structures of the human IL-11 signalling complex, including the complex containing the complete extracellular domains of the shared IL-6 family β-receptor, gp130. We show that complex formation requires conformational reorganisation of IL-11 and that the membrane-proximal domains of gp130 are dynamic. We demonstrate that the cytokine mutant, IL-11 Mutein, competitively inhibits signalling in human cell lines. Structural shifts in IL-11 Mutein underlie inhibition by altering cytokine binding interactions at all three receptor-engaging sites and abrogating the final gp130 binding step. Our results reveal the structural basis of IL-11 signalling, define the molecular mechanisms of an inhibitor, and advance understanding of gp130-containing receptor complexes, with potential applications in therapeutic development.

Foxp3 orchestrates reorganization of chromatin architecture to establish regulatory T cell identity

Chromatin conformation reorganization is emerging as an important layer of regulation for gene expression and lineage specification. Yet, how lineage-specific transcription factors contribute to the establishment of cell type-specific 3D chromatin architecture in the immune cells remains unclear, especially for the late stages of T cell subset differentiation and maturation. Regulatory T cells (Treg) are mainly generated in the thymus as a subpopulation of T cells specializing in suppressing excessive immune responses. Here, by comprehensively mapping 3D chromatin organization during Treg cell differentiation, we show that Treg-specific chromatin structures were progressively established during its lineage specification, and highly associated with Treg signature gene expression. Additionally, the binding sites of Foxp3, a Treg lineage specifying transcription factor, were highly enriched at Treg-specific chromatin loop anchors. Further comparison of the chromatin interactions between wide-type Tregs versus Treg cells from Foxp3 knock-in/knockout or newly-generated Foxp3 domain-swap mutant mouse revealed that Foxp3 was essential for the establishment of Treg-specific 3D chromatin architecture, although it was not dependent on the formation of the Foxp3 domain-swapped dimer. These results highlighted an underappreciated role of Foxp3 in modulating Treg-specific 3D chromatin structure formation.

Biochemical, structural and dynamical characterizations of the lactate dehydrogenase from Selenomonas ruminantium provide information about an intermediate evolutionary step prior to complete allosteric regulation acquisition in the super family of lactate and malate dehydrogenases

In this work, we investigated the lactate dehydrogenase (LDH) from Selenomonas ruminantium (S. rum), an enzyme that differs at key amino acid positions from canonical allosteric LDHs. The wild type (Wt) of this enzyme recognises pyuvate as all LDHs. However, introducing a single point mutation in the active site loop (I85R) allows S. Rum LDH to recognize the oxaloacetate substrate as a typical malate dehydrogenase (MalDH), whilst maintaining homotropic activation as an LDH. We report the tertiary structure of the Wt and I85RLDH mutant.The Wt S. rum enzyme structure binds NADH and malonate, whilst also resembling the typical compact R-active state of canonical LDHs. The structure of the mutant with I85R was solved in the Apo State (without ligand), and shows no large conformational reorganization such as that observed with canonical allosteric LDHs in Apo state. This is due to a local structural feature typical of S. rum LDH that prevents large-scale conformational reorganization. The S. rum LDH was also studied using Molecular Dynamics simulations, probing specific local deformations of the active site that allow the S. rum LDH to sample the T-inactive state. We propose that, with respect to the LDH/MalDH superfamily, the S. rum enzyme possesses a specificstructural and dynamical way to ensure homotropic activation.

Structure of a ribonucleotide reductase R2 protein radical.

Aerobic ribonucleotide reductases (RNRs) initiate synthesis of DNA building blocks by generating a free radical within the R2 subunit; the radical is subsequently shuttled to the catalytic R1 subunit through proton-coupled electron transfer (PCET). We present a high-resolution room temperature structure of the class Ie R2 protein radical captured by x-ray free electron laser serial femtosecond crystallography. The structure reveals conformational reorganization to shield the radical and connect it to the translocation path, with structural changes propagating to the surface where the protein interacts with the catalytic R1 subunit. Restructuring of the hydrogen bond network, including a notably short O-O interaction of 2.41 angstroms, likely tunes and gates the radical during PCET. These structural results help explain radical handling and mobilization in RNR and have general implications for radical transfer in proteins.

Chemical-induced phase transition and global conformational reorganization of chromatin

Chemicals or drugs can accumulate within biomolecular condensates formed through phase separation in cells. Here, we use super-resolution imaging to search for chemicals that induce phase transition within chromatin at the microscale. This microscopic screening approach reveals that adriamycin (doxorubicin) — a widely used anticancer drug that is known to interact with chromatin — specifically induces visible local condensation and global conformational change of chromatin in cancer and primary cells. Hi-C and ATAC-seq experiments systematically and quantitatively demonstrate that adriamycin-induced chromatin condensation is accompanied by weakened chromatin interaction within topologically associated domains, compartment A/B switching, lower chromatin accessibility, and corresponding transcriptomic changes. Mechanistically, adriamycin complexes with histone H1 and induces phase transition of H1, forming fibrous aggregates in vitro. These results reveal a phase separation-driven mechanism for a chemotherapeutic drug.

Instant Macrocyclizations via Multicomponent Reactions.

Macrocycles fascinate chemists due to both their structure and their applications. However, we still lack efficient and sustainable synthetic methods, giving us straightforward access to them. Herein, a rapid macrocyclization utilizing a two-step, one-pot approach based on orthogonal multicomponent reaction (MCR) tactics is introduced. This synthetic protocol, which is based on Ugi and Groebke-Blackburn-Bienaymé reactions with isocyanides tethered to alkyl tosylates, yields medium sized macrocycles that are otherwise difficult to achieve. Single crystal structures reveal conformational reorganization via intramolecular hydrogen bonding, and modeling studies profile the synthesized libraries.

Fusogenic structural changes in arenavirus glycoproteins are associated with viroporin activity.

Many enveloped viruses enter host cells by fusing with acidic endosomes. The fusion activity of multiple viral envelope glycoproteins does not generally affect viral membrane permeability. However, fusion induced by the Lassa virus (LASV) glycoprotein complex (GPc) is always preceded by an increase in viral membrane permeability and the ensuing acidification of the virion interior. Here, systematic investigation of this LASV fusion phenotype using single pseudovirus tracking in live cells reveals that the change in membrane barrier function is associated with the fusogenic conformational reorganization of GPc. We show that a small-molecule fusion inhibitor or mutations that impair viral fusion by interfering with GPc refolding into the post-fusion structure prevent the increase in membrane permeability. We find that the increase in virion membrane permeability occurs early during endosomal maturation and is facilitated by virus-cell contact. This increase is observed using diverse arenavirus glycoproteins, whether presented on lentivirus-based pseudoviruses or arenavirus-like particles, and in multiple different cell types. Collectively, these results suggest that conformational changes in GPc triggered by low pH and cell factor binding are responsible for virion membrane permeabilization and acidification of the virion core prior to fusion. We propose that this viroporin-like activity may augment viral fusion and/or post-fusion steps of infection, including ribonucleoprotein release into the cytoplasm.

Carrier Dynamics Mechanisms in Bi‐Based Photocatalytic Materials

Bi‐based nanomaterials have unique physicochemical and structural properties, allowing them to respond to visible light effectively. The Bi‐based semiconductors can be used for degradation of pollutants, reduction of CO2, nitrogen fixation, and decomposition of water, having the strong potential to address environmental pollution and energy shortages. However, the large‐scale application is still limited by the separation and transfer of electron–hole pairs. Herein this review article, carrier dynamics are studied from three perspectives, including structural modulation, atomic conformational reorganization, and surface modification. The relevant mechanisms of different strategies are mainly highlighted, the variations of carrier motion or lifetime are explored, and the progress in practical studies is elucidated. The structural modulation strategy is first introduced, including different dimensional structures and heterogeneous junctions. Further, the atomic conformational reorganization strategy is presented, including Bi‐rich strategies, doping, and vacancy defects. Additionally, the surface modification strategy is outlined, including facet engineering, loading of semiconductor cocatalysts, precious metals, or single atoms. Finally, these strategies are summarized for regulating carrier dynamics and the outlook with the main research trend, providing more inspiration for designing desirable Bi‐based semiconductors.

Elucidating the origins of acyltransferase selectivity in primary and secondary metabolism.

Carrier-protein-S-acyltransferases (ATs) are enzymes that load acyl groups onto carrier proteins (CP). These catalysts can exist as components of larger megasynth(et)ases that act in-cis (i.e., intramolecularly) or as so-called trans-ATs that act “in-trans”, forming intermolecular complexes with a corresponding CP or CP domain. A growing number of crystal structures of trans-ATs, including have been resolved in recent years. In many cases, these structures feature a CP covalently tethered to its cognate AT, revealing the specific protein-protein interactions and conformational reorganization that mediate CP-AT binding and catalysis. To complement insights offered by these experimental structures, we have employed long-timescale molecular dynamics simulations to provide a dynamical understanding of biomolecular recognition and acyltransferase function. These simulations reveal key differences that may explain the distinct specificities of trans-AT that operate in primary and secondary metabolism, while also demonstrating that certain internal rigid body motions of the AT that modulate active site access are common among all trans-ATs examined herein.

Pressure-based mapping of Arf1 switch transition.

Biomolecular functions are often associated with complex conformational reorganization. These concerted, nontrivial rearrangements among various regions of proteins are essential for their biological functions. Although crystal structures of proteins can provide snapshots of end states essential for their function, a thorough understanding of the structural mechanics of conformational ensemble associated with protein function remains elusive for most proteins. A combination of experimental and computational techniques can assist in understanding these processes. ADP-ribosylation factor 1 (Arf1) is a small GTPase that is implicated in cellular trafficking from the endoplasmic reticulum to the Golgi complex. The transition of Arf1 from its GDP-bound inactive state to the GTP-bound, active state involves a series of conformational transitions resulting in GDP dissociation, a shift in the register of the β-strands in the inter-switch region, and reorganization of the N-terminal helix, and the switch 1 and switch 2 regions. Here we use a combination of molecular modelling, extensive molecular dynamic simulations, pressure perturbation NMR-constrained simulations and pressure perturbation SAXS guided ensemble optimization methods to obtain a comprehensive picture of the functional dynamics of Arf1. Our approach illuminates the events associated with the allosteric communication required for the activation of Arf1.

Relationship between enhancement of PEDOT:PSS conductivity by solvent treatment and PSS chain reorganization

AbstractThe treatment of water‐soluble PEDOT:PSS copolymer film by DMSO or DMF considerably enhances its electrical conductivity. The mechanism leading to a new molecular conformation in the PEDOT:PSS layer was investigated by in situ ATR, PM‐IRRAS and XPS spectroscopies. The release of PSS chains containing excess [Na+, (−Φ‐SO3−)‐] and (−Φ‐SO3H)‐] moieties by the solvent treatment and the implications of this process are discussed. By comparison with a NaPSS/HPSS adduct, the essential role of the conformational reorganization of the poly(styrene sulfonic/sulfonate) chains via hydrogen bonds between SO3H functions is demonstrated. Residual water molecules play a fundamental role both in the removal of excess sulfonates or sulfonic groups, the separation of the (Na, H, PSS) chains, which can then be extracted, and the establishment of the suitable PEDOT conformation.

Taming multiple binding poses in alchemical binding free energy prediction: the β-cyclodextrin host-guest SAMPL9 blinded challenge.

We apply the Alchemical Transfer Method (ATM) and a bespoke fixed partial charge force field to the SAMPL9 bCD host-guest binding free energy prediction challenge that comprises a combination of complexes formed between five phenothiazine guests and two cyclodextrin hosts. Multiple chemical forms, competing binding poses, and computational modeling challenges pose significant obstacles to obtaining reliable computational predictions for these systems. The phenothiazine guests exist in solution as racemic mixtures of enantiomers related by nitrogen inversions that bind the hosts in various binding poses, each requiring an individual free energy analysis. Due to the large size of the guests and the conformational reorganization of the hosts, which prevent a direct absolute binding free energy route, binding free energies are obtained by a series of absolute and relative binding alchemical steps for each chemical species in each binding pose. Metadynamics-accelerated conformational sampling was found to be necessary to address the poor convergence of some numerical estimates affected by conformational trapping. Despite these challenges, our blinded predictions quantitatively reproduced the experimental affinities for the β-cyclodextrin host and, to a lesser extent, those with a methylated derivative. The work illustrates the challenges of obtaining reliable free energy data in in silico drug design for even seemingly simple systems and introduces some of the technologies available to tackle them.

Decoding the spatial chromatin organization and dynamic epigenetic landscapes of macrophage cells during differentiation and immune activation

Immunocytes dynamically reprogram their gene expression profiles during differentiation and immunoresponse. However, the underlying mechanism remains elusive. Here, we develop a single-cell Hi-C method and systematically delineate the 3D genome and dynamic epigenetic atlas of macrophages during these processes. We propose “degree of disorder” to measure genome organizational patterns inside topologically-associated domains, which is correlated with the chromatin epigenetic states, gene expression, and chromatin structure variability in individual cells. Furthermore, we identify that NF-κB initiates systematic chromatin conformation reorganization upon Mycobacterium tuberculosis infection. The integrated Hi-C, eQTL, and GWAS analysis depicts the atlas of the long-range target genes of mycobacterial disease susceptible loci. Among these, the SNP rs1873613 is located in the anchor of a dynamic chromatin loop with LRRK2, whose inhibitor AdoCbl could be an anti-tuberculosis drug candidate. Our study provides comprehensive resources for the 3D genome structure of immunocytes and sheds insights into the order of genome organization and the coordinated gene transcription during immunoresponse.

Sensitive temperature-dependent phase resolution of polyethylene-clay nanocomposites

With the help of spectroscopic and differential scanning calorimetry (DSC) techniques, we have studied conformational and phase reorganizations as function of temperature in intercalated polymer-clay nanocomposites based on low-, middle-, and high-density polyethylene (PE) matrices. We show that Raman spectroscopy is sensitive to structural changes appearing during heating at much lower temperatures (about 47 °C) in comparison to DSC measurements. In fact, in the melting region where DSC traces show endotherms, Raman spectra reveal dramatic changes in the phase and conformational compositions of PE and PE-matrices within PE-clay nanocomposites. Noteworthy, the structural reorganization pathway, through which the semicrystalline nanocomposites transform into a melt state, depends primarily on the PE density and weakly on the filler (nanoclay). Moreover, the temperature-dependent crystallinity degree and the total amount of trans-conformers of the PE system are determined, and the evidence for the formation of intermediate crystal-like phase during heating is shown.

Metalloceneincorporated Hybrid Singly N-Methyl N-Confused Calixphyrins: Synthesis, Characterization, Protonation and Deprotonation Studies.

Rational design and isolation of two hitherto unknown highly stable single conformer of ferrocene incorporated meso-aryl substituted singly N-methyl N-confused-calixphyrins have been achieved in quantitative yields. The solid-state crystal structure reveals the obvious trans-geometry for the meso-protons with the possibility for both the macrocycles to exist either racemic or enantiomer forms. However, thorough solution-state spectroscopic characterization strongly concludes the experimental isolation of a single isomer for both the macrocycles. The drastic modification of UV-vis spectral patterns upon imine pyrrole N protonation and amine pyrrole NH deprotonation of both the calixphyrins could pave way for these macrocycles to act as opto-electronic materials. The conformational preorganization and protonation and deprotonation induced conformational reorganization have been extensively studied by solution state spectroscopic techniques, solid state X-ray crystal structure and in depth DFT level theoretical calculations.