Elongated Conformation Research Articles

Interaction of chondroitin sulfate with zwitterionic lipid membranes

Chondroitin sulfates (CSs) are important components of the extracellular matrix and side chains of membrane proteoglycans. These polysaccharides are, therefore, likely to interact with plasma membranes and play a significant role in modulating cellular functions. So far, the details of the processes occurring at the interface between the extracellular matrix and cellular membranes are not fully understood. In this study, we used experimental methods and atomic-scale molecular dynamics (MD) simulations to reveal the molecular picture of the interactions between CS and phosphocholine (PC) membranes, used as a simplified model of cell membranes. MD simulations reveal that the polysaccharide associates to the PC bilayer as a result of electrostatic interactions between the positively charged quaternary ammonium groups of choline and the negatively charged sulfate groups of CS. Compared to an aqueous medium, the adsorbed polysaccharide chains adopt more elongated conformations, which facilitates the electrostatic interactions with the membrane, and have a high degree of freedom to change their conformations and to adhere to and detach from the membrane surface. Penetrating slightly between the polar groups of the bilayer, they form a loosely anchored layer, but do not intrude into the hydrophobic region of the PC bilayer. The CS adsorption spread the PC headgroups apart, which is manifested by an increase in the value of the area pre lipid. The expansion of the lipid polar groups weakens the dispersion interactions between the lipid acyl chains. As a result, the lipid membrane in the membrane-polysaccharide contact areas becomes more fluid. Our outcomes may help to understand in detail the interaction of chondroitin sulfate with zwitterionic membranes at the molecular level, which is of biological interest since many biological processes depend on lipid-CS interactions.

Beyond the VSG layer: Exploring the role of intrinsic disorder in the invariant surface glycoproteins of African trypanosomes.

In the bloodstream of mammalian hosts, African trypanosomes face the challenge of protecting their invariant surface receptors from immune detection. This crucial role is fulfilled by a dense, glycosylated protein layer composed of variant surface glycoproteins (VSGs), which undergo antigenic variation and provide a physical barrier that shields the underlying invariant surface glycoproteins (ISGs). The protective shield's limited permeability comes at the cost of restricted access to the extracellular host environment, raising questions regarding the specific function of the ISG repertoire. In this study, we employ an integrative structural biology approach to show that intrinsically disordered membrane-proximal regions are a common feature of members of the ISG super-family, conferring the ability to switch between compact and elongated conformers. While the folded, membrane-distal ectodomain is buried within the VSG layer for compact conformers, their elongated counterparts would enable the extension beyond it. This dynamic behavior enables ISGs to maintain a low immunogenic footprint while still allowing them to engage with the host environment when necessary. Our findings add further evidence to a dynamic molecular organization of trypanosome surface antigens wherein intrinsic disorder underpins the characteristics of a highly flexible ISG proteome to circumvent the constraints imposed by the VSG coat.

Altering Conformational States of Dynamic Ion Populations using Traveling Wave Structures for Lossless Ion Manipulations.

With its capacity to store and translate ions across considerable distances and times, traveling wave structures for lossless ion manipulations (TW-SLIM) provide the foundation to expand the scope of ion mobility spectrometry (IMS) experiments. While promising, the dynamic electric fields and consequential ion-neutral collisions used to realize extensive degrees of separation have a considerable impact on the empirical results and the fundamental interpretation of observed arrival time distributions. Using a custom-designed set of TW-SLIM boards (∼9 m) coupled with a time-of-flight mass spectrometer (SLIM-ToF), we detail the capacity to systematically alter the gas-phase distribution of select peptide conformers. In addition to discussing the role charge-transfer may play in TW-SLIM experiments that occur at extended time scales, the ability of the SLIM-ToF to perform tandem IMS was leveraged to confirm that both the compact and elongated conformers of bradykinin2+ undergo interconversion within the SLIM. Storage experiments in which ions are confined within SLIM using static potential wells suggest that factors aside from TW-induced ion motion contribute to interconversion. Further investigation into this matter suggests that the use of radio frequency (RF) fields to confine ions within SLIM may play a role in ion heating. Aside from interconversion, storage experiments also provide insight into charge transfer behavior over the course of extended periods. The results of the presented experiments suggest that considerations should be taken when analyzing labile species and inform strategies for the TW-SLIM design and method development.

Structural Insights into Linkage-Specific Ubiquitin Chains Using Ion Mobility Mass Spectrometry.

The structural characterization and differentiation of four types of oligoubiquitin conjugates [linear (Met1)-, Lys11-, Lys48-, Lys63-linked di-, tri-, and tetraubiquitin chains] using ion mobility mass spectrometry are reported. A comparison of collision cross sections for the same linkage of di-, tri-, and tetraubiquitin chains shows differences in conformational elongation for higher charge states due to the interplay of linkage-derived structure and Coulombic repulsion. For di- and triubiquitin chains, this elongation results in a single narrow feature representing an elongated conformation type for multiple higher charge state species. In contrast, higher charge state tetraubiquitin species do not form a single conformer type as readily. A comparison of different linkages in tetraubiquitin chains reveals greater similarity in conformation type at lower charge states; with increasing charge state, the four linkage types diverge in the relative proportions of elongated conformer types with Met1- ≥ Lys11- > Lys63- > Lys48-linkage. These differences in conformational trends could be discussed with respect to biological functions of linkage-specific polyubiquitinated proteins.

Active motion of tangentially driven polymers in periodic array of obstacles

One key question about transport of active polymers within crowded environments is how spatial order of obstacles influences their conformation and dynamics when compared to disordered media. To this end, we computationally investigate the active transport of tangentially driven polymers with varying degrees of flexibility and activity in two-dimensional square lattices of obstacles. Tight periodic confinement induces notable conformational changes and distinct modes of transport for flexible and stiff active filaments. It leads to caging of low activity flexible polymers inside the inter-obstacle pores while promoting more elongated conformations and enhanced diffusion for stiff polymers at low to moderate activity levels. The migration of flexible active polymers occurs via hopping events, where they unfold to move from one cage to another, similar to their transport in disordered media. However, in ordered media, polymers are more compact and their long-time dynamics is significantly slower. In contrast, stiff chains travel mainly in straight paths within periodic inter-obstacle channels while occasionally changing their direction of motion. This mode of transport is unique to periodic environment and leads to more extended conformation and substantially enhanced long-time dynamics of stiff filaments with low to moderate activity levels compared to disordered media. At high active forces, polymers overcome confinement effects and move through inter-obstacle pores just as swiftly as in open spaces, regardless of the spatial arrangement of obstacles. We explain the center of mass dynamics of semiflexible polymers in terms of active force and obstacle packing fraction by developing an approximate analytical theory.

Three small partner proteins facilitate the type VII-dependent secretion of an antibacterial nuclease.

Staphylococcus aureus is an opportunistic human pathogen associated with severe infections and antimicrobial resistance. S. aureus strains utilize a type VII secretion system to secrete toxins targeting competitor bacteria, likely facilitating colonization. EsaD is a nuclease toxin secreted by the type VII secretion system in many strains of S. aureus as well as other related bacterial species. Here, we identify three small proteins of previously unknown function as export factors, required for efficient secretion of EsaD. We show that these proteins bind to the transport domain of EsaD, forming a complex with a striking cane-like conformation.

The Reliability of PCL/Anti-VEGF Electrospun Scaffolds to Support Limbal Stem Cells for Corneal Repair.

Since only few reported studies propose anti-vascular endothelial growth factor (anti-VEGF) delivery through electrospun scaffolds, this study greatly contributes to the potential prevention of patient's vision loss, as it explores electrospun polycaprolactone (PCL) coated with anti-VEGF for the blockage of abnormal cornea vascularization. In terms of physicochemical properties, the biological component increased the PCL scaffold fiber diameter (by ~24%) and pore area (by ~82%), while ut slightly reduced its total porosity as the anti-VEGF solution filled the voids of the microfibrous structure. The addition of the anti-VEGF increased the scaffold stiffness almost three-fold at both strains of 5 and 10%, as well as its biodegradation rate (~36% after 60 days) with a sustained release profile after Day 4 of phosphate buffered saline incubation. In terms of scaffold application function, the PCL/Anti-VEGF scaffold proved to be more favorable for the adhesion of cultured limbal stem cells (LSCs); this was confirmed by the SEM images, where the cells showed flat and elongated conformations. Further support of the LSC growth and proliferation was confirmed by the identified p63 and CK3 markers after cell staining. These results demonstrate the advantageous effect of the surface-adsorbed anti-VEGF to stop vision loss and help damaged corneal tissue repair.

Structures of apo Cas12a and its complex with crRNA and DNA reveal the dynamics of ternary complex formation and target DNA cleavage.

Cas12a is a programmable nuclease for adaptive immunity against invading nucleic acids in CRISPR-Cas systems. Here, we report the crystal structures of apo Cas12a from Lachnospiraceae bacterium MA2020 (Lb2) and the Lb2Cas12a+crRNA complex, as well as the cryo-EM structure and functional studies of the Lb2Cas12a+crRNA+DNA complex. We demonstrate that apo Lb2Cas12a assumes a unique, elongated conformation, whereas the Lb2Cas12a+crRNA binary complex exhibits a compact conformation that subsequently rearranges to a semi-open conformation in the Lb2Cas12a+crRNA+DNA ternary complex. Notably, in solution, apo Lb2Cas12a is dynamic and can exist in both elongated and compact forms. Residues from Met493 to Leu523 of the WED domain undergo major conformational changes to facilitate the required structural rearrangements. The REC lobe of Lb2Cas12a rotates 103° concomitant with rearrangement of the hinge region close to the WED and RuvC II domains to position the RNA-DNA duplex near the catalytic site. Our findings provide insight into crRNA recognition and the mechanism of target DNA cleavage.

Crystal structures of the NAD+-II riboswitch reveal two distinct ligand-binding pockets.

We present crystal structures of a new NAD+-binding riboswitch termed NAD+-II, bound to nicotinamide mononucleotide (NMN), nicotinamide adenine dinucleotide (NAD+) and nicotinamide riboside (NR). The RNA structure comprises a number of structural features including three helices, one of which forms a triple helix by interacting with an A5 strand in its minor-groove, and another formed from a long-range pseudoknot. The core of the structure (centrally located and coaxial with the triplex and the pseudoknot) includes two consecutive quadruple base interactions. Unusually the riboswitch binds two molecules of ligand, bound at distinct, non-overlapping sites in the RNA. Binding occurs primarily through the nicotinamide moiety of each ligand, held by specific hydrogen bonding and stacking interactions with the pyridyl ring. The mode of binding is the same for NMN, NR and the nicotinamide moiety of NAD+. In addition, when NAD+ is bound into one site it adopts an elongated conformation such that its diphosphate linker occupies a groove on the surface of the RNA, following which the adenine portion inserts into a pocket and makes specific hydrogen bonding interactions. Thus the NAD+-II riboswitch is distinct from the NAD+-I riboswitch in that it binds two molecules of ligand at separate sites, and that binding occurs principally through the nicotinamide moiety.

Molecular Dynamics of Lysine Dendrigrafts in Methanol–Water Mixtures

The molecular dynamics method was used to study the structure and properties of dendrigrafts of the first and second generations in methanol-water mixtures with various volume fractions of methanol. At a small volume fraction of methanol, the size and other properties of both dendrigrafts are very similar to those in pure water. A decrease in the dielectric constant of the mixed solvent with an increase in the methanol fraction leads to the penetration of counterions into the dendrigrafts and a reduction of the effective charge. This leads to a gradual collapse of dendrigrafts: a decrease in their size, and an increase in the internal density and the number of intramolecular hydrogen bonds inside them. At the same time, the number of solvent molecules inside the dendrigraft and the number of hydrogen bonds between the dendrigraft and the solvent decrease. At small fractions of methanol in the mixture, the dominant secondary structure in both dendrigrafts is an elongated polyproline II (PPII) helix. At intermediate volume fractions of methanol, the proportion of the PPII helix decreases, while the proportion of another elongated β-sheet secondary structure gradually increases. However, at a high fraction of methanol, the proportion of compact α-helix conformations begins to increase, while the proportion of both elongated conformations decreases.

Native Mass Spectrometry of BRD4 Bromodomains Linked to a Long Disordered Region.

The contribution of disordered regions to protein function and structure is a relatively new field of study and of particular significance as their function has been implicated in some human diseases. Our objective was to analyze various deletion mutants of the bromodomain-containing protein 4 (BRD4) using native mass spectrometry to characterize the gas-phase behavior of the disordered region connected to the folded domain. A protein with a single bromodomain but no long disordered linker displayed a narrow charge distribution at low charge states, suggesting a compact structure. In contrast, proteins containing one or two bromodomains connected to a long disordered region exhibited multimodal charge distributions, suggesting the presence of compact and elongated conformers. In the presence of a pan-BET-bromodomain inhibitor, JQ1, the protein-JQ1 complex ions had relatively small numbers of positive charges, corresponding to compact conformers. In contrast, the ions with extremely high charge states did not form a complex with JQ1. This suggests that all of the JQ1-bound BRD4 proteins in the gas phase are in a compact conformation, including the linker region, while the unbound forms are considerably elongated. Although these are gas-phase phenomena, it is possible that the long disordered linker connected to the bromodomain causes the denaturation of the folded domain, which, in turn, affects its JQ1 recognition.

Atomic-Resolution Structure of the Protein Encoded by Gene V of fd Bacteriophage in Complex with Viral ssDNA Determined by Magic-Angle Spinning Solid-State NMR.

F-specific filamentous phages, elongated particles with circular single-stranded DNA encased in a symmetric protein capsid, undergo an intermediate step, where thousands of homodimers of a non-structural protein, gVp, bind to newly synthesized strands of DNA, preventing further DNA replication and preparing the circular genome in an elongated conformation for assembly of a new virion structure at the membrane. While the structure of the free homodimer is known, the ssDNA-bound conformation has yet to be determined. We report an atomic-resolution structure of the gVp monomer bound to ssDNA of fd phage in the nucleoprotein complex elucidated via magic-angle spinning solid-state NMR. The model presents significant conformational changes with respect to the free form. These modifications facilitate the binding mechanism and possibly promote cooperative binding in the assembly of the gVp-ssDNA complex.

Structure and stability of polydiacetylene membrane systems: Molecular dynamics simulation studies

We have performed full atomistic molecular dynamics (MD) simulations to investigate structure and stability of bilayer membrane systems consisting of monomeric or polymeric 10,12-pentacosadiynoic acid (PCDA) units connected with lysine groups by amide bonds. The PCDA monomer molecules show a twisted three-rod-domain structure with two kinks but upon polymerization, they possess more elongated conformation. The resulting polydiacetylene (PDA) membrane systems have stable membrane structures at room temperature, which is similar to biological lipid bilayer membranes and maintain their gel-like membrane integrity even up to as high as 370 K. Structural properties such as area per monomer, membrane thickness, density profile, 2D pair distribution function, and orientational correlation function are also calculated to understand the membrane structure and check its stability upon thermal fluctuation with atomistic resolution. This study is expected to provide the understanding about PDA membrane systems in atomistic details as well as significant insights into designing new novel PDA sensors.

Conformations of Three Types of Ultra-Long-Chain Fatty Acids in Multicomponent Lipid Bilayers.

Ultra-long-chain fatty acids (ULCFAs) are biosynthesized in certain types of tissues, but their biological roles remain unknown. Here, we report how the conformation of ULCFAs depends on the length and unsaturated-bond ratio of the ultra-long chains and the composition of the host bilayer membrane using molecular dynamics simulations. The ultra-long chain of ULCFAs flips between the two leaflets and fluctuates among three conformations: elongated, L-shaped, and turned. Furthermore, we found that the saturated ultra-long chain exhibited an elongated conformation more frequently than the unsaturated chain. In addition, the truncation of the ultra-long chain at C26 had little effect on the remaining ULCFAs. ULCFAs respond to lipid-density differences in the two leaflets, and the ratio of the elongated and turned conformations changed to reduce this difference. However, in cholesterol-containing membranes, ULCFAs exhibit no density difference after the flip-flop of cholesterol removes the difference.

Study on internal structure of casein micelles in reconstituted skim milk powder

In the current study, the internal structure of casein micelles (CMs), primary casein cluster, has been studied by size-exclusion chromatography (SEC) coupled with small-angle X-ray scattering (SAXS), isothermal titration calorimetry (ITC), transmission electron microscopy (TEM) and molecular dynamics (MD) simulations. The casein cluster featured a hierarchical structure predominately consisting of αs-CN and β-CN molecules and a trace of κ-CN. ITC profile showed a typical enthalpogram of CMs with a critical micelle concentration (CMC) of ~0.85 μg/mL. The casein cluster exhibited apparent characteristics of intrinsically disordered proteins (IDPs) with a secondary structure content of 24 % in α-helix, 35.4 % in antiparallel-parallel, 20.2 % in β-turn and 20.4 % in random coil. SAXS results revealed a slightly elongated and tortuous worm-like conformation for the casein cluster in solution with an aspect ratio of 1.36 and an estimated molecular weight of 162.7 kDa. Further scattering data analysis proposed three coexisted species (αs1-β-αs2-CN, αs1-CN and αs1-β-αs2-CN dimer) with a volume fraction of 57.4 %, 30.1 %, and 12.5 % respectively in the casein cluster. TEM observation was consistent with the results of spectra, SAXS and MD that calcium sequestration resulted in a more extended and loose structure, instead, EDTA chelation induced a more compact conformation of the casein cluster.

Helical remodeling augments 5-lipoxygenase activity in the synthesis of proinflammatory mediators

The synthesis of proinflammatory leukotrienes implicated in asthma, allergic rhinitis, and atherosclerosis is initiated by the enzyme 5-lipoxygenase (5-LOX). The crystal structure of human Stable-5-LOX revealed a conformation where the catalytic iron was inaccessible to bulk solvent as two aromatic residues on a conserved helix-α2 (Hα2) plugged the substrate access portal. Whether 5-LOX can also adopt a more open conformation has not been resolved. Here, we present a new conformation of 5-LOX where Hα2 adopts an elongated conformation equivalent to that described in other animal lipoxygenase structures. Our observation of the sigmoidal kinetic behavior of 5-LOX, which is indicative of positive cooperativity, is consistent with a substrate-induced conformational change that shifts the ensemble of enzyme populations to favor the catalytically competent state. Strategic point mutations along Hα2 designed to unlock the closed conformation and elongate Hα2 resulted in improved kinetic parameters, altered limited proteolysis data, and a drastic reduction in the length of the lag phase yielding the most active Stable-5-LOX to date. Structural predictions by AlphaFold2 of these variants statistically favor an elongated Hα2 and reinforce a model in which improved kinetic parameters correlate with a more readily adopted open conformation. Taken together, these data provide valuable insights into the synthesis of leukotrienes.

Structural analysis of crosstalk between Type I and Type III CRISPR systems

CRISPR‐Cas comprise adaptive immune systems against foreign nucleic acids in prokaryotes. However, little is known about how the expression of CRISPR‐Cas genes is regulated in response to viral infection. Type‐III CRISPR interference results in the production of cyclic oligoadenylate (cOA) second messengers, which are known to bind various CRISPR Associated Rossmann Fold (CARF) ‐containing nuclease receptors to regulate their functions. Despite conservation of the CARF domain across Csa3 transcription factors, the basis for cOA binding specificity was unclear. In this study, we extend the known receptor repertoire of cOAs to include transcriptional factors by demonstrating specific binding of cA4 to Saccharolobus solfataricus Csa3 (Csa3Sso) (KD of 5.8 ± 0.03 μM). We determined a 2.0 Å resolution X‐ray crystal structure of cA4‐bound Csa3Sso, which reveals the binding of the Csa3Sso CARF domain to an elongated conformation of cA4. Binding affinity analyses of Csa3Sso mutants targeting the observed Csa3Sso•cA4 structural interface identified Csa3Sso residues that are required for ligand binding and specificity. We determined an essential role of a conserved glutamate (Glu122) for the low affinity interaction with cA4; a mutation of this residue to Ala and Gln resulted in a ~145 and ~125‐fold increase in cA4 binding affinity by Csa3Sso. Our complementary SAXS analyses detected a cA4‐induced conformational change in Csa3Sso, involving an asymmetric quaternary rearrangement of the C‐terminal winged helix‐turn‐helix (wHTH) domains supporting an allosteric mode of Csa3 regulation by cA4. Overall, our results support cA4 and Csa3‐mediated cross‐talk between type‐III and type‐I CRISPR systems that co‐exist in several prokaryotes.

The structure of the Clostridium thermocellum RsgI9 ectodomain provides insight into the mechanism of biomass sensing.

Clostridium thermocellum is actively being developed as a microbial platform to produce biofuels and chemicals from renewable plant biomass. An attractive feature of this bacterium is its ability to efficiently degrade lignocellulose using surface-displayed cellulosomes, large multi-protein complexes that house different types of cellulase enzymes. Clostridium thermocellum tailors the enzyme composition of its cellulosome using nine membrane-embedded anti-σ factors (RsgI1-9), which are thought to sense different types of extracellular carbohydrates and then elicit distinct gene expression programs via cytoplasmic σ factors. Here we show that the RsgI9 anti-σ factor interacts with cellulose via a C-terminal bi-domain unit. A 2.0Å crystal structure reveals that the unit is constructed from S1C peptidase and NTF2-like protein domains that contain a potential binding site for cellulose. Small-angle X-ray scattering experiments of the intact ectodomain indicate that it adopts a bi-lobed, elongated conformation. In the structure, a conserved RsgI extracellular (CRE) domain is connected to the bi-domain via a proline-rich linker, which is expected to project the carbohydrate-binding unit ~160 Å from the cell surface. The CRE and proline-rich elements are conserved in several other C. thermocellum anti-σ factors, suggesting that they will also form extended structures that sense carbohydrates.

Exploring the structural dynamics of DNA using fluorescence polarization microscopy and optical tweezers

DNA structural transitions facilitate genomic processes, mediate drug-DNA interactions, and inform the development of emerging DNA-based biotechnology. Here, we use concurrent fluorescence polarization imaging and DNA manipulation experiments to probe the structure of DNA conformations that form under high tension and topological constraints. First, we employ polarization imaging of DNA intercalators (small dye molecules that slide between the DNA base-pairs) to probe the structure of S-DNA, an elongated conformation that can be accessed by mechanical overstretching of (relaxed) B-DNA.

Structural basis of cyclic oligoadenylate binding to the transcription factor Csa3 outlines cross talk between type III and type I CRISPR systems

RNA interference by type III CRISPR systems results in the synthesis of cyclic oligoadenylate (cOA) second messengers, which are known to bind and regulate various CARF domain–containing nuclease receptors. The CARF domain–containing Csa3 family of transcriptional factors associated with the DNA-targeting type I CRISPR systems regulate expression of various CRISPR and DNA repair genes in many prokaryotes. In this study, we extend the known receptor repertoire of cOA messengers to include transcriptional factors by demonstrating specific binding of cyclic tetra-adenylate (cA4) to Saccharolobus solfataricus Csa3 (Csa3Sso). Our 2.0-Å resolution X-ray crystal structure of cA4-bound full-length Csa3Sso reveals the binding of its CARF domain to an elongated conformation of cA4. Using cA4 binding affinity analyses of Csa3Sso mutants targeting the observed Csa3Sso•cA4 structural interface, we identified a Csa3-specific cA4 binding motif distinct from a more widely conserved cOA-binding CARF motif. Using a rational surface engineering approach, we increased the cA4 binding affinity of Csa3Sso up to ∼145-fold over the wildtype, which has potential applications for future second messenger-driven CRISPR gene expression and editing systems. Our in-solution Csa3Sso structural analysis identified cA4-induced allosteric and asymmetric conformational rearrangement of its C-terminal winged helix-turn-helix effector domains, which could potentially be incompatible to DNA binding. However, specific in vitro binding of the purified Csa3Sso to its putative promoter (PCas4a) was found to be cA4 independent, suggesting a complex mode of Csa3Sso regulation. Overall, our results support cA4-and Csa3-mediated cross talk between type III and type I CRISPR systems.