Helix Bundle Research Articles

Role and structure of the small subunit forming heterodimers with laccase-like enzymes.

Unlike laccases sensu stricto, which are usually monomeric enzymes, laccase-like enzymes recently re-classified as Novel Laccases (NLACs) are characterized by the formation of heterodimers with small proteins (subunits) of unknown function. Here the NLAC from Pleurotus eryngii (PeNL) and a small protein selected from the fungal genome, that is homologous to reported POXA3 from Pleurotus ostreatus, were produced in Aspergillus oryzae separately or together. The two proteins interacted regardless of whether the small subunit was co-expressed or exogenously added to the enzyme. The stability and catalytic activity of PeNL was significantly enhanced in the presence of the small subunit. SEC-MALS analysis confirmed that the complex PeNL-ss is a heterodimer of 77.4 kDa. The crystallographic structure of the small protein expressed in Escherichia coli was solved at 1.6 Å resolution. This is the first structure elucidated of a small subunit of a NLAC. The helix bundle structure of the small subunit accommodates well with the enzyme model structure, including interactions with specific regions of NLACs and some amino acid residues of the substrate-binding loops. This article is protected by copyright. All rights reserved.

Crystal structure of domain of unknown function 507 (DUF507) reveals a new protein fold

The crystal structure of the domain of unknown function family 507 protein from Aquifex aeolicus is reported (AaDUF507, UniProt O67633, 183 residues). The structure was determined in two space groups (C2221 and P3221) at 1.9 Å resolution. The phase problem was solved by molecular replacement using an AlphaFold model as the search model. AaDUF507 is a Y-shaped α-helical protein consisting of an anti-parallel 4-helix bundle base and two helical arms that extend 30-Å from the base. The two crystal structures differ by a 25° rigid body rotation of the C-terminal arm. The tertiary structure exhibits pseudo-twofold symmetry. The structural symmetry mirrors internal sequence similarity: residues 11–57 and 102–148 are 30% identical and 53% similar with an E-value of 0.002. In one of the structures, electron density for an unknown ligand, consistent with nicotinamide or similar molecule, may indicate a functional site. Docking calculations suggest potential ligand binding hot spots in the region between the helical arms. Structure-based query of the Protein Data Bank revealed no other protein with a similar tertiary structure, leading us to propose that AaDUF507 represents a new protein fold.

Inhibitory mechanism of CRISPR-Cas9 by AcrIIC4.

CRISPR-Cas systems act as the adaptive immune systems of bacteria and archaea, targeting and destroying invading foreign mobile genetic elements (MGEs) such as phages. MGEs have also evolved anti-CRISPR (Acr) proteins to inactivate the CRISPR-Cas systems. Recently, AcrIIC4, identified from Haemophilus parainfluenzae phage, has been reported to inhibit the endonuclease activity of Cas9 from Neisseria meningitidis (NmeCas9), but the inhibition mechanism is not clear. Here, we biochemically and structurally investigated the anti-CRISPR activity of AcrIIC4. AcrIIC4 folds into a helix bundle composed of three helices, which associates with the REC lobe of NmeCas9 and sgRNA. The REC2 domain of NmeCas9 is locked by AcrIIC4, perturbing the conformational dynamics required for the target DNA binding and cleavage. Furthermore, mutation of the key residues in the AcrIIC4-NmeCas9 and AcrIIC4-sgRNA interfaces largely abolishes the inhibitory effects of AcrIIC4. Our study offers new insights into the mechanism of AcrIIC4-mediated suppression of NmeCas9 and provides guidelines for the design of regulatory tools for Cas9-based gene editing applications.

Tail engagement of arrestin at the glucagon receptor.

Arrestins have pivotal roles in regulating G protein-coupled receptor (GPCR) signalling by desensitizing G protein activation and mediating receptor internalization1,2. It has been proposed that the arrestin binds to the receptor in two different conformations, 'tail' and 'core', which were suggested to govern distinct processes of receptor signalling and trafficking3,4. However, little structural information is available for the tail engagement of the arrestins. Here we report two structures of the glucagon receptor (GCGR) bound to β-arrestin 1 (βarr1) in glucagon-bound and ligand-free states. These structures reveal a receptor tail-engaged binding mode of βarr1 with many unique features, to our knowledge, not previously observed. Helix VIII, instead of the receptor core, has a major role in accommodating βarr1 by forming extensive interactions with the central crest of βarr1. The tail-binding pose is further defined by a close proximity between the βarr1 C-edge and the receptor helical bundle, and stabilized by a phosphoinositide derivative that bridges βarr1 with helices I and VIII of GCGR. Lacking any contact with the arrestin, the receptor core is in an inactive state and loosely binds to glucagon. Further functional studies suggest that the tail conformation of GCGR-βarr governs βarr recruitment at the plasma membrane and endocytosis of GCGR, and provides a molecular basis for the receptor forming a super-complex simultaneously with G protein and βarr to promote sustained signalling within endosomes. These findings extend our knowledge about the arrestin-mediated modulation of GPCR functionalities.

Cellulose assembles into helical bundles of uniform handedness in cell walls with abnormal pectin composition.

Plant cells and organs grow into a remarkable diversity of shapes, as directed by cell walls composed primarily of polysaccharides such as cellulose and multiple structurally distinct pectins. The properties of the cell wall that allow for precise control of morphogenesis are distinct from those of the individual polysaccharide components. For example, cellulose, the primary determinant of cell morphology, is a chiral macromolecule that can self-assemble in vitro into larger-scale structures of consistent chirality, and yet most plant cells do not display consistent chirality in their growth. One interesting exception is the Arabidopsis thaliana rhm1 mutant, which has decreased levels of the pectin rhamnogalacturonan-I and causes conical petal epidermal cells to grow with a left-handed helical twist. Here, we show that in rhm1 the cellulose is bundled into large macrofibrils, unlike the evenly distributed microfibrils of the wild type. This cellulose bundling becomes increasingly severe over time, consistent with cellulose being synthesized normally and then self-associating into macrofibrils. We also show that in the wild type, cellulose is oriented transversely, whereas in rhm1 mutants, the cellulose forms right-handed helices that can account for the helical morphology of the petal cells. Our results indicate that when the composition of pectin is altered, cellulose can form cellular-scale chiral structures in vivo, analogous to the helicoids formed in vitro by cellulose nano-crystals. We propose that an important emergent property of the interplay between rhamnogalacturonan-I and cellulose is to permit the assembly of nonbundled cellulose structures, providing plants flexibility to orient cellulose and direct morphogenesis.

Subunit gating resulting from individual protonation events in Kir2 channels

Inwardly rectifying potassium (Kir) channels open at the ‘helix bundle crossing’ (HBC), formed by the M2 helices at the cytoplasmic end of the transmembrane pore. Introduced negative charges at the HBC (G178D) in Kir2.2 channels forces opening, allowing pore wetting and free movement of permeant ions between the cytoplasm and the inner cavity. Single-channel recordings reveal striking, pH-dependent, subconductance behaviors in G178D (or G178E and equivalent Kir2.1[G177E]) mutant channels, with well-resolved non-cooperative subconductance levels. Decreasing cytoplasmic pH shifts the probability towards lower conductance levels. Molecular dynamics simulations show how protonation of Kir2.2[G178D], or the D173 pore-lining residues, changes solvation, K+ ion occupancy, and K+ conductance. Ion channel gating and conductance are classically understood as separate processes. The present data reveal how individual protonation events change the electrostatic microenvironment of the pore, resulting in step-wise alterations of ion pooling, and hence conductance, that appear as ‘gated’ substates.

An expandable, modular de novo protein platform for precision redox engineering

The electron-conducting circuitry of life represents an as-yet untapped resource of exquisite, nanoscale biomolecular engineering. Here, we report the characterization and structure of a de novo diheme "maquette" protein, 4D2, which we subsequently use to create an expanded, modular platform for heme protein design. A well-folded monoheme variant was created by computational redesign, which was then utilized for the experimental validation of continuum electrostatic redox potential calculations. This demonstrates how fundamental biophysical properties can be predicted and fine-tuned. 4D2 was then extended into a tetraheme helical bundle, representing a 7 nm molecular wire. Despite a molecular weight of only 24 kDa, electron cryomicroscopy illustrated a remarkable level of detail, indicating the positioning of the secondary structure and the heme cofactors. This robust, expressible, highly thermostable and readily designable modular platform presents a valuable resource for redox protein design and the future construction of artificial electron-conducting circuitry.

FKBP25 regulates myoblast viability and migration and is differentially expressed in in vivo models of muscle adaptation.

FKBP25 (FKBP3 gene) is a dual-domain PPIase protein that consists of a C-terminal PPIase domain and an N-terminal basic tilted helix bundle (BTHB). The PPIase domain of FKBP25 has been shown to bind to microtubules, which has impacts upon microtubule polymerisation and cell cycle progression. Using quantitative proteomics, it was recently found that FKBP25 was expressed in the top 10% of the mouse skeletal muscle proteome. However, to date there have been few studies investigating the role ofFKBP25 in non-transformed systems. As such, this study aimed to investigate potential roles for FKBP25 in myoblast viability, migration and differentiation and in adaptation of mature skeletal muscle. Doxycycline-inducible FKBP25 knockdown in C2C12 myoblasts revealed an increase in cell accumulation/viability and migration in vitro that was independent of alterations in tubulin dynamics; however, FKBP25 knockdown had no discernible impact on myoblast differentiation into myotubes. Finally, a series of in vivo models of muscle adaptation were assessed, where it was observed that FKBP25 protein expression was increased in hypertrophy and regeneration conditions (chronic mechanical overload and the mdx model of Duchenne muscular dystrophy) but decreased in an atrophy model (denervation). Overall, the findings of this study establish FKBP25 as a regulator of myoblast viability and migration, with possible implications for satellite cell proliferation and migration and muscle regeneration, and as a potential regulator of in vivo skeletal muscle adaptation.

Design of Heme Enzymes with a Tunable Substrate Binding Pocket Adjacent to an Open Metal Coordination Site.

The catalytic versatility of pentacoordinated iron is highlighted by the broad range of natural and engineered activities of heme enzymes such as cytochrome P450s, which position a porphyrin cofactor coordinating a central iron atom below an open substrate binding pocket. This catalytic prowess has inspired efforts to design de novo helical bundle scaffolds that bind porphyrin cofactors. However, such designs lack the large open substrate binding pocket of P450s, and hence, the range of chemical transformations accessible is limited. Here, with the goal of combining the advantages of the P450 catalytic site geometry with the almost unlimited customizability of de novo protein design, we design a high-affinity heme-binding protein, dnHEM1, with an axial histidine ligand, a vacant coordination site for generating reactive intermediates, and a tunable distal pocket for substrate binding. A 1.6 Å X-ray crystal structure of dnHEM1 reveals excellent agreement to the design model with key features programmed as intended. The incorporation of distal pocket substitutions converted dnHEM1 into a proficient peroxidase with a stable neutral ferryl intermediate. In parallel, dnHEM1 was redesigned to generate enantiocomplementary carbene transferases for styrene cyclopropanation (up to 93% isolated yield, 5000 turnovers, 97:3 e.r.) by reconfiguring the distal pocket to accommodate calculated transition state models. Our approach now enables the custom design of enzymes containing cofactors adjacent to binding pockets with an almost unlimited variety of shapes and functionalities.

Ordered-domain unfolding of thermophilic isolated β subunit ATP synthase.

The flexibility of the ATP synthase's β subunit promotes its role in the ATP synthase rotational mechanism, but its domains stability remains unknown. A reversible thermal unfolding of the isolated β subunit (Tβ) of the ATP synthase from Bacillus thermophilus PS3, tracked through circular dichroism and molecular dynamics, indicated that Tβ shape transits from an ellipsoid to a molten globule through an ordered unfolding of its domains, preserving the β-sheet residual structure at high temperature. We determined that part of the stability origin of Tβ is due to a transversal hydrophobic array that crosses the β-barrel formed at the N-terminal domain and the Rossman fold of the nucleotide-binding domain (NBD), while the helix bundle of the C-terminal domain is the less stable due to the lack of hydrophobic residues, and thus the more flexible to trigger the rotational mechanism of the ATP synthase.

Structural and dynamic characterization of the hexa-coordinated globin from Spisula solidissima

High energy consumption in the nervous system requires a continuous supply of O2. This role is assisted by proteins from the globin super-family in the nerve cells of invertebrates, where ‘nerve hemoglobins’ (nHbs) are mainly present at mM concentrations and exhibit oxygen affinities comparable to those of vertebrate myoglobins. To gain insight into the structural bases of this function, we report the crystal structure of nHb from the Atlantic surf clam Spisula solidissima (SsHb), previously suggested to display a bis-histidyl hexa-coordinated heme in the deoxy state, high O2 affinity, and ligand binding cooperativity when assayed in situ. The crystallized protein forms a dimer through packing of a 4-helix bundle involving helices E and F of each subunit. The SsHb ‘classic’ globin fold displays bis-histidyl (His71(E7) and His103(F8)) hexa-coordination of the heme-Fe atom, with structural and dynamics variations found in the inter-helix hinge regions. Molecular Dynamics simulations of both monomeric and dimeric species in the bis-histidyl hexa-coordinated, deoxy penta-coordinated, and O2-bound hexa-coordinated states reveal distinct structural rearrangements at the interface between subunits in the dimer; these would affect the magnitude of the conformational fluctuations observed between monomer and dimer, and the topology of cavities within the protein matrix and at the interface. These results point to a distal site opening mechanism allowing access of the exogenous ligand to the heme and cast hypotheses on the dimer interface structural and dynamic properties that may support ligand binding cooperativity in dimeric SsHb.-

Isolation of recombinant apolipoprotein E4 N-terminal domain by foam fractionation

Apolipoprotein (apo) E functions in lipoprotein metabolism as a low density lipoprotein receptor ligand. ApoE is comprised of two structural domains, a 22 kDa N-terminal (NT) domain that adopts a helix bundle conformation and a 10 kDa C-terminal domain with strong lipid binding affinity. The NT domain is capable of transforming aqueous phospholipid dispersions into discoidal reconstituted high density lipoprotein (rHDL) particles. Given the utility of apoE-NT as a structural component of rHDL, expression studies were conducted. A plasmid construct encoding a pelB leader sequence fused to the N-terminus of human apoE4 (residues 1–183) was transformed into Escherichia coli. Upon expression, the fusion protein is directed to the periplasmic space where leader peptidase cleaves the pelB sequence, generating mature apoE4-NT. In shaker flask expression cultures, apoE4-NT escapes the bacteria and accumulates in the medium. In a bioreactor setting, however, apoE4-NT was found to combine with gas and liquid components in the culture medium to generate large quantities of foam. When this foam was collected in an external vessel and collapsed into a liquid foamate, analysis revealed that apoE4-NT was the sole major protein present. The product protein was further isolated by heparin affinity chromatography (60–80 mg/liter bacterial culture), shown to be active in rHDL formulation, and documented to serve as an acceptor of effluxed cellular cholesterol. Thus, foam fractionation provides a streamlined process to produce recombinant apoE4-NT for biotechnology applications.

Polyproline type II helical antifreeze proteins are widespread in Collembola and likely originated over 400 million years ago in the Ordovician Period

Antifreeze proteins (AFPs) bind to ice crystals to prevent organisms from freezing. A diversity of AFP folds has been found in fish and insects, including alpha helices, globular proteins, and several different beta solenoids. But the variety of AFPs in flightless arthropods, like Collembola, has not yet been adequately assessed. Here, antifreeze activity was shown to be present in 18 of the 22 species of Collembola from cold or temperate zones. Several methods were used to characterize these AFPs, including isolation by ice affinity purification, MALDI mass spectrometry, amino acid composition analysis, tandem mass spectrometry sequencing, transcriptome sequencing, and bioinformatic investigations of sequence databases. All of these AFPs had a high glycine content and were predicted to have the same polyproline type II helical bundle fold, a fold unique to Collembola. These Hexapods arose in the Ordovician Period with the two orders known to produce AFPs diverging around 400 million years ago during the Andean-Saharan Ice Age. Therefore, it is likely that the AFP arose then and persisted in many lineages through the following two ice ages and intervening warm periods, unlike the AFPs of fish which arose independently during the Cenozoic Ice Age beginning ~ 30 million years ago.

Design and Selection of Heterodimerizing Helical Hairpins for Synthetic Biology.

Synthetic biology applications would benefit from protein modules of reduced complexity that function orthogonally to cellular components. As many subcellular processes depend on peptide-protein or protein-protein interactions, de novo designed polypeptides that can bring together other proteins controllably are particularly useful. Thanks to established sequence-to-structure relationships, helical bundles provide good starting points for such designs. Typically, however, such designs are tested in vitro and function in cells is not guaranteed. Here, we describe the design, characterization, and application of de novo helical hairpins that heterodimerize to form 4-helix bundles in cells. Starting from a rationally designed homodimer, we construct a library of helical hairpins and identify complementary pairs using bimolecular fluorescence complementation in E.coli. We characterize some of the pairs using biophysics and X-ray crystallography to confirm heterodimeric 4-helix bundles. Finally, we demonstrate the function of an exemplar pair in regulating transcription in both E.coli and mammalian cells.

Genetic Variations of Fourth Exon Region of Prolactin Gene and Its Associations with Growth Traits in European Sea Bass Dicentrarchus labrax (Moronidae)

European sea bass Dicentrarchus labrax (Moronidae), which has a high commercial value, is mainly produced in the Mediterranean region. Prolactin (PRL) is a member of the helix bundle protein family that is secreted in the anterior pituitary gland and has a significant role in osmoregulation and growth in teleost fish. In this study, we investigated the genetic variation of the partial sequence of 3rd, 4th introns, and 4th exon regions of the PRL gene in Dicentrarchus labrax and its association with growth traits that were reared in cage conditions. Two SNPs (g.484 A C, g.529 T C) and one 15 bp indel (insertion/deletion) have been found in the 3rd intron region of the PRL gene of Dicentrarchus labrax. A significant association has been identified between the total body weight and the genotypes for PRL g.529 T C locus (р 0.05). The heterozygote genotype of the PRL g.529 T C locus has a significantly higher total body weight than the other genotypes in the population (p 0.05). Association analysis indicated that this SNP in the PRL gene is correlated with growth traits and could be used for genomic selection programs in Dicentrarchus labrax breeding.

A heme pocket aromatic quadrupole modulates gas binding to cytochrome c′-β: Implications for NO sensors

The structural basis by which gas-binding heme proteins control their interactions with NO, CO, and O2 is fundamental to enzymology, biotechnology, and human health. Cytochromes c' (cyts c') are a group of putative NO-binding heme proteins that fall into two families: the well-characterized four alpha helix bundle fold (cyts c'-α) and an unrelated family with a large beta-sheet fold (cyts c'-β) resembling that of cytochromes P460. A recent structure of cyt c'-β from Methylococcus capsulatus Bath revealed two heme pocket phenylalanine residues (Phe 32 and Phe 61) positioned near the distal gas-binding site. This feature, dubbed the "Phe cap," is highly conserved within the sequences of other cyts c'-β but is absent in their close homologs, the hydroxylamine-oxidizing cytochromes P460, although some do contain a single Phe residue. Here, we report an integrated structural, spectroscopic, and kinetic characterization of cyt c'-β from Methylococcus capsulatus Bath complexes with diatomic gases, focusing on the interaction of the Phe cap with NO and CO. Significantly, crystallographic and resonance Raman data show that orientation of the electron-rich aromatic ring face of Phe 32 toward distally bound NO or CO is associated with weakened backbonding and higher off rates. Moreover, we propose that an aromatic quadrupole also contributes to the unusually weak backbonding reported for some heme-based gas sensors, including the mammalian NO sensor, soluble guanylate cyclase. Collectively, this study sheds light on the influence of highly conserved distal Phe residues on heme-gas complexes of cytochrome c'-β, including the potential for aromatic quadrupoles to modulate NO and CO binding in other heme proteins.

Keto-form directed hierarchical chiral self-assembly of Schiff base derivatives with amplified circularly polarized luminescence

While enol-keto tautomerism has attracted great interest in Schiff bases and related compounds in solution and crystal states, the self-assembly of energy-unfavored keto form were scarcely investigated. Here, we report a keto-form directed self-assembly of a naphthalene-attached enantiomeric N-salicylideneanil analog L/DGG-Nap accompanied with a significantly amplified circularly polarized luminescence (CPL). It was found that LGG-Nap exists as a mixture of enol and keto form in monomer at a diluted toluene solution. The increment of the concentrations leads to the formation of predominated keto form, which subsequently triggers the self-assembly. Cryo-transmission electron microscopy (Cryo-TEM) revealed that a hierarchical assembly process happened upon increasing the concentration of LGG-Nap in toluene. Individual nanofibers formed at 1 × 10−4 mol/L and transferred into helical nanofiber bundles in 5 × 10−3 mol/L. Interestingly, while these is nearly no circular dichroism (CD) or CPL in the monomeric solution, the assembly showed strong CD and CPL. Remarkably, the dissymmetry factor (glum) was significantly amplified from zero in solution through the 0.005 in individual nanofiber to 0.1 in nanofiber bundles. This work demonstrates that the enol-keto tautomerism can be broken and trigger the self-assembly upon increasing the concentration, which can subsequently direct the chiral self-assembly and significantly amplify the dissymmetry factor of assembled CPL materials.

The Trypanosoma brucei MISP family of invariant proteins is co-expressed with BARP as triple helical bundle structures on the surface of salivary gland forms, but is dispensable for parasite development within the tsetse vector.

Trypanosoma brucei spp. develop into mammalian-infectious metacyclic trypomastigotes inside tsetse salivary glands. Besides acquiring a variant surface glycoprotein (VSG) coat, little is known about the metacyclic expression of invariant surface antigens. Proteomic analyses of saliva from T. brucei-infected tsetse flies identified, in addition to VSG and Brucei Alanine-Rich Protein (BARP) peptides, a family of glycosylphosphatidylinositol (GPI)-anchored surface proteins herein named as Metacyclic Invariant Surface Proteins (MISP) because of its predominant expression on the surface of metacyclic trypomastigotes. The MISP family is encoded by five paralog genes with >80% protein identity, which are exclusively expressed by salivary gland stages of the parasite and peak in metacyclic stage, as shown by confocal microscopy and immuno-high resolution scanning electron microscopy. Crystallographic analysis of a MISP isoform (MISP360) and a high confidence model of BARP revealed a triple helical bundle architecture commonly found in other trypanosome surface proteins. Molecular modelling combined with live fluorescent microscopy suggests that MISP N-termini are potentially extended above the metacyclic VSG coat, and thus could be tested as a transmission-blocking vaccine target. However, vaccination with recombinant MISP360 isoform did not protect mice against a T. brucei infectious tsetse bite. Lastly, both CRISPR-Cas9-driven knock out and RNAi knock down of all MISP paralogues suggest they are not essential for parasite development in the tsetse vector. We suggest MISP may be relevant during trypanosome transmission or establishment in the vertebrate's skin.

DESIGN SCENARIO AND ANALYSIS FOR PRELIMINARY SPECIFICATION OF STEAM GENERATOR IN THE PELUIT-40

The helical steam generator is connected to an HTGR-type nuclear reactor called PeLUIt-40 for steam production. Steam is used to generate electricity and hydrogen. A once-through helical tube bundle was employed because of its ability to endure mechanical stress due to thermal expansion, high resistance to flow-induced vibrations, and better thermal performance compared to a straight tube one. To produce the targeted steam, a design analysis of the once-through helical steam generator needs to be conducted. A quick evaluation method was used to predict the preliminary specifications required for steam production. Simple thermodynamic calculations combined with empirical heat transfer coefficients covering convective and boiling processes at constant pressure were used to carry out the analysis. Two scenarios were conducted to evaluate the design choice based on the previous design of RDE-10.

Two gates mediate NMDA receptor activity and are under subunit-specific regulation

Kinetics of NMDA receptor (NMDAR) ion channel opening and closing contribute to their unique role in synaptic signaling. Agonist binding generates free energy to open a canonical gate at the M3 helix bundle crossing. Single channel activity is characterized by clusters, or periods of rapid opening and closing, that are separated by long silent periods. A conserved glycine in the outer most transmembrane helices, the M4 helices, regulates NMDAR function. Here we find that the GluN1 glycine mainly regulates single channel events within a cluster, whereas the GluN2 glycine mainly regulates entry and exit from clusters. Molecular dynamics simulations suggest that, whereas the GluN2 M4 (along with GluN2 pre-M1) regulates the gate at the M3 helix bundle crossing, the GluN1 glycine regulates a ‘gate’ at the M2 loop. Subsequent functional experiments support this interpretation. Thus, the distinct kinetics of NMDARs are mediated by two gates that are under subunit-specific regulation.