Α-helical Coils Research Articles

Invasive mussels fashion silk-like byssus via mechanical processing of massive horizontally acquired coiled coils.

Zebra and quagga mussels (Dreissena spp.) are invasive freshwater biofoulers that perpetrate devastating economic and ecological impact. Their success depends on their ability to anchor onto substrates with protein-based fibers known as byssal threads. Yet, compared to other mussel lineages, little is understood about the proteins comprising their fibers or their evolutionary history. Here, we investigated the hierarchical protein structure of Dreissenid byssal threads and the process by which they are fabricated. Unique among bivalves, we found that threads possess a predominantly β-sheet crystalline structure reminiscent of spider silk. Further analysis revealed unexpectedly that the Dreissenid thread protein precursors are mechanoresponsive α-helical proteins that are mechanically processed into β-crystallites during thread formation. Proteomic analysis of the byssus secretory organ and byssus fibers revealed a family of ultrahigh molecular weight (354 to 467 kDa) asparagine-rich (19 to 20%) protein precursors predicted to form α-helical coiled coils. Moreover, several independent lines of evidence indicate that the ancestral predecessor of these proteins was likely acquired via horizontal gene transfer. This chance evolutionary event that transpired at least 12 Mya has endowed Dreissenids with a distinctive and effective fiber formation mechanism, contributing significantly to their success as invasive species and possibly, inspiring new materials design.

CC+ : A searchable database of validated coiled coils in PDB structures and AlphaFold2 models.

α-Helical coiled coils are common tertiary and quaternary elements of protein structure. In coiled coils, two or more α helices wrap around each other to form bundles. This apparently simple structural motif can generate many architectures and topologies. Coiled coil-forming sequences can be predicted from heptad repeats of hydrophobic and polar residues, hpphppp, although this is not always reliable. Alternatively, coiled-coil structures can be identified using the program SOCKET, which finds knobs-into-holes (KIH) packing between side chains of neighboring helices. SOCKET also classifies coiled-coil architecture and topology, thus allowing sequence-to-structure relationships to be garnered. In 2009, we used SOCKET to create a relational database of coiled-coil structures, CC+ , from the RCSB Protein Data Bank (PDB). Here, we report an update of CC+ following an update of SOCKET (to Socket2) and the recent explosion of structural data and the success of AlphaFold2 in predicting protein structures from genome sequences. With the most-stringent SOCKET parameters, CC+ contains ≈12,000 coiled-coil assemblies from experimentally determined structures, and ≈120,000 potential coiled-coil structures within single-chain models predicted by AlphaFold2 across 48 proteomes. CC+ allows these and other less-stringently defined coiled coils to be searched at various levels of structure, sequence, and side-chain interactions. The identified coiled coils can be viewed directly from CC+ using the Socket2 application, and their associated data can be downloaded for further analyses. CC+ is available freely at http://coiledcoils.chm.bris.ac.uk/CCPlus/Home.html. It will be updated automatically. We envisage that CC+ could be used to understand coiled-coil assemblies and their sequence-to-structure relationships, and to aid protein design and engineering.

Cooperative Assembly of Self-Adjusting α-Helical Coiled Coils along the Length of an mRNA Chain to Form a Thermodynamically Stable Nanotube Carrier.

Although mRNA delivery technology is very promising, problems in safety and transport arise due to the intrinsically low thermodynamic stability of the current mRNA carriers. Considering that mRNAs are filamentous and a nanotube is one of the most thermodynamically stable shapes among nanoassemblies, a nanotube is one of the most stable supramolecular structures that can be assembled with mRNA. Here, we develop a nanotube-shaped filamentous mRNA delivery platform that shows exceptionally high thermodynamic stability. The key to the development of the mRNA nanotube is the design of self-adjusting supramolecular building blocks (SABs) that have two disparate properties, i.e., dynamic property and stiffness, in a single molecule. The counterbalance of the dynamic property and stiffness in SABs enables the coating of mRNA by winding its way through the flexible and irregular mRNA chain via cooperative interactions. SAB nanotubes with targeting ligands installed show a high uptake efficiency in mammalian cells and controllable gene expression behavior. Thus, the mRNA nanotube provides an enabling technology toward the development of safe and stable mRNA vaccines and therapeutics.

Chain Sliding versus β-Sheet Formation upon Shearing Single α-Helical Coiled Coils

Chain Sliding versus β-Sheet Formation upon Shearing Single α-Helical Coiled Coils

Chain Sliding versus β-Sheet Formation upon Shearing Single α-Helical Coiled Coils

Chain Sliding versus β-Sheet Formation upon Shearing Single α-Helical Coiled Coils

Structural Determinants and Functional Significance of Dimerization for Osmosensing Transporter ProP in Escherichia coli.

Osmosensing transporter ProP forestalls cellular dehydration by detecting environments with high osmotic pressure and mediating the accumulation of organic osmolytes by bacterial cells. It is composed of 12 transmembrane helices with cytoplasmic N- and C-termini. In Escherichia coli, dimers form when the C-terminal domains of ProP molecules form homodimeric, antiparallel, α-helical coiled coils. No dominant negative effect was detected when inactive and active ProP molecules formed heterodimers in vivo. Purification of ProP in detergent dodecylmaltoside yielded monomers, which were functional after reconstitution in proteoliposomes. With other evidence, this suggests that ProP monomers function independently whether in the monomeric or dimeric state. Amino acid replacements that disrupted or reversed the coiled coil did not prevent in vivo dimerization of ProP detected with a bacterial two-hybrid system. Maleimide labeling detected no osmolality-dependent variation in the reactivities of cysteine residues introduced to transmembrane helix (TM) XII. In contrast, coarse-grained molecular dynamic simulations detected deformation of the lipid around TMs III and VI, on the lipid-exposed protein surface opposite to TM XII. This suggests that the dimer interface of ProP includes the surfaces of TMs III and VI, not of TM XII as previously suggested by crosslinking data. Homology modeling suggested that coiled-coil formation and dimerization via such an interface are not mutually exclusive. In previous work, alterations to the C-terminal coiled coil blocked co-localization of ProP with phospholipid cardiolipin at E. coli cell poles. Thus, dimerization may contribute to ProP targeting, adjust its lipid environment, and hence indirectly modify its osmotic stress response.

Secondary structure and stability of a gel-forming tardigrade desiccation-tolerance protein.

Protein-based pharmaceuticals are increasingly important, but their inherent instability necessitates a "cold chain" requiring costly refrigeration during production, shipment, and storage. Drying can overcome this problem, but most proteins need the addition of stabilizers, and some cannot be successfully formulated. Thus, there is a need for new, more effective protective molecules. Cytosolically, abundant heat-soluble proteins from tardigrades are both fundamentally interesting and a promising source of inspiration; these disordered, monodisperse polymers form hydrogels whose structure may protect client proteins during drying. We used attenuated total reflectance Fourier transform infrared spectroscopy, differential scanning calorimetry, and small-amplitude oscillatory shear rheometry to characterize gelation. A 5% (wt/vol) gel has a strength comparable with human skin, and melts cooperatively and reversibly near body temperature with an enthalpy comparable with globular proteins. We suggest that the dilute protein forms α-helical coiled coils and increasing their concentration drives gelation via intermolecular β-sheet formation.

Molecular architecture of the autoinhibited kinesin-1 lambda particle.

Despite continuing progress in kinesin enzyme mechanochemistry and emerging understanding of the cargo recognition machinery, it is not known how these functions are coupled and controlled by the α-helical coiled coils encoded by a large component of kinesin protein sequences. Here, we combine computational structure prediction with single-particle negative-stain electron microscopy to reveal the coiled-coil architecture of heterotetrameric kinesin-1 in its compact state. An unusual flexion in the scaffold enables folding of the complex, bringing the kinesin heavy chain–light chain interface into close apposition with a tetrameric assembly formed from the region of the molecule previously assumed to be the folding hinge. This framework for autoinhibition is required to uncover how engagement of cargo and other regulatory factors drives kinesin-1 activation.

Structural guidelines for stabilization of α-helical coiled coils via PEG stapling.

Macrocyclization or stapling is one of the most well-known and generally applicable strategies for enhancing peptide/protein conformational stability and target binding affinity. However, there are limited structure- or sequence-based guidelines for the incorporation of optimal interhelical staples within coiled coils: the location and length of an interhelical staple is either arbitrarily chosen or requires significant optimization. Here we explore the impact of interhelical PEG stapling on the conformational stability and proteolytic resistance of a model disulfide-bound heterodimeric coiled coil. We demonstrate that (1) interhelical PEG staples are more stabilizing when placed farther from an existing disulfide crosslink; (2) e/g′ staples are more stabilizing than f/b′ or b/c′ staples; (3) PEG staples between different positions have different optimal staple lengths; (4) PEG stapling tolerates variation in the structure of the PEG linker and in the mode of conjugation; and (5) the guidelines developed here enable the rational design of a stabilized PEG-stapled HER-2 affibody with enhanced conformational stability and proteolytic resistance.

Self-assembling systems comprising intrinsically disordered protein polymers like elastin-like recombinamers.

Despite lacking cooperatively folded structures under native conditions, numerous intrinsically disordered proteins (IDPs) nevertheless have great functional importance. These IDPs are hybrids containing both ordered and intrinsically disordered protein regions (IDPRs), the structure of which is highly flexible in this unfolded state. The conformational flexibility of these disordered systems favors transitions between disordered and ordered states triggered by intrinsic and extrinsic factors, folding into different dynamic molecular assemblies to enable proper protein functions. Indeed, prokaryotic enzymes present less disorder than eukaryotic enzymes, thus showing that this disorder is related to functional and structural complexity. Protein-based polymers that mimic these IDPs include the so-called elastin-like polypeptides (ELPs), which are inspired by the composition of natural elastin. Elastin-like recombinamers (ELRs) are ELPs produced using recombinant techniques and which can therefore be tailored for a specific application. One of the most widely used and studied characteristic structures in this field is the pentapeptide (VPGXG)n . The structural disorder in ELRs probably arises due to the high content of proline and glycine in the ELR backbone, because both these amino acids help to keep the polypeptide structure of elastomers disordered and hydrated. Moreover, the recombinant nature of these systems means that different sequences can be designed, including bioactive domains, to obtain specific structures for each application. Some of these structures, along with their applications as IDPs that self-assemble into functional vesicles or micelles from diblock copolymer ELRs, will be studied in the following sections. The incorporation of additional order- and disorder-promoting peptide/protein domains, such as α-helical coils or β-strands, in the ELR sequence, and their influence on self-assembly, will also be reviewed. In addition, chemically cross-linked systems with controllable order-disorder balance, and their role in biomineralization, will be discussed. Finally, we will review different multivalent IDPs-based coatings and films for different biomedical applications, such as spatially controlled cell adhesion, osseointegration, or biomaterial-associated infection (BAI).

FT-Raman Spectroscopy as a Tool to Study the Secondary Structures of Wheat Gliadin Proteins.

Raman spectroscopy is a useful method in biological, biomedical, food, and agricultural studies, allowing the simultaneous examination of various chemical compounds and evaluation of molecular changes occurring in tested objects. The purpose of our research was to explain how the elimination of ω-fractions from the wheat gliadin complex influences the secondary structures of the remaining αβγ-gliadins. To this aim, we analyzed the endosperm of wheat kernels as well as gliadin proteins extracted from two winter wheat genotypes: wasko.gl+ (control genotype containing the full set of gliadins) and wasko.gl− (modified genotype lacking all ω-gliadins). Based on the decomposition of the amide I band, we observed a moderate increase in β-forms (sheets and turns) at the expense of α-helical and random coil structures for gliadins isolated from the flour of the wasko.gl− line. Since ω-gliadins contain no cysteine residues, they do not participate in the formation of the disulfide bridges that stabilize the protein structure. However, they can interact with other proteins via weak, low-energetic hydrogen bonds. We conclude that the elimination of ω-fractions from the gliadin complex causes minor modifications in secondary structures of the remaining gliadin proteins. In our opinion, these small, structural changes of proteins may lead to alterations in gliadin allergenicity.

How Coiled-Coil Assemblies Accommodate Multiple Aromatic Residues.

Rational protein design requires understanding the contribution of each amino acid to a targeted protein fold. For a subset of protein structures, namely, α-helical coiled coils (CCs), knowledge is sufficiently advanced to allow the rational de novo design of many structures, including entirely new protein folds. Current CC design rules center on using aliphatic hydrophobic residues predominantly to drive the folding and assembly of amphipathic α helices. The consequences of using aromatic residues-which would be useful for introducing structural probes, and binding and catalytic functionalities-into these interfaces are not understood. There are specific examples of designed CCs containing such aromatic residues, e.g., phenylalanine-rich sequences, and the use of polar aromatic residues to make buried hydrogen-bond networks. However, it is not known generally if sequences rich in tyrosine can form CCs, or what CC assemblies these would lead to. Here, we explore tyrosine-rich sequences in a general CC-forming background and resolve new CC structures. In one of these, an antiparallel tetramer, the tyrosine residues are solvent accessible and pack at the interface between the core and the surface. In another more complex structure, the residues are buried and form an extended hydrogen-bond network.

Comprehensive predictions of secondary structures for comparative analysis in different species

Protein structures are directly linked to biological functions. However, there is a gap of knowledge between the decoded genome and the structure. To bridge the gap, we focused on the secondary structure (SS). From a comprehensive analysis of predicted SS of proteins in different types of organisms, we have arrived at the following findings: The proportions of SS in genomes were different among phylogenic domains. The distributions of strand lengths indicated structural limitations in all of the species. Different from bacteria and archaea, eukaryotes have an abundance of α-helical and random coil proteins. Interestingly, there was a relationship between SS and post-translational modifications. By calculating hydrophobicity moments of helices and strands, highly amphipathic fragments of SS were found, which might be related to the biological functions. In conclusion, comprehensive predictions of SS will provide valuable perspectives to understand the entire protein structures in genomes and will help one to discover or design functional proteins.

A Model of bZIP Protein/DNA Recognition using Knob-Socket Analysis

The basic leucine zipper protein (bZIP) family binds specific DNA sequences through interactions between the individual α-helical coils and respective half-sites. Despite previous studies on target sequences and affinities of both homo- and heterodimers, the recognition and binding mechanism of bZIP dimers are not fully understood. Despite the relatively high homology between the subfamilies, dimers of bZIP coils have shown high variability in target sequences, ranging from conjoined and variably-spaced target sequences to novel and emergent sequences. To help elucidate the determinants of protein-DNA recognition, a novel Knob-Socket analysis of the packing at the protein and nucleic acid interface is able to produce an insightful model of half-site recognition specificity between a protein α-helix and the DNA double helix. The model not only characterizes the protein sequence motifs that recognize DNA bases, but uniquely is able to describe the DNA motifs that specifically pack protein residues. The bZIP coils studied include Jun, Fos, CREB, and C/EBP, through the analyses of their respective PDB files. For each half-site, the protein ɑ-helix splits recognition on both the positive and negative strands of the DNA double helix. Half of the α-helix packs into the first two nucleotides of the positive DNA strand, and the other packs into the last two nucleotides on the negative DNA strand. Along the i+4 ridge, a set of three conserved residues provides a dividing line on the protein helix between positive and negative DNA strand recognition. These three residues are also involved in a quadripartite region that specifically packs and recognizes the methyl group on a thymidine. Recognition of other bases are more complex and involve characterization of the DNA sequence motifs that pack and recognize residues on the protein α-helix.

Optically Active Polymers with Cationic Units Connected through Neutral Spacers: Helical Conformation and Chirality Transfer to External Molecules

Cationic chiral polymers consisting of a chiral “Arg monomeric unit” consisting of two l-arginine moieties joined together through an oxyhexane-1,6-diyloxy spacer unit and achiral “Sp monomeric unit” based on the spermine moiety where the two units are connected through a 1,4-dioxobutane-1,4-diyl spacer unit (“C2 monomeric unit”) were prepared in the cationic form as a salt of p-toluenesulfonic acid. Polymer conformation could not be directly deduced from circular dichroism (CD) spectra based on the amide group bands (200–240 nm), which are generally used to quantify α-helical, β-sheet, and random coil residues because of spectral overlapping with the p-toluenesulfonate bands. On the other hand, a helical conformation was proposed on the basis of the relation between CD intensity and Arg monomeric unit ratio of polymers, while the proposed conformation may not be highly ordered but rather fragmented and partial. The helix-forming propensity of a polymer consisting of a higher Arg monomeric unit ratio was supported by MD simulations in water and in vacuum. The copolymer consisting of 100% Arg monomeric units [poly(Arg100-C2)], the one consisting of 42% Arg monomeric units and 58% Sp monomeric units [poly(Arg50-C2–Sp50-C2)], and the one consisting of 20% Arg monomeric units and 80% Sp monomeric units [poly(Arg25-C2–Sp75-C2)] exhibited chirality transfer to methyl orange (MO), where transfer was most efficient with poly(Arg50-C2–Sp50-C2). Chirality transfer efficiency also remarkably varied depending on the standing time of polymer solution in H2O before it is mixed with MO, the time after mixing, and the concentration of the polymer. Theoretical calculations of absorbance and CD spectra of polymer–MO complexes suggested that the induced CD spectra mostly having bisignet spectral patterns are not based on exciton coupling between two MO molecules bound to the polymer chain but are composed of independent excitation bands. The design of chiral macromolecules introduced in this work based on a chain composed of charged chiral and achiral units separated by neutral achiral linker (spacer) units has not been studied from a view of chiral polymeric materials. Such a design of polymer chain may be applicable to a wider variety of chemical structures and will largely widen the scope of optically active polymers with helical conformation.

Silk from Indian paper wasp: Structure prediction and secondary conformational analysis

The pupae of Indian paper wasp, Ropalidia marginata, produce silk that covers the opening of each cells in their hives, which probably helps in thermoregulation and providing mechanical stability. In this study an attempt has been made to investigate the amino acid composition and protein structure of Indian wasp silk using bioinformatics and experimental tools. The amino acid analysis of the regenerated solubilized wasp silk revealed high alanine and serine content that increases the propensity of α-helical and random coil and/or β-sheet structure respectively. The Matrix Assisted Laser Desorption/Ionization-Time Of Flight/Time Of Flight based protein fingerprinting analysis of the regenerated solubilized wasp silk revealed its short-range sequences. The protein modeling studies indicated the presence of predominant coiled coil α-helical and random coil structure in the protein isolated from wasp hive caps, similar to previous reports from the Vespoidea family. Fourier transform infrared spectroscopic analysis and X-ray diffraction analysis of the native paper wasp cap revealed the presence of mix of coiled coil α-helix and β-sheet structure in outer regions; whereas inner region contains tannin/intermolecularly bonded hydroxyl groups of polyphenols. The present study is first report to generate valuable insights about the chemical composition and secondary structure prediction of Indian paper wasp silk and highlight on nature's novel modus operandi to engineer unique functional fibrous matrix.

Structural Requirements of the Phytoplasma Effector Protein SAP54 for Causing Homeotic Transformation of Floral Organs.

Phytoplasmas are intracellular bacterial plant pathogens that cause devastating diseases in crops and ornamental plants by the secretion of effector proteins. One of these effector proteins, termed SECRETED ASTER YELLOWS WITCHES' BROOM PROTEIN 54 (SAP54), leads to the degradation of a specific subset of floral homeotic proteins of the MIKC-type MADS-domain family via the ubiquitin-proteasome pathway. In consequence, the developing flowers show the homeotic transformation of floral organs into vegetative leaf-like structures. The molecular mechanism of SAP54 action involves binding to the keratin-like domain of MIKC-type proteins and to some RAD23 proteins, which translocate ubiquitylated substrates to the proteasome. The structural requirements and specificity of SAP54 function are poorly understood, however. Here, we report, based on biophysical and molecular biological analyses, that SAP54 folds into an α-helical structure. Insertion of helix-breaking mutations disrupts correct folding of SAP54 and compromises SAP54 binding to its target proteins and, concomitantly, its ability to evoke disease phenotypes in vivo. Interestingly, dynamic light scattering data together with electrophoretic mobility shift assays suggest that SAP54 preferentially binds to multimeric complexes of MIKC-type proteins rather than to dimers or monomers of these proteins. Together with data from literature, this finding suggests that MIKC-type proteins and SAP54 constitute multimeric α-helical coiled coils. Our investigations clarify the structure-function relationship of an important phytoplasma effector protein and may thus ultimately help to develop treatments against some devastating plant diseases.

Intrahelical Interactions in an α-Helical Coiled Coil Determine the Structural Stability of Tropomyosin.

Tropomyosin (Tpm) is a two-stranded parallel α-helical coiled-coil protein, and studying its structure is crucial for understanding the nature of coiled coils. Previously, we found that the N-terminal half of the human skeletal muscle α-Tpm (α-Tpm 140) was less structurally stable in the presence of phosphate ions than the coiled-coil protein carrier (CCPC) 140 variant with 18 mutated residues, in which all amino acid residues located at the interface between the two α-helices were completely conserved. A classical hypothesis explains that interhelical interactions stabilize the coiled-coil structure. In this study, we tested the hypothesis that the structural stability of Tpm and its variant is governed by the binding of multivalent ions that form a bridge between charged side chains located at positions b, c, and f of the heptad repeat on a single α-helical chain. We found that the structural stability of α-Tpm 140 and CCPC 140 markedly increased upon addition of divalent cations and divalent anions, respectively. We also clarified that the structural stability of the α-Tpm 140/CCPC 140 heteromeric coiled-coil molecule was governed by the stability of a less stable α-helical chain. These results demonstrated that the entire structural stability of Tpm is determined by the stability of a single α-helix. Our findings provide new insights into the study of the structure of coiled-coil proteins.

Highly Stabilized α-Helical Coiled Coils Kill Gram-Negative Bacteria by Multicomplementary Mechanisms under Acidic Condition.

Although antimicrobial peptides (AMPs) hold tremendous promise in overcoming the threats of multidrug resistance, the main obstacle to successful therapeutic applications is their poor stability. Various synthetic strategies such as unnatural amino acids and chemical modifications have made advances for improving this problem. However, this complicated synthesis often greatly increases the cost of production. Here, we show that a series of novel peptides, designed by combining an α-helical coiled coil model, knowledge of the specificity of proteolysis and major parameters of AMPs, exhibited efficient activity against all tested Gram-negative bacteria under acidic condition and demonstrate low toxicity. Of these α-helical coiled coil peptides, 3IH3 displayed the highest average therapeutic index (GMTI = 294.25) with high stability toward salts, serum, extreme pH, heat, and proteases. Electron microscopy and biological analytical technique analyses showed that 3IH3 killed bacterial cells via a multicomplementary mechanism at pH 6.0, with physical membrane disruption as the dominant bactericidal mechanism. These results suggest that 3IH3 shows great stability as an inexpensive and effective antimicrobial activity agent and has the potential for clinical application in the treatment of infections occurring in body sites with acidic pH.

Navigating the Structural Landscape of De Novo α-Helical Bundles.

The association of amphipathic α helices in water leads to α-helical-bundle protein structures. However, the driving force for this-the hydrophobic effect-is not specific and does not define the number or the orientation of helices in the associated state. Rather, this is achieved through deeper sequence-to-structure relationships, which are increasingly being discerned. For example, for one structurally extreme but nevertheless ubiquitous class of bundle-the α-helical coiled coils-relationships have been established that discriminate between all-parallel dimers, trimers, and tetramers. Association states above this are known, as are antiparallel and mixed arrangements of the helices. However, these alternative states are less well understood. Here, we describe a synthetic-peptide system that switches between parallel hexamers and various up-down-up-down tetramers in response to single-amino-acid changes and solution conditions. The main accessible states of each peptide variant are characterized fully in solution and, in most cases, to high resolution with X-ray crystal structures. Analysis and inspection of these structures helps rationalize the different states formed. This navigation of the structural landscape of α-helical coiled coils above the dimers and trimers that dominate in nature has allowed us to design rationally a well-defined and hyperstable antiparallel coiled-coil tetramer (apCC-Tet). This robust de novo protein provides another scaffold for further structural and functional designs in protein engineering and synthetic biology.