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The intermitochondrial cement (IMC) is a prominent germ granule that locates among clustered mitochondria in mammalian germ cells. Serving as a key platform for Piwi-interacting RNA (piRNA) biogenesis; however, how the IMC assembles among mitochondria remains elusive. Here, we identify that Tudor domain-containing 1 (TDRD1) triggers IMC assembly via phase separation. TDRD1 phase separation is driven by the cooperation of its tetramerized coiled-coil domain and dimethylarginine-binding Tudor domains but is independent of its intrinsically disordered region. TDRD1 is recruited to mitochondria by MILI and sequentially enhances mitochondrial clustering and triggers IMC assembly via phase separation to promote piRNA processing. TDRD1 phase separation deficiency in mice disrupts IMC assembly and piRNA biogenesis, leading to transposon de-repression and spermatogenic arrest. Moreover, TDRD1 phase separation is conserved in vertebrates but not in invertebrates. Collectively, our findings demonstrate a role of phase separation in germ granule formation and establish a link between membrane-bound organelles and membrane-less organelles. [Display omitted] • TDRD1 undergoes phase separation via oligomerization of coiled-coil domain • Tudor domains facilitate TDRD1 phase separation by recognizing sDMA • TDRD1 phase separation deficiency disrupts IMC assembly and piRNA biogenesis in mice • TDRD1 phase separation is conserved in vertebrates but not in invertebrates Gao et al. address the mechanism governing the assembly of mitochondrial-associated germ granules (IMCs) in mammalian germ cells. The IMC component TDRD1 undergoes phase separation to trigger IMC assembly, which is essential for piRNA biogenesis, transposon silencing, and male fertility in mice. [ABSTRACT FROM AUTHOR]

Self‐assembly of designed molecules has enabled the construction of a variety of functional nanostructures. Specifically, adaptable self‐assembly has demonstrated several advantageous features for smart materials. Here, we demonstrate that an α‐helical coiled coil conjugated with a dendrimer can adapt to spatial restriction due to the strong steric repulsion between dendrimer chains. The adaptable transformation of a tetrameric coiled coil to a trimeric coiled coil can be confirmed using analytical ultracentrifugation upon conjugation of the dendrimer to the coiled coil‐forming building block. Interestingly, circular dichroism spectroscopy analysis of the dendrimer conjugate revealed an unconventional trend: the multimerization of the coiled coil is inversely dependent on concentration. This result implies that the spatial crowding between the bulky dendritic chains is significantly stronger than that between linear chains, thereby affecting the overall assembly process. We further illustrated the application potential by decorating the surface of gold nanorods (AuNRs) with the adaptable coiled coil. The dendrimer‐coiled coil peptide conjugate can be utilized to fabricate organic‐inorganic nanohybrids with enhanced colloidal and thermal stabilities. This study demonstrates that the coiled coil can engage in the adaptable mode of self‐assembly with the potential to form dynamic peptide‐based materials. [ABSTRACT FROM AUTHOR]

Soluble N‐ethylmaleimide‐sensitive factor Attachment protein REceptor (SNARE) proteins catalyze the fusion process of vesicles with target membranes in eukaryotic cells. To do this, they assemble in a zipper‐like fashion into stable complexes between the membranes. Structural studies have shown that the complexes consist of four different helices, which we subdivide into Qa‐, Qb‐, Qc‐, and R‐helix on the basis of their sequence signatures. Using a combination of biochemistry, modeling and molecular dynamics, we investigated how the four different types are arranged in a complex. We found that there is a matching pattern in the core of the complex that dictates the position of the four fundamental SNARE types in the bundle, resulting in a QabcR complex. In the cell, several different cognate QabcR‐SNARE complexes catalyze the different transport steps between the compartments of the endomembrane system. Each of these cognate QabcR complexes is compiled from a repertoire of about 20 SNARE subtypes. Our studies show that exchange within the four types is largely tolerated structurally, although some non‐cognate exchanges lead to structural imbalances. This suggests that SNARE complexes have evolved for a catalytic mechanism, a mechanism that leaves little scope for selectivity beyond the QabcR rule. [ABSTRACT FROM AUTHOR]

Efforts to develop an HIV-1 vaccine include those focusing on conserved structural elements as the target of broadly neutralizing monoclonal antibodies. MAb D5 binds to a highly conserved hydrophobic pocket on the gp41 N-heptad repeat (NHR) coiled coil and neutralizes through prevention of viral fusion and entry. Assessment of 17-mer and 36-mer NHR peptides presenting the D5 epitope in rodent immunogenicity studies showed that the longer peptide elicited higher titers of neutralizing antibodies, suggesting that neutralizing epitopes outside of the D5 pocket may exist. Although the magnitude and breadth of neutralization elicited by NHR-targeting antigens are lower than that observed for antibodies directed to other epitopes on the envelope glycoprotein complex, it has been shown that NHR-directed antibodies are potentiated in TZM-bl cells containing the FcγRI receptor. Herein, we report the design and evaluation of covalently stabilized trimeric 51-mer peptides encompassing the complete gp41 NHR. We demonstrate that these peptide trimers function as effective antiviral entry inhibitors and retain the ability to present the D5 epitope. We further demonstrate in rodent and nonhuman primate immunization studies that our 51-mer constructs elicit a broader repertoire of neutralizing antibody and improved cross-clade neutralization of primary HIV-1 isolates relative to 17-mer and 36-mer NHR peptides in A3R5 and FcγR1-enhanced TZM-bl assays. These results demonstrate that sensitive neutralization assays can be used for structural enhancement of moderately potent neutralizing epitopes. Finally, we present expanded trimeric peptide designs which include unique low-molecular-weight scaffolds that provide versatility in our immunogen presentation strategy. [ABSTRACT FROM AUTHOR]

Introduction: The therapeutic potential of bispecific antibodies is becoming widely recognised, with over a hundred formats already described. For many applications, enhanced tissue penetration is sought, so bispecifics with low molecular weight may offer a route to enhanced potency. Here we report the design of bi- and tri-specific antibody-based constructs with molecular weights as low as 14.5 and 22 kDa respectively. Methods: Autonomous bovine ultra-long CDR H3 (knob domain peptide) modules have been engineered with artificial coiled-coil stalks derived from Sin Nombre orthohantavirus nucleocapsid protein and human Beclin-1, and joined in series to produce bi- and tri-specific antibody-based constructs with exceptionally low molecular weights. Results: Knob domain peptides with coiled-coil stalks retain high, independent antigen binding affinity, exhibit exceptional levels of thermal stability, and can be readily joined head-to-tail yielding the smallest described multi-specific antibody format. The resulting constructs are able to bind simultaneously to all their targets with no interference. Discussion: Compared to existing bispecific formats, the reduced molecular weight of the knob domain fusions may enable enhanced tissue penetration and facilitate binding to cryptic epitopes that are inaccessible to conventional antibodies. Furthermore, they can be easily produced at high yield as recombinant products and are free from the heavy-light chain mispairing issue. Taken together, our approach offers an efficient route to modular construction of minimalistic bi- and multi-specifics, thereby further broadening the therapeutic scope for knob domain peptides. [ABSTRACT FROM AUTHOR]

The designability of orthogonal coiled coil (CC) dimers, which draw on well‐established design rules, plays a pivotal role in fueling the development of CCs as synthetically versatile assembly‐directing motifs for the fabrication of bionanomaterials. Here, we aim to expand the synthetic CC toolkit through establishing a "minimalistic" set of orthogonal, de novo CC peptides that comprise 3.5 heptads in length and a single buried Asn to prescribe dimer formation. The designed sequences display excellent partner fidelity, confirmed via circular dichroism (CD) spectroscopy and Ni‐NTA binding assays, and are corroborated in silico using molecular dynamics (MD) simulation. Detailed analysis of the MD conformational data highlights the importance of interhelical E@g‐N@a interactions in coordinating an extensive 6‐residue hydrogen bonding network that "locks" the interchain Asn‐Asn′ contact in place. The enhanced stability imparted to the Asn‐Asn′ bond elicits an increase in thermal stability of CCs up to ~15°C and accounts for significant differences in stability within the collection of similarly designed orthogonal CC pairs. The presented work underlines the utility of MD simulation as a tool for constructing de novo, orthogonal CCs, and presents an alternative handle for modulating the stability of orthogonal CCs via tuning the number of interhelical E@g‐N@a contacts. Expansion of CC design rules is a key ingredient for guiding the design and assembly of more complex, intricate CC‐based architectures for tackling a variety of challenges within the fields of nanomedicine and bionanotechnology. [ABSTRACT FROM AUTHOR]

IntroductionThe therapeutic potential of bispecific antibodies is becoming widely recognised, with over a hundred formats already described. For many applications, enhanced tissue penetration is sought, so bispecifics with low molecular weight may offer a route to enhanced potency. Here we report the design of bi- and tri-specific antibody-based constructs with molecular weights as low as 14.5 and 22 kDa respectively.MethodsAutonomous bovine ultra-long CDR H3 (knob domain peptide) modules have been engineered with artificial coiled-coil stalks derived from Sin Nombre orthohantavirus nucleocapsid protein and human Beclin-1, and joined in series to produce bi- and tri-specific antibody-based constructs with exceptionally low molecular weights.ResultsKnob domain peptides with coiled-coil stalks retain high, independent antigen binding affinity, exhibit exceptional levels of thermal stability, and can be readily joined head-to-tail yielding the smallest described multi-specific antibody format. The resulting constructs are able to bind simultaneously to all their targets with no interference.DiscussionCompared to existing bispecific formats, the reduced molecular weight of the knob domain fusions may enable enhanced tissue penetration and facilitate binding to cryptic epitopes that are inaccessible to conventional antibodies. Furthermore, they can be easily produced at high yield as recombinant products and are free from the heavy-light chain mispairing issue. Taken together, our approach offers an efficient route to modular construction of minimalistic bi- and multi-specifics, thereby further broadening the therapeutic scope for knob domain peptides.

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 a-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 a-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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary: Nucleotide‐binding domain and leucine‐rich repeat (NLR) proteins are important intracellular immune receptors that activate robust plant immune responses upon detecting pathogens. Canonical NLRs consist of a conserved tripartite architecture that includes a central regulatory nucleotide‐binding domain, C‐terminal leucine‐rich repeats, and variable N‐terminal domains that directly participate in immune execution. In flowering plants, the vast majority of NLR N‐terminal domains belong to the coiled‐coil, Resistance to Powdery Mildew 8, or Toll/interleukin‐1 receptor subfamilies, with recent structural and biochemical studies providing detailed mechanistic insights into their functions. In this insight review, we focus on the immune‐related biochemistries of known plant NLR N‐terminal domains and discuss the evolutionary diversity of atypical NLR domains in nonflowering plants. We further contrast these observations against the known diversity of NLR‐related receptors from microbes to metazoans across the tree of life. [ABSTRACT FROM AUTHOR]

We report the preparation and spectroscopic characterization of a highly elusive copper site bound exclusively to oxygen donor atoms within a protein scaffold. Despite copper generally being considered unsuitable for use in MRI contrast agents, which in the clinic are largely Gd(III) based, the designed copper coiled coil displays relaxivity values equal to, or superior than, those of the Gd(III) analog at clinical field strengths. The creation of this new-to-biology proteinaceous CuOx-binding site demonstrates the power of the de novo peptide design approach to access chemistry for abiological applications, such as for the development of MRI contrast agents. [ABSTRACT FROM AUTHOR]

Human immunodeficiency virus type 1 (HIV-1) is characterized by high variability and drug resistance. This has necessitated the development of antivirals with a new chemotype and therapy. We previously identified an artificial peptide with non-native protein sequence, AP3, with the potential to inhibit HIV-1 fusion through targeting hydrophobic grooves on the N-terminal heptad repeat trimer of viral glycoprotein gp41. Here, a small-molecule HIV-1 inhibitor targeting chemokine coreceptor CCR5 on the host cell was integrated into the AP3 peptide, producing a novel dual-target inhibitor with improved activity against multiple HIV-1 strains including those resistant to the currently used anti-HIV-1 drug enfuvirtide. Its superior antiviral potency in comparison with the respective pharmacophoric moieties is in consonance with the dual binding of viral gp41 and host factor CCR5. Therefore, our work provides a potent artificial peptide-based bifunctional HIV-1 entry inhibitor and highlights the multitarget-directed ligands approach in the development of novel therapeutic anti-HIV-1 agents. [ABSTRACT FROM AUTHOR]

Neurodegenerative disorders, such as Huntington's diseases and spinocerebellar ataxias (SCAs), are driven by proteins with expanded polyglutamine (polyQ) tracts. Recently, coiled-coil structures in polyQ regions of such proteins were shown to facilitate aggregate formation and ultimately lead to cell death. However, the molecular mechanism linking these structural domains to neuronal toxicity of polyQ proteins remains elusive. Here, we demonstrate that coiled-coil structures in the Q repeat region of SCA type 3 (SCA3) polyQ proteins confer protein toxicity in Drosophila neurons. To functionally characterize coiled-coil structures in the Q repeat regions, we generated three structural variants of SCA3 polyQ proteins: (i) MJDtr-76Q, containing both α-helical coiled-coil and β-sheet hairpin structures in the Q repeat region; (ii) MJDtr-70Q_cc0, possessing only α-helical coiled-coil structures due to the incorporation of β-sheet-breaking residues (Q-to-N or Q-to-E mutations); and (iii) MJDtr-70Q_pQp, with no secondary structure due to the introduced proline residues (Q-to-P mutations). Through comparative analysis of these variants, we found that coiled-coil structures facilitated nuclear localization of SCA3 polyQ proteins and induced dendrite defects in Drosophila dendritic arborization neurons. Furthermore, genetic and functional screening identified the transcription factor Foxo as a target of polyQ proteins, and coiled-coil-mediated interactions of Foxo and polyQ proteins in the nucleus resulted in the observed dendrite and behavioral defects in Drosophila These results demonstrate that coiled-coil structures of polyQ proteins are crucial for their neuronal toxicity, which is conferred through coiled-coil to coiled-coil interactions with the nuclear targets of these proteins.

Four insect orders have flight muscles that are both asynchronous and indirect; they are asynchronous in that the wingbeat frequency is decoupled from the frequency of nervous stimulation and indirect in that the muscles attach to the thoracic exoskeleton instead of directly to the wing. Flight muscle thick filaments from two orders, Hemiptera and Diptera, have been imaged at a subnanometer resolution, both of which revealed a myosin tail arrangement referred to as "curved molecular crystalline layers". Here, we report a thick filament structure from the indirect flight muscles of a third insect order, Hymenoptera, the Asian bumble bee Bombus ignitus. The myosin tails are in general agreement with previous determinations from Lethocerus indicus and Drosophila melanogaster. The Skip 2 region has the same unusual structure as found in Lethocerus indicus thick filaments, an α-helix discontinuity is also seen at Skip 4, but the orientation of the Skip 1 region on the surface of the backbone is less angled with respect to the filament axis than in the other two species. The heads are disordered as in Drosophila, but we observe no non-myosin proteins on the backbone surface that might prohibit the ordering of myosin heads onto the thick filament backbone. There are strong structural similarities among the three species in their non-myosin proteins within the backbone that suggest how one previously unassigned density in Lethocerus might be assigned. Overall, the structure conforms to the previously observed pattern of high similarity in the myosin tail arrangement, but differences in the non-myosin proteins. [ABSTRACT FROM AUTHOR]

NEMO is an essential component in the activation of the canonical nuclear factor κB (NF-κB) pathway and exerts its function by recruiting the IκB kinases (IKK) to the IKK complex. Inhibition of the NEMO/IKKs interaction is an attractive therapeutic paradigm for diseases related to NF-κB mis-regulation, but a difficult endeavor because of the extensive protein-protein interface. Here we report the design and characterization of novel engineered constructs of the IKK-binding domain of NEMO, programmed to render this difficult protein domain amenable to NMR measurements and crystallization, while preserving its biological function. ZipNEMO binds IKKβ with nanomolar affinity, is amenable to heteronuclear nuclear magnetic resonance (NMR) techniques and structure determination by X-ray crystallography. We show that NMR spectra of zipNEMO allow to detect inhibitor binding in solution and resonance assignment. The crystal structure of zipNEMO reveals a novel ligand binding motif and the adaptability of the binding pocket and inspired the design of new peptide inhibitors., Competing Interests: Declaration of interests A.E.K. is affiliated to Oxford Cryosystems, Hanover, NH, US. A.H.B. is affiliated to Incyte, Wilmington, DE, US. G.G. is affiliated to Generate Biomedicines, Inc., Arlington, MA, US. M.P., G.G., and A.H.B. have a patent application related to this work “ENGINEERED NEMO AND USES THEREOF” US Patent Application No. 63/196,217., (Copyright © 2025 Elsevier Inc. All rights reserved.)

A de novo lanthanide-binding coiled-coil polypeptide (MB1-2) was previously reported to self-assemble into a trimeric complex upon addition of Tb 3+ with a micromolar range dissociation constant. This study examines the effect of substitution of hydrophobic residues in heptad repeats of MB1-2 on the thermodynamic stability of the resulting Tb-peptide complex. Substitution of isoleucine to norleucine in each heptad repeat was assessed considering the greater accessible surface area of the latter and predicted increased hydrophobic interaction. Job's method of continuous variation using circular dichroism spectroscopy suggests a trimeric structure for the analog complex equivalent to that formed by MB1-2. The dissociation constant and CD spectra suggest that complex formation in the analog is more favorable as a result of ligand preorganization. In addition, thermal denaturation suggests greater stability of the Tb-MB1-2 Nle complex in comparison to the parent Tb-MB1-2. These results indicate improved stability of the complex class can be achieved through heptad repeat amino acid substitutions that increase peptide interchain interaction., (© 2024 European Peptide Society and John Wiley & Sons Ltd.)

α-helical coiled-coils are ubiquitous protein structures in all living organisms. For decades, modified coiled-coils sequences have been used in biotechnology, vaccine development, and biochemical research to induce protein oligomerization, and form self-assembled protein scaffolds. A prominent model for the versatility of coiled-coil sequences is a peptide derived from the yeast transcription factor, GCN4. In this work, we show that its trimeric variant, GCN4-pII, binds bacterial lipopolysaccharides (LPS) from different bacterial species with picomolar affinity. LPS molecules are highly immunogenic, toxic glycolipids that comprise the outer leaflet of the outer membrane of Gram-negative bacteria. Using scattering techniques and electron microscopy, we show how GCN4-pII breaks down LPS micelles in solution. Our findings suggest that the GCN4-pII peptide and derivatives thereof could be used for novel LPS detection and removal solutions with high relevance to the production and quality control of biopharmaceuticals and other biomedical products, where even minuscule amounts of residual LPS can be lethal.

Intermediate filaments (IFs) form an essential part of the metazoan cytoskeleton. Despite a long history of research, a proper understanding of their molecular architecture and assembly process is still lacking. IFs self‐assemble from elongated dimers, which are defined by their central "rod" domain. This domain forms an α‐helical coiled coil consisting of three segments called coil1A, coil1B, and coil2. It has been hypothesized that the structural plasticity of the dimer, including the unraveling of some coiled‐coil regions, is essential for the assembly process. To systematically explore this possibility, we have studied six 50‐residue fragments covering the entire rod domain of human vimentin, a model IF protein. After creating in silico models of these fragments, their evaluation using molecular dynamics was performed. Large differences were seen across the six fragments with respect to their structural variability during a 100 ns simulation. Next, the fragments were prepared recombinantly, whereby their correct dimerization was promoted by adding short N‐ or C‐terminal capping motifs. The capped fragments were subjected to circular dichroism measurements at varying temperatures. The obtained melting temperatures reveal the relative stabilities of individual fragments, which correlate well with in silico results. We show that the least stable regions of vimentin rod are coil1A and the first third of coil2, while the structures of coil1B and the rest of coil2 are significantly more robust. These observations are in line with the data obtained using other experimental approaches, and contribute to a better understanding of the molecular mechanisms driving IF assembly. [ABSTRACT FROM AUTHOR]

Septins are cytoskeletal proteins and their interaction with membranes is crucial for their role in various cellular processes. Septins have polybasic regions (PB1 and PB2) which are important for lipid interaction. Earlier, we and others have highlighted the role of the septin C-terminal domain (CTD) to membrane interaction. However, detailed information on residues/group of residues important for such feature is lacking. In this study, we investigate the lipid-binding profile of Schistosoma mansoni Septin10 (Sm SEPT10) using PIP strip and Langmuir monolayer adsorption assays. Our findings highlight the CTD as the primary domain responsible for lipid interaction in Sm SEPT10, showing binding to phosphatidylinositol phosphates. Sm SEPT10 CTD contains a conserved polybasic region (PB3) present in both animals and fungi septins, and a Lys (K367) within its putative amphipathic helix (AH) that we demonstrate as important for lipid binding. PB3 deletion or mutation of this Lys (K367A) strongly impairs lipid interaction. Remarkably, we observe that the AH within a construct lacking the final 43 amino acid residues is insufficient for lipid binding. Furthermore, we investigate the homocomplex formed by Sm SEPT10 CTD in solution by cross-linking experiments, CD spectroscopy, SEC-MALS and SEC-SAXS. Taken together, our studies define the lipid-binding region in Sm SEPT10 and offer insights into the molecular basis of septin-membrane binding. This information is particularly relevant for less-studied non-human septins, such as Sm SEPT10. [Display omitted] • The amphipathic helix of Sm SEPT10 is not sufficient to drive lipid interactions. • Instead, the last 46 residues within Sm SEPT10 CTD are crucial for this. • We highlight the role of K367 and a novel polybasic region (PB3) for lipid binding. • PB3 is present and conserved across specific septin groups in both fungi and animals. [ABSTRACT FROM AUTHOR]

The cohesin complex is required for sister chromatid cohesion and genome compaction. Cohesin coiled coils (CCs) can fold at break sites near midpoints to bring head and hinge domains, located at opposite ends of coiled coils, into proximity. Whether ATPase activities in the head play a role in this conformational change is yet to be known. Here, we dissected functions of cohesin ATPase activities in cohesin dynamics in Schizosaccharomyces pombe. Isolation and characterization of cohesin ATPase temperature-sensitive (ts) mutants indicate that both ATPase domains are required for proper chromosome segregation. Unbiased screening of spontaneous suppressor mutations rescuing the temperature lethality of cohesin ATPase mutants identified several suppressor hotspots in cohesin that located outside of ATPase domains. Then, we performed comprehensive saturation mutagenesis targeted to these suppressor hotspots. Large numbers of the identified suppressor mutations indicated several different ways to compensate for the ATPase mutants: 1) Substitutions to amino acids with smaller side chains in coiled coils at break sites around midpoints may enable folding and extension of coiled coils more easily; 2) substitutions to arginine in the DNA binding region of the head may enhance DNA binding; or 3) substitutions to hydrophobic amino acids in coiled coils, connecting the head and interacting with other subunits, may alter conformation of coiled coils close to the head. These results reflect serial structural changes in cohesin driven by its ATPase activities potentially for packaging DNAs. [ABSTRACT FROM AUTHOR]

A coiled coil is a structural motif in proteins that consists of at least two α-helices wound around each other. For structural stabilization, these α-helices form interhelical contacts via their amino acid side chains. However, there are restrictions as to the distances along the amino acid sequence at which those contacts occur. As the spatial period of the α-helix is 3.6, the most frequent distances between hydrophobic contacts are 3, 4, and 7. Up to now, the multitude of possible decompositions of α-helices participating in coiled coils at these distances has not been explored systematically. Here, we present an algorithm that computes all non-redundant decompositions of sequence periods of hydrophobic amino acids into distances of 3, 4, and 7. Further, we examine which decompositions can be found in nature by analyzing the available data and taking a closer look at correlations between the properties of the coiled coil and its decomposition. We find that the availability of decompositions allowing for coiled-coil formation without putting too much strain on the α-helix geometry follows an oscillatory pattern in respect of period length. Our algorithm supplies the basis for exploring the possible decompositions of coiled coils of any period length. [ABSTRACT FROM AUTHOR]

The TRIM5α protein from rhesus macaques (rhTRIM5α) mediates a potent inhibition of HIV-1 infection via a mechanism that involves the abortive disassembly of the viral core. We have demonstrated that alpha-helical elements within the Linker 2 (L2) region, which lies between the SPRY domain and the Coiled-Coil domain, influence the potency of restriction. Here, we utilize single-molecule FRET analysis to reveal that the L2 region of the TRIM5α dimer undergoes dynamic conformational changes, which results in the displacement of L2 regions by 25 angstroms relative to each other. Analysis of restriction enhancing or abrogating mutations in the L2 region reveal that restriction defective mutants are unable to undergo dynamic conformational changes and do not assume compact, alpha-helical conformations in the L2 region. These data suggest a model in which conformational changes in the L2 region mediate displacement of CA bound SPRY domains to induce the destabilization of assembled capsid during restriction.

The sequences of M proteins, the major surface-associated virulence factors of the widespread bacterial pathogen group A Streptococcus, are antigenically variable but have in common a strong propensity to form coiled coils. Paradoxically, these sequences are also replete with coiled-coil destabilizing residues. These features are evident in the irregular coiled-coil structure and thermal instability of M proteins. We present an explanation for this paradox through studies of the B repeats of the medically important M1 protein. The B repeats are required for interaction of M1 with fibrinogen (Fg) and consequent proinflammatory activation. The B repeats sample multiple conformations, including intrinsically disordered, dissociated, as well as two alternate coiled-coil conformations: a Fg-nonbinding register 1 and a Fg-binding register 2. Stabilization of M1 in the Fg-nonbinding register 1 resulted in attenuation of Fg binding as expected, but counterintuitively, so did stabilization in the Fg-binding register 2. Strikingly, these register-stabilized M1 proteins gained the ability to bind Fg when they were destabilized by a chaotrope. These results indicate that M1 stability is antithetical to Fg interaction and that M1 conformational dynamics, as specified by destabilizing residues, are essential for interaction. A "capture-and-collapse" model of association accounts for these observations, in which M1 captures Fg through a dynamic conformation and then collapses into a register 2-coiled coil as a result of stabilization provided by binding energy. Our results support the general conclusion that destabilizing residues are evolutionarily conserved in M proteins to enable functional interactions necessary for pathogenesis.

Abstract Background Amyloids are ordered, insoluble protein aggregates, characterized by a cross-β sheet quaternary structure in which molecules in a β-strand conformation are stacked along the filament axis via intermolecular interactions. While amyloids are typically associated with pathological conditions, functional amyloids have also been identified and are present in a wide variety of organisms ranging from bacteria to humans. The cytoplasmic polyadenylation element-binding (CPEB) prion-like protein is an mRNA-binding translation regulator, whose neuronal isoforms undergo activity-dependent aggregation, a process that has emerged as a plausible biochemical substrate for memory maintenance. CPEB aggregation is driven by prion-like domains (PLD) that are divergent in sequence across species, and it remains unknown whether such divergent PLDs follow a similar aggregating assembly pathway. Here, we describe the amyloid-like features of the neuronal Aplysia CPEB (ApCPEB) PLD and compare them to those of the Drosophila ortholog, Orb2 PLD. Results Using in vitro single-molecule and bulk biophysical methods, we find transient oligomers and mature amyloid-like filaments that suggest similarities in the late stages of the assembly pathway for both ApCPEB and Orb2 PLDs. However, while prior to aggregation the Orb2 PLD monomer remains mainly as a random coil in solution, ApCPEB PLD adopts a diversity of conformations comprising α-helical structures that evolve to coiled-coil species, indicating structural differences at the beginning of their amyloid assembly pathways. Conclusion Our results indicate that divergent PLDs of CPEB proteins from different species retain the ability to form a generic amyloid-like fold through different assembly mechanisms.

Abstract Background CP12 is a small chloroplast protein that is widespread in various photosynthetic organisms and is an actor of the redox signaling pathway involved in the regulation of the Calvin Benson Bassham (CBB) cycle. The gene encoding this protein is conserved in many diatoms, but the protein has been overlooked in these organisms, despite their ecological importance and their complex and still enigmatic evolutionary background. Methods A combination of biochemical, bioinformatics and biophysical methods including electrospray ionization-mass spectrometry, circular dichroism, nuclear magnetic resonance spectroscopy and small X ray scattering, was used to characterize a diatom CP12. Results Here, we demonstrate that CP12 is expressed in the marine diatom Thalassiosira pseudonana constitutively in dark-treated and in continuous light-treated cells as well as in all growth phases. This CP12 similarly to its homologues in other species has some features of intrinsically disorder protein family: it behaves abnormally under gel electrophoresis and size exclusion chromatography, has a high net charge and a bias amino acid composition. By contrast, unlike other known CP12 proteins that are monomers, this protein is a dimer as suggested by native electrospray ionization-mass spectrometry and small angle X-ray scattering. In addition, small angle X-ray scattering revealed that this CP12 is an elongated cylinder with kinks. Circular dichroism spectra indicated that CP12 has a high content of α-helices, and nuclear magnetic resonance spectroscopy suggested that these helices are unstable and dynamic within a millisecond timescale. Together with in silico predictions, these results suggest that T. pseudonana CP12 has both coiled coil and disordered regions. Conclusions These findings bring new insights into the large family of dynamic proteins containing disordered regions, thus increasing the diversity of known CP12 proteins. As it is a protein that is more abundant in many stresses, it is not devoted to one metabolism and in particular, it is not specific to carbon metabolism. This raises questions about the role of this protein in addition to the well-established regulation of the CBB cycle. Choregraphy of metabolism by CP12 proteins in Viridiplantae and Heterokonta. While the monomeric CP12 in Viridiplantae is involved in carbon assimilation, regulating phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) through the formation of a ternary complex, in Heterokonta studied so far, the dimeric CP12 is associated with Ferredoxin-NADP reductase (FNR) and GAPDH. The Viridiplantae CP12 can bind metal ions and can be a chaperone, the Heterokonta CP12 is more abundant in all stresses (C, N, Si, P limited conditions) and is not specific to a metabolism. Video Abstract

Wag31, or DivIVA, is an essential protein and a drug target in the human pathogen Mycobacterium tuberculosis that self-assembles at the negatively curved membrane surface to form a higher-order structural scaffold, maintains rod-shaped cellular morphology and localizes key cell-wall synthesizing enzymes at the pole for exclusive polar growth. The crystal structure of the N-terminal lipid-binding domain of mycobacterial Wag31 was determined at 2.3 Å resolution. The structure revealed a highly polar surface lined with several conserved charged residues that suggest probable sites for interactions with membrane lipids. Crystal-packing analysis revealed a previously unseen `dimer-of-dimers' assembly state of N-terminal Wag31, which is formed by antiparallel stacking of two coiled-coil dimers. Size-exclusion column-chromatography-coupled small-angle solution X-ray scattering data revealed a tetrameric form as a major assembly state of N-terminal Wag31 in solution, further supporting the crystal structure. The results suggest that, in addition to lipid binding, the N-terminal Wag31 can participate in self-assembly to form filamentous structures. Plausible models of linear self-assembly and branching of Wag31 filaments consistent with available data are suggested.

The Mre11 complex comprises Mre11, Rad50 and Nbs1 (Xrs2 in S. cerevisiae ). The core components, Mre11 and Rad50 are highly conserved, with readily identifiable orthologs in all clades of life, whereas Nbs1/Xrs2 are present only in eukaryotes. In eukaryotes, the complex is integral to the DNA damage response, acting in DNA double strand break (DSB) detection and repair, and the activation of DNA damage signaling. We present here a 3.2 Å cryo-EM structure of the S. cerevisiae Mre11-Rad50 complex with bound dsDNA. The structure provided a foundation for detailed mutational analyses regarding homo and heterotypic protein interfaces, as well as DNA binding properties of Rad50. We define several conserved residues in Rad50 and Mre11 that are critical to complex assembly as well as for DNA binding. In addition, the data reveal that the Rad50 coiled coil domain influences ATP hydrolysis over long distances., Competing Interests: DECLARATION OF INTERESTS The authors declare no competing interests.

In recent years, protein engineering efforts have yielded a diverse set of binding proteins that hold promise for various therapeutic applications. Despite this, their inability to reach intracellular targets limits their applications to cell surface or soluble targets. To address this challenge, we previously reported a protein carrier that binds antibodies and delivers them to therapeutic targets inside cancer cells. This carrier, known as the Hex carrier, is comprised of a self-assembling coiled coil hexamer at the core, with each alpha helix fused to a linker, an antibody binding domain, and a six Histidine-tag (His-tag). In this work, we designed different versions of the carrier to determine the role of each building block in cytosolic protein delivery. We found that increasing exposure of the Hex coiled coil on the carriers, through molecular design or removing antibodies, increased internalization, pointing to a role of the coiled coil in promoting endocytosis. We observed a clear increase in endosomal disruption events when His-tags were present on the carrier relative to when they were removed, due to an endosomal buffering effect. Finally, we found that the antibody binding domains of the Hex carrier could be replaced with monomeric ultra-stable GFP for intracellular delivery and endosomal escape. Our results demonstrate that the Hex coiled coil, in conjunction with His-tags, could be a generalizable vehicle for delivering small and large proteins to intracellular targets. This work also highlights new biological applications for oligomeric coiled coils and shows the direct and quantifiable impact of histidine residues on endosomal disruption. These findings could inform the design of future drug delivery vehicles in applications beyond intracellular protein delivery. [Display omitted] • Protein drug carrier can deliver different proteins inside of cells. • Exposure of the central domain of the carrier increases delivery to cells. • Presence of histidine tags on the carrier increases escape from endosomes. • Delivery platform could deliver other therapeutic proteins to new targets. [ABSTRACT FROM AUTHOR]

Cohesin is key to eukaryotic genome organization and acts throughout the cell cycle in an ATP-dependent manner. The mechanisms underlying cohesin ATPase activity are poorly understood. Here, we characterize distinct steps of the human cohesin ATPase cycle and show that the SMC1A and SMC3 ATPase domains undergo specific but concerted structural rearrangements along this cycle. Specifically, whereas the proximal coiled coil of the SMC1A ATPase domain remains conformationally stable, that of the SMC3 displays an intrinsic flexibility. The ATP-dependent formation of the heterodimeric SMC1A/SMC3 ATPase module (engaged state) favors this flexibility, which is counteracted by NIPBL and DNA binding (clamped state). Opening of the SMC3/RAD21 interface (open-engaged state) stiffens the SMC3 proximal coiled coil, thus constricting together with that of SMC1A the ATPase module DNA-binding chamber. The plasticity of the ATP-dependent interface between the SMC1A and SMC3 ATPase domains enables these structural rearrangements while keeping the ATP gate shut. [Display omitted] [Display omitted] • SMC1A and SMC3 ATPase domains show specific structural dynamics • SMC3 ATPase domain proximal coiled coil has a specific intrinsic flexibility • SMC3/RAD21 interface dissociation constricts SMC1A/SMC3 ATPase DNA-binding chamber • SMC1A/SMC3 ATP interface plasticity allows dynamic moves but keeps the ATP gate shut Vitoria Gomes et al. show that SMC1A and SMC3 ATPase domains have distinct, specific, but concerted conformational dynamics during the human cohesin ATPase cycle. Whereas the SMC1A proximal coiled coil is stable, that of the SMC3 has an intrinsic flexibility that modifies the size of the SMC1A/SMC3 ATPase module DNA-binding chamber. [ABSTRACT FROM AUTHOR]

Coiled-coil proteins consisting of multiple copies of helices take part in transmembrane transportation and oligomerization, and are used for drug delivery. Cross-alpha amyloid-like coiled-coil structures, in which tens of short helices align perpendicular to the fibril axis, often resist molecular replacement due to the uncertainty to position each helix. Eight coiled-coil structures already solved and posted in the protein data bank are reconstructed ab initio to demonstrate the direct phasing results. Non-crystallographic symmetry and intermediate-resolution diffraction data are considered for direct phasing. The retrieved phases have a mean phase error around 30∼40°. The calculated density map is ready for model building, and the reconstructed model agrees with the deposited structure. The results indicate that direct phasing is an efficient approach to construct the protein envelope from scratch, build each helix without model bias which is also used to confirm the prediction of AlphaFold and RosettaFold, and solve the whole structure of coiled-coil proteins.

CENP-E (centromere protein E) and CENP-F (centromere protein F), also known as mitosin, are large, multi-functional proteins associated with the outer kinetochore. CENP-E features a well-characterized kinesin motor domain at its N-terminus and a second microtubule-binding domain at its C-terminus of unknown function. CENP-F is important for the formation of proper kinetochore-microtubule attachment and, similar to CENP-E, contains two microtubule-binding domains at its termini. While the importance of these proteins is known, the details of their interactions with microtubules have not yet been investigated. We have biochemically and structurally characterized the microtubule-binding properties of the amino- and carboxyl-terminal domains of CENP-F as well as the carboxyl-terminal (non-kinesin) domain of CENP-E. CENP-E's C-terminus and CENP-F's N-terminus bind microtubules with similar affinity to the well-characterized Ndc80 complex, while CENP-F's C-terminus shows much lower affinity. Electron microscopy analysis reveals that all of these domains engage the microtubule surface in a disordered manner, suggesting that these factors have no favored binding geometry and may allow for initial side-on attachments early in mitosis.

Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor (SNARE) proteins catalyze the fusion process of vesicles with target membranes in eukaryotic cells. To do this, they assemble in a zipper-like fashion into stable complexes between the membranes. Structural studies have shown that the complexes consist of four different helices, which we subdivide into Qa-, Qb-, Qc-, and R-helix on the basis of their sequence signatures. Using a combination of biochemistry, modeling and molecular dynamics, we investigated how the four different types are arranged in a complex. We found that there is a matching pattern in the core of the complex that dictates the position of the four fundamental SNARE types in the bundle, resulting in a QabcR complex. In the cell, several different cognate QabcR-SNARE complexes catalyze the different transport steps between the compartments of the endomembrane system. Each of these cognate QabcR complexes is compiled from a repertoire of about 20 SNARE subtypes. Our studies show that exchange within the four types is largely tolerated structurally, although some non-cognate exchanges lead to structural imbalances. This suggests that SNARE complexes have evolved for a catalytic mechanism, a mechanism that leaves little scope for selectivity beyond the QabcR rule., (© 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

The synthetic biology toolkit contains a growing number of parts for regulating transcription and translation, but very few that can be used to control protein association. Here we report characterization of 22 previously published heterospecific synthetic coiled-coil peptides called SYNZIPs. We present biophysical analysis of the oligomerization states, helix orientations, and affinities of 27 SYNZIP pairs. SYNZIP pairs were also tested for interaction in two cell-based assays. In a yeast two-hybrid screen, >85% of 253 comparable interactions were consistent with prior in vitro measurements made using coiled-coil microarrays. In a yeast-signaling assay controlled by coiled-coil mediated scaffolding, 12 SYNZIP pairs were successfully used to down-regulate the expression of a reporter gene following treatment with α-factor. Characterization of these interaction modules dramatically increases the number of available protein interaction parts for synthetic biology and should facilitate a wide range of molecular engineering applications. Summary characteristics of 27 SYNZIP peptide pairs are reported in specification sheets available in the Supporting Information and at the SYNZIP Web site [http://keatingweb.mit.edu/SYNZIP/].

Brain’s functions, such as memory and learning, rely on synapses that are highly specialized cellular junctions connecting neurons. Functional synapses orchestrate the assembly of ion channels, receptors, enzymes, and scaffold proteins in both pre- and post-synapse. Liprin-α proteins are master scaffolds in synapses and coordinate various synaptic proteins to assemble large protein complexes. The functions of liprin-αs in synapse formation have been largely uncovered by genetic studies in diverse model systems. Recently, emerging structural and biochemical studies on liprin-α proteins and their binding partners begin to unveil the molecular basis of the synaptic assembly. This review summarizes the recent structural findings on liprin-αs, proposes the assembly mechanism of liprin-α-mediated complexes, and discusses the liprin-α-organized assemblies in the regulation of synapse formation and function.

Neuronal granules are biomolecular condensates that concentrate high quantities of RNAs and RNA-related proteins within neurons. These dense packets of information are trafficked from the soma to distal sites rich in polysomes, where local protein synthesis can occur. Movement of neuronal granules to distal sites, and local protein synthesis, play a critical role in synaptic plasticity. The formation of neuronal granules is intriguing; these granules lack a membrane and instead phase separate due to protein and RNA interactions. Low complexity motifs and RNA binding domains are highly prevalent in these proteins. Here, we introduce the role that coiled-coil motifs play in neuronal granule proteins, and investigate the structure-function relationship of coiled-coil proteins in RNA regulation. Interestingly, low complexity domains and coiled-coil motifs are highly dynamic, allowing for increased functional response to environmental influences. Finally, biomolecular condensates have been suggested to drive the formation of toxic, neurodegenerative proteins such as TDP-43 and tau. Here, we review the conversion of coiled-coil motifs to amyloid structures, and speculate a role that neuronal granules play in coiled-coil to amyloid conversions of neurodegenerative proteins.

Biological materials combine stress relaxation and self-healing with non-linear stress-strain responses. These characteristic features are a direct result of hierarchical self-assembly, which often results in fiber-like architectures. Even though structural knowledge is rapidly increasing, it has remained a challenge to establish relationships between microscopic and macroscopic structure and function. Here, we focus on understanding how network topology determines the viscoelastic properties, i.e., stress relaxation, of biomimetic hydrogels. We have dynamically crosslinked two different synthetic polymers with one and the same crosslink. The first polymer, a polyisocyanopeptide (PIC), self-assembles into semi-flexible, fiber-like bundles, and thus displays stress-stiffening, similar to many biopolymer networks. The second polymer, 4-arm poly(ethylene glycol) (starPEG), serves as a reference network with well-characterized structural and viscoelastic properties. Using one and the same coiled coil crosslink allows us to decouple the effects of crosslink kinetics and network topology on the stress relaxation behavior of the resulting hydrogel networks. We show that the fiber-containing PIC network displays a relaxation time approximately two orders of magnitude slower than the starPEG network. This reveals that crosslink kinetics is not the only determinant for stress relaxation. Instead, we propose that the different network topologies determine the ability of elastically active network chains to relax stress. In the starPEG network, each elastically active chain contains exactly one crosslink. In the absence of entanglements, crosslink dissociation thus relaxes the entire chain. In contrast, each polymer is crosslinked to the fiber bundle in multiple positions in the PIC hydrogel. The dissociation of a single crosslink is thus not sufficient for chain relaxation. This suggests that tuning the number of crosslinks per elastically active chain in combination with crosslink kinetics is a powerful design principle for tuning stress relaxation in polymeric materials. The presence of a higher number of crosslinks per elastically active chain thus yields materials with a slow macroscopic relaxation time but fast dynamics at the microscopic level. Using this principle for the design of synthetic cell culture matrices will yield materials with excellent long-term stability combined with the ability to locally reorganize, thus facilitating cell motility, spreading, and growth.

Background: CP12 is a small chloroplast protein that is widespread in various photosynthetic organisms and is an actor of the redox signaling pathway involved in the regulation of the Calvin Benson Bassham (CBB) cycle. The gene encoding this protein is conserved in many diatoms, but the protein has been overlooked in these organisms, despite their ecological importance and their complex and still enigmatic evolutionary background. Methods: A combination of biochemical, bioinformatics and biophysical methods including electrospray ionization-mass spectrometry, circular dichroism, nuclear magnetic resonance spectroscopy and small X ray scattering, was used to characterize a diatom CP12. Results: Here, we demonstrate that CP12 is expressed in the marine diatom Thalassiosira pseudonana constitutively in dark-treated and in continuous light-treated cells as well as in all growth phases. This CP12 similarly to its homologues in other species has some features of intrinsically disorder protein family: it behaves abnormally under gel electrophoresis and size exclusion chromatography, has a high net charge and a bias amino acid composition. By contrast, unlike other known CP12 proteins that are monomers, this protein is a dimer as suggested by native electrospray ionization-mass spectrometry and small angle X-ray scattering. In addition, small angle X-ray scattering revealed that this CP12 is an elongated cylinder with kinks. Circular dichroism spectra indicated that CP12 has a high content of α-helices, and nuclear magnetic resonance spectroscopy suggested that these helices are unstable and dynamic within a millisecond timescale. Together with in silico predictions, these results suggest that T. pseudonana CP12 has both coiled coil and disordered regions. Conclusions: These findings bring new insights into the large family of dynamic proteins containing disordered regions, thus increasing the diversity of known CP12 proteins. As it is a protein that is more abundant in many stresses, it is not devoted to one metabolism and in particular, it is not specific to carbon metabolism. This raises questions about the role of this protein in addition to the well-established regulation of the CBB cycle. Choregraphy of metabolism by CP12 proteins in Viridiplantae and Heterokonta. While the monomeric CP12 in Viridiplantae is involved in carbon assimilation, regulating phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) through the formation of a ternary complex, in Heterokonta studied so far, the dimeric CP12 is associated with Ferredoxin-NADP reductase (FNR) and GAPDH. The Viridiplantae CP12 can bind metal ions and can be a chaperone, the Heterokonta CP12 is more abundant in all stresses (C, N, Si, P limited conditions) and is not specific to a metabolism. 5h3nzM1M4bjv4RCQxTYJAC Video Abstract [ABSTRACT FROM AUTHOR]

Background: Amyloids are ordered, insoluble protein aggregates, characterized by a cross-ß sheet quaternary structure in which molecules in a ß-strand conformation are stacked along the filament axis via intermolecular interactions. While amyloids are typically associated with pathological conditions, functional amyloids have also been identified and are present in a wide variety of organisms ranging from bacteria to humans. The cytoplasmic polyadenylation element-binding (CPEB) prion-like protein is an mRNA-binding translation regulator, whose neuronal isoforms undergo activity-dependent aggregation, a process that has emerged as a plausible biochemical substrate for memory maintenance. CPEB aggregation is driven by prion-like domains (PLD) that are divergent in sequence across species, and it remains unknown whether such divergent PLDs follow a similar aggregating assembly pathway. Here, we describe the amyloid-like features of the neuronal Aplysia CPEB (ApCPEB) PLD and compare them to those of the Drosophila ortholog, Orb2 PLD. Results: Using in vitro single-molecule and bulk biophysical methods, we find transient oligomers and mature amyloid-like filaments that suggest similarities in the late stages of the assembly pathway for both ApCPEB and Orb2 PLDs. However, while prior to aggregation the Orb2 PLD monomer remains mainly as a random coil in solution, ApCPEB PLD adopts a diversity of conformations comprising a-helical structures that evolve to coiled-coil species, indicating structural differences at the beginning of their amyloid assembly pathways. Conclusion: Our results indicate that divergent PLDs of CPEB proteins from different species retain the ability to form a generic amyloid-like fold through different assembly mechanisms. [ABSTRACT FROM AUTHOR]

The development of tailorable and biocompatible three‐dimensional (3D) substrates or molecular networks that reliably mimic the extracellular matrix (ECM) and influence cell behavior and growth in vitro is of increasing interest for cell‐based applications in the field of tissue engineering and regenerative medicine. In this context, we present a novel coiled coil‐based peptide that self‐assembles into a 3D‐α‐helical fibril network and functions as a self‐supporting hydrogel. By functionalizing distinct coiled‐coil peptides with cellular binding motifs or carbohydrate ligands (mannose), and by utilizing the multivalency and modularity of coiled‐coil assemblies, tailored artificial ECMs are obtained. Fibrillar network and ligand density, as well as ligand composition can readily be adjusted by changes in water content or peptide concentrations, respectively. Mesoscopic structure of these networks was assessed by rheology and small‐angle neutron scattering experiments. Initial cell viability studies using NIH/3T3 cells showed comparable or even superior cell viability using the presented artificial ECMs, compared to commercially available 3D‐cell culture scaffold Matrigel. The herein reported approach presents a reliable (low batch‐to‐batch variation) and modular pathway toward biocompatible and tailored artificial ECMs. [ABSTRACT FROM AUTHOR]