Search

During HIV-1 infection, the envelope glycoprotein subunit gp41 folds into a six-helix bundle structure (6HB) formed by the interaction between its N-terminal (NHR) and C-terminal (CHR) heptad-repeats, promoting viral and cell membranes fusion. A highly preserved, hydrophobic pocket (HP) on the NHR surface is crucial in 6HB formation and, therefore, HP-binding compounds constitute promising therapeutics against HIV-1. Here, we investigated the conformational and dynamic properties of the HP using a rationally designed single-chain protein (named covNHR) that mimics the gp41 NHR structure. We found that the fluorescent dye 8-anilino-naphtalene- 1-sulfonic acid (ANS) binds specifically to the HP, suggesting that ANS derivatives may constitute lead compounds to inhibit 6HB formation. ANS shows different binding modes to the HP, depending on the occupancy of other NHR pockets. Moreover, in presence of a CHR peptide bound to the N-terminal pockets in gp41, two ANS molecules can occupy the HP showing cooperative behavior. This binding mode was assessed using molecular docking and molecular dynamics simulations. The results show that the HP is conformationally flexible and connected allosterically to other NHR regions, which strongly influence the binding of potential ligands. These findings could guide the development of small-molecule HIV-1 inhibitors targeting the HP., Spanish State Research Agency, SRA/ 10.13039/501100011033 (grants BIO2016-76640-R and PID2019.107515RB.C21), ERDF/ESF

In bacteria, Lon is a large hexameric ATP-dependent protease that targets misfolded and also folded substrates, some of which are involved in cell division and survival of cellular stress. The N-terminal domain of Lon facilitates substrate recognition, but how the domains confer such activity has remained unclear. Here, we report the full-length structure of Lon protease from Thermus thermophilus at 3.9 Å resolution in a substrate-engaged state. The six N-terminal domains are arranged in three pairs, stabilized by coiled-coil segments and forming an additional channel for substrate sensing and entry into the AAA+ ring. Sequence conservation analysis and proteolysis assays confirm that this architecture is required for the degradation of both folded and unfolded substrates in bacteria.

Surface layer proteins perform multiple functions in prokaryotic cells, including cellular defense, cell-shape maintenance, and regulation of import and export of materials. However, mimicking the complex and dynamic behavior of such two-dimensional biochemical systems is challenging, and hence research has so far focused mainly on the design and manipulation of the structure and functionality of protein assemblies in solution. Motivated by the new opportunities that dynamic surface layer proteins may offer for modern technology, we herein demonstrate that immobilization of coiled coil proteins onto an inorganic surface facilitates complex behavior, manifested by reversible chemical reactions that can be rapidly monitored as digital surface readouts. Using multiple chemical triggers as inputs and several surface characteristics as outputs, we can realize reversible switching and logic gate operations that are read in parallel. Moreover, using the same coiled coil protein monolayers for derivatization of nanopores drilled into silicon nitride membranes facilitates control over ion and mass transport through the pores, thereby expanding the applicability of the dynamic coiled coil system for contemporary stochastic biosensing applications.

Transmembrane receptors are central components of the chemosensory systems by which motile bacteria detect and respond to chemical gradients. An attractant bound to the receptor periplasmic domain generates conformational signals that regulate a histidine kinase interacting with its cytoplasmic domain. Ligand-induced signaling through the periplasmic and transmembrane domains of the receptor involves a piston-like helical displacement, but the nature of this signaling through the >200 A four-helix coiled coil of the cytoplasmic domain had not yet been identified. We performed single-molecule Forster resonance energy transfer measurements on Escherichia coli aspartate receptor homodimers inserted into native phospholipid bilayers enclosed in nanodiscs. The receptors were labeled with fluorophores at diagnostic positions near the middle of the cytoplasmic coiled coil. At these positions, we found that the two N-helices of the homodimer were more distant, that is, less tightly packed and more dynamic than the companion C-helix pair, consistent with previous deductions that the C-helices form a stable scaffold and the N-helices are dynamic. Upon ligand binding, the scaffold pair compacted further, while separation and dynamics of the dynamic pair increased. Thus, ligand binding had asymmetric effects on the two helical pairs, shifting mean distances in opposite directions and increasing the dynamics of one pair. We suggest that this reflects a conformational change in which differential alterations to the packing and dynamics of the two helical pairs are coupled. These coupled changes could represent a previously unappreciated mode of conformational signaling that may well occur in other coiled-coil signaling proteins.

Copper nitrite reductase (CuNiR) is a copper enzyme that converts nitrite to nitric oxide and is an important part of the global nitrogen cycle in bacteria. The relatively simple CuHis3 binding site of the CuNiR active site has made it an enticing target for small molecule modeling and de novo protein design studies. We have previously reported symmetric CuNiR models within parallel three stranded coiled coil systems, with activities that span a range of three orders of magnitude. In this report, we investigate the same CuHis3 binding site within an antiparallel three helical bundle scaffold, which allows the design of asymmetric constructs. We determine that a simple CuHis3 binding site can be designed within this scaffold with enhanced activity relative to the comparable construct in parallel coiled coils. Incorporating more complex designs or repositioning this binding site can decrease this activity as much as 15 times. Comparing these constructs, we reaffirm a previous result in which a blue shift in the 1s to 4p transition energy determined by Cu(I) X-ray absorption spectroscopy is correlated with an enhanced activity within imidazole-based constructs. With this step and recent successful electron transfer site designs within this scaffold, we are one step closer to a fully functional de novo designed nitrite reductase.

The protein stromal interaction molecule 1 (STIM1) plays a pivotal role in mediating store-operated calcium entry (SOCE) into cells, which is essential for adaptive immunity. It acts as a calcium sensor in the endoplasmic reticulum (ER) and extends into the cytosol, where it changes from an inactive (tight) to an active (extended) oligomeric form upon calcium store depletion. NMR studies of this protein are challenging due to its membrane-spanning and aggregation properties. Therefore follow the divide-and-conquer approach, focusing on individual domains first is in order. The cytosolic part is predicted to have a large content of coiled-coil (CC) structure. We report the 1H, 13C, 15N chemical shift assignments of the CC3 domain. This domain is crucial for the stabilisation of the tight quiescent form of STIM1 as well as for activating the ORAI calcium channel by direct contact, in the extended active form. Supplementary Information The online version contains supplementary material available at 10.1007/s12104-021-10042-7.

Coiled-coil domain-containing 124 (CCDC124) is a recently discovered ribosome-binding protein conserved in eukaryotes. CCDC124 has regulatory functions on the mediation of reversible ribosomal hibernation and translational recovery by direct attachment to large subunit ribosomal protein uL5, 25S rRNA backbone, and tRNA binding P/A-site major groove. Moreover, it independently mediates cell division and cellular stress response by facilitating cytokinetic abscission and disulfide stress-dependent transcriptional regulation, respectively. However, the structural characterization and intracellular physiological status of CCDC124 remain unknown. In this study, we employed advanced in silico protein modeling and characterization tools to generate a native-like tertiary structure of CCDC124 and examine the disorder, low sequence complexity, and aggregation propensities, as well as high-order dimeric/oligomeric states. Subsequently, dimerization of CCDC124 was investigated with co-immunoprecipitation (CO-IP) analysis, immunostaining, and a recent live-cell protein-protein interaction method, bimolecular fluorescence complementation (BiFC). Results revealed CCDC124 as a highly disordered protein consisting of low complexity regions at the N-terminus and an aggregation sequence (151-IAVLSV-156) located in the middle region. Molecular docking and post-docking binding free energy analyses highlighted a potential involvement of V153 residue on the generation of high-order dimeric/oligomeric structures. Co-IP, immunostaining, and BiFC analyses were used to further confirm the dimeric state of CCDC124 predominantly localized at the cytoplasm. In conclusion, our findings revealed in silico structural characterization and in vivo subcellular physiological state of CCDC124, suggesting low complexity regions located at the N-terminus of disordered CCDC124 may regulate the formation of aggregates or high-order dimeric/oligomeric states. This article is protected by copyright. All rights reserved.

TMEM120A (Transmembrane protein 120A) was recently identified as a mechanical pain sensing ion channel named as TACAN, while its homologue TMEM120B has no mechanosensing property1. Here, we report the cryo-EM structures of both human TMEM120A and TMEM120B. The two structures share the same dimeric assembly, mediated by extensive interactions through the transmembrane domain (TMD) and the N-terminal coiled coil domain (CCD). However, the nearly identical structures cannot provide clues for the difference in mechanosensing between TMEM120A and TMEM120B. Although TMEM120A could mediate conducting currents in a bilayer system, it does not mediate mechanical-induced currents in a heterologous expression system, suggesting TMEM120A is unlikely a mechanosensing channel. Instead, the TMDs of TMEM120A and TMEM120B resemble the structure of a fatty acid elongase, ELOVL7, indicating their potential role of an enzyme in lipid metabolism.

In bacteria, the co-chaperone GrpE acts as a nucleotide exchange factor and plays an important role in controlling the chaperone cycle of DnaK. The functional form of GrpE is an asymmetric dimer, consisting of a non-ideal coiled coil. Partial unfolding of this region during heat stress results in reduced nucleotide exchange and disrupts protein folding by DnaK. In this study, we elucidate the role of non-ideality in the coiled-coil domain of Escherichia coli GrpE in controlling its co-chaperone activity. The presence of a four-residue stutter introduces nonheptad periodicity in the GrpE coiled coil, resulting in global structural changes in GrpE and regulating its interaction with DnaK. Introduction of hydrophobic residues at the stutter core increased the structural stability of the protein. Using an in vitro FRET assay, we show that the enhanced stability of GrpE resulted in an increased affinity for DnaK. However, these mutants were unable to support bacterial growth at 42°C in a grpE-deleted E. coli strain. This work provides valuable insights into the functional role of a stutter in GrpE in regulating the DnaK-chaperone cycle during heat stress. More generally, our findings illustrate how stutters in a coiled-coil domain regulate structure-function trade-off in proteins.

Swallow, a 62 kDa multidomain protein, is required for the proper localization of several mRNAs involved in the development of Drosophila oocytes. The dimerization of Swallow depends on a 71-residue self-association domain in the center of the protein sequence, and is significantly stabilized by a binding interaction with dynein light chain (LC8). Here, we detail the use of solution-state nuclear magnetic resonance spectroscopy to characterize the structure of this self-association domain, thereby establishing that this domain forms a parallel coiled-coil and providing insight into how the stability of the dimerization interaction is regulated.

We previously reported a family of hydrocarbon-stapled peptides designed to interact with the epidermal growth factor receptor (EGFR) juxtamembrane (JM) segment, blocking its ability to form a coiled coil dimer that is essential for receptor activation. These hydrocarbon-stapled peptides, most notably E1S, decreased the proliferation of cell lines that express wild-type EGFR (H2030 and A431) as well as those expressing the oncogenic mutants EGFR L858R (H3255) and L858R/T790M (H1975). Although our previous investigations provided evidence that E1S interacted with EGFR directly, the location and details of these interactions were not established. Here we apply biochemical and cross-linking mass spectrometry tools to better define the interactions between E1S and EGFR. Taken with previously reported structure-activity relationships, our results support a model in which E1S interacts simultaneously with both the JM and the C-lobe of the activator kinase, effectively displacing the JM of the receiver kinase. Our results also reveal potential interactions between E1S and the N-terminal region of the C-terminal tail. We propose a model in which E1S inhibits EGFR by both mimicking and inhibiting JM coiled coil formation. This model could be used to design novel, allosteric (and perhaps nonpeptidic) EGFR inhibitors.

Plant nucleotide-binding, leucine-rich repeat receptors (NLRs) perceive pathogen avirulence effectors and activate defense responses. Nucleotide-binding, leucine-rich repeat receptors are classified into coiled-coil (CC)-containing and Toll/interleukin-1 receptor (TIR)-containing NLRs. Recent advances suggest that NLR CC domains often function in signaling activation, especially for induction of cell death. In this review, we outline our current understanding of NLR CC domains, including their diversity/classification and structure, their roles in cell death induction, disease resistance, and interaction with other proteins. Furthermore, we provide possible directions for future work.

Coiled-coil protein origami (CCPO) polyhedra are designed self-assembling nanostructures constructed from coiled coil (CC)-forming modules connected into a single chain. For testing new CCPO building modules, simpler polyhedra could be used that should maintain most features relevant to larger scaffolds. We show the design and characterization of nanoscale single-chain triangles, composed of six concatenated parallel CC dimer-forming segments connected by flexible linker peptides. The polypeptides self-assembled in bacteria in agreement with the design, and the shape of the polypeptides was confirmed with small-angle X-ray scattering. Fusion with split-fluorescent protein domains was used as a functional assay in bacteria, based on the discrimination between the correctly folded and misfolded nanoscale triangles comprising correct, mismatched, or truncated modules. This strategy was used to evaluate the optimal size of linkers between CC segments which comprised eight amino acid residues.

To date, most research into the inhibition of oncogenic transcriptional regulator, Activator Protein 1 (AP-1), has focused on heterodimers of cJun and cFos. However, the Fra1 homologue remains an important cancer target. Here we describe library design coupled with computational and intracellular screening as an effective methodology to derive an antagonist that is selective for Fra1 relative to Jun counterparts. To do so the isCAN computational tool was used to rapidly screen >75 million peptide library members, narrowing the library size by >99.8% to one accessible to intracellular PCA selection. The resulting 131 072-member library was predicted to contain high quality binders with both a high likelihood of target engagement, while simultaneously avoiding homodimerization and off-target interaction with Jun homologues. PCA screening was next performed to enrich those members that meet these criteria. In particular, optimization was achieved via inclusion of options designed to generate the potential for compromised intermolecular contacts in both desired and non-desired species. This is an often-overlooked prerequisite in the conflicting design requirement of libraries that must be selective for their target in the context of a range of alternative potential interactions. Here we demonstrate that specificity is achieved via a combination of both hydrophobic and electrostatic contacts as exhibited by the selected peptide (Fra1W). In vitro analysis of the desired Fra1–Fra1W interaction further validates high Fra1 affinity (917 nM) yet selective binding relative to Fra1W homodimers or affinity for cJun. The isCAN → PCA based multidisciplinary approach provides a robust screening pipeline in generating target-specific hits, as well as new insight into rational peptide design in the search for novel bZIP family inhibitors., Here we describe library design coupled with computational and intracellular screening as an effective methodology to derive an antagonist that is selective for Fra1 relative to Jun counterparts.

The envelope glycoprotein, termed the spike protein, of severe acute respiratory syndrome coronavirus (SARS-CoV) is known to mediate viral entry. Similar to other class 1 viral fusion proteins, the heptad repeat regions of SARS-CoV spike are thought to undergo conformational changes from a prefusion form to a subsequent post-fusion form that enables fusion of the viral and host membranes. Recently, the structure of a post-fusion form of SARS-CoV spike, which consists of isolated domains of heptad repeats 1 and 2 (HR1 and HR2), has been determined by x-ray crystallography. To date there is no structural information for the prefusion conformations of SARS-CoV HR1 and HR2. In this work we present the NMR structure of the HR2 domain (residues 1141-1193) from SARS-CoV (termed S2-HR2) in the presence of the co-solvent trifluoroethanol. We find that in the absence of HR1, S2-HR2 forms a coiled coil symmetric trimer with a complex molecular mass of 18 kDa. The S2-HR2 structure, which is the first example of the prefusion form of coronavirus envelope, supports the current model of viral membrane fusion and gives insight into the design of structure-based antagonists of SARS.

[Image: see text] Antibiotic-resistant microbes have become a global health threat. New delivery systems that enhance the efficacy of antibiotics and/or overcome the resistances can help combat them. In this context, we present FF03, a fibril-forming α-helical coiled-coil peptide that functions as an efficient scaffold for the multivalent presentation of the weakly cationic antimicrobial peptide (AMP) IN4. The resulting IN4-decorated FF03 coiled-coil fibrils (FF03 + IN4) are nonhemolytic and noncytotoxic and show enhanced antimicrobial activity relative to unconjugated IN4 and standard antibiotics against several bacterial strains. Scanning electron microscopy analysis shows that FF03 + IN4 nanofibers disrupt methicillin-resistant Staphylococcus aureus membranes, indicating a surface-level mode of action. Furthermore, transmission electron microscopy and circular dichroism studies indicate that decoration of the FF03 scaffold with IN4 does not alter the secondary-structure propensity or fibril-forming properties of FF03. Thus, the approach reported herein provides a new peptidic scaffold for the multivalent presentation of AMPs to obtain nonhemolytic and noncytotoxic antimicrobial systems with improved efficacy relative to the unconjugated AMP analogues.

Heme‐nitric oxide/oxygen binding (H‐NOX) domains bind gaseous ligands for signal transduction in organisms spanning prokaryotic and eukaryotic kingdoms. In the bioluminescent marine bacterium Shewanella woodyi (Sw), H‐NOX proteins regulate quorum sensing and biofilm formation. In higher animals, soluble guanylyl cyclase (sGC) binds nitric oxide with an H‐NOX domain to induce cyclase activity and regulate vascular tone, wound healing and memory formation. sGC also binds stimulator compounds targeting cardiovascular disease. The molecular details of stimulator binding to sGC remain obscure but involve a binding pocket near an interface between H‐NOX and coiled‐coil domains. Here, we report the full NMR structure for CO‐ligated Sw H‐NOX in the presence and absence of stimulator compound IWP‐051, and its backbone dynamics. Nonplanar heme geometry was retained using a semi‐empirical quantum potential energy approach. Although IWP‐051 binding is weak, a single binding conformation was found at the interface of the two H‐NOX subdomains, near but not overlapping with sites identified in sGC. Binding leads to rotation of the subdomains and closure of the binding pocket. Backbone dynamics are similar across both domains except for two helix‐connecting loops, which display increased dynamics that are further enhanced by compound binding. Structure‐based sequence analyses indicate high sequence diversity in the binding pocket, but the pocket itself appears conserved among H‐NOX proteins. The largest dynamical loop lies at the interface between Sw H‐NOX and its binding partner as well as in the interface with the coiled coil in sGC, suggesting a critical role for the loop in signal transduction.

Motivation Coiled coils are widespread protein domains involved in diverse processes ranging from providing structural rigidity to the transduction of conformational changes. They comprise two or more α-helices that are wound around each other to form a regular supercoiled bundle. Owing to this regularity, coiled-coil structures can be described with parametric equations, thus enabling the numerical representation of their properties, such as the degree and handedness of supercoiling, rotational state of the helices, and the offset between them. These descriptors are invaluable in understanding the function of coiled coils and designing new structures of this type. The existing tools for such calculations require manual preparation of input and are therefore not suitable for the high-throughput analyses. Results To address this problem, we developed SamCC-Turbo, a software for fully automated, per-residue measurement of coiled coils. By surveying Protein Data Bank with SamCC-Turbo, we generated a comprehensive atlas of ∼50 000 coiled-coil regions. This machine learning-ready dataset features precise measurements as well as decomposes coiled-coil structures into fragments characterized by various degrees of supercoiling. The potential applications of SamCC-Turbo are exemplified by analyses in which we reveal general structural features of coiled coils involved in functions requiring conformational plasticity. Finally, we discuss further directions in the prediction and modeling of coiled coils. Availability and implementation SamCC-Turbo is available as a web server (https://lbs.cent.uw.edu.pl/samcc_turbo) and as a Python library (https://github.com/labstructbioinf/samcc_turbo), whereas the results of the Protein Data Bank scan can be browsed and downloaded at https://lbs.cent.uw.edu.pl/ccdb. Supplementary information Supplementary data are available at Bioinformatics online.

The crystal structure of the N-terminal membrane anchoring domain of mycobacterial DivIVA/Wag31 reveals a filament-compatible ‘dimer-of-dimers’ assembly state. The results suggest that, in addition to lipid binding, the N-terminal of Wag31 can participate in self-assembly to form filamentous structures., 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.

Coronin proteins are widely expressed among eukaryotic organisms. Most coronins consist of a WD repeat domain followed by a C-terminal coiled coil. Dictyostelium discoideum expresses a single short coronin coronin A, which has been implicated in both actin modulation as well as multicellular differentiation. Whether coronin A's coiled coil is important for functionality, as well as the oligomeric state of coronin A is not known. Here, we show that the coiled-coil domain in Dictyostelium coronin A functions in homodimerization, is dispensable for coronin A stability and localization but essential for multicellular differentiation. These results allow a better understanding of the role for the coiled-coil domain of coronin A in oligomerization and demonstrate that its presence is essential for multicellular differentiation.

The relationship between GTP binding and hydrolysis on the one hand and septin filament assembly and function on the other has yet to be fully elucidated. The crystal structures of SEPT3 subgroup members bound to either GDP or to a GTP analogue shed light on this question by indicating how the squeezing of one of the inter-subunit interfaces may lead to the exposure of a polybasic region important for membrane association., Human septins 3, 9 and 12 are the only members of a specific subgroup of septins that display several unusual features, including the absence of a C-terminal coiled coil. This particular subgroup (the SEPT3 septins) are present in rod-like octameric protofilaments but are lacking in similar hexameric assemblies, which only contain representatives of the three remaining subgroups. Both hexamers and octamers can self-assemble into mixed filaments by end-to-end association, implying that the SEPT3 septins may facilitate polymerization but not necessarily function. These filaments frequently associate into higher order complexes which associate with biological membranes, triggering a wide range of cellular events. In the present work, a complete compendium of crystal structures for the GTP-binding domains of all of the SEPT3 subgroup members when bound to either GDP or to a GTP analogue is provided. The structures reveal a unique degree of plasticity at one of the filamentous interfaces (dubbed NC). Specifically, structures of the GDP and GTPγS complexes of SEPT9 reveal a squeezing mechanism at the NC interface which would expel a polybasic region from its binding site and render it free to interact with negatively charged membranes. On the other hand, a polyacidic region associated with helix α5′, the orientation of which is particular to this subgroup, provides a safe haven for the polybasic region when retracted within the interface. Together, these results suggest a mechanism which couples GTP binding and hydrolysis to membrane association and implies a unique role for the SEPT3 subgroup in this process. These observations can be accounted for by constellations of specific amino-acid residues that are found only in this subgroup and by the absence of the C-terminal coiled coil. Such conclusions can only be reached owing to the completeness of the structural studies presented here.

The M protein is the major surface-associated virulence factor of Group A Streptococcus (GAS). M protein is predicted to be a parallel α-helical coiled coil, as identified by a heptad repeat pattern with small, hydrophobic residues at the first and fourth positions. To date, over 200 M variants have been identified, however a common trend among M proteins is that they contain regions characterized as non-ideal coiled coils, with both destabilizing residues in the putative dimer interface and skips and insertions in the heptad pattern. Crystallographic studies on M1 revealed two distinct registers at the fibrinogen (Fg) binding sites: a Fg non-binding register 1 and a Fg-binding register 2. These conformations are related by a rotation of one helical face, resulting in the exposure of adjacent faces of the coiled coil. It was previously found that mutations stabilizing register 1 understandably reduced interaction with Fg. However, we now report that mutations stabilizing register 2 also reduce Fg binding. These results suggest that dynamic motion in M1 is required for association with Fg, in a “lure-and-catch” model. This requirement for binding offers a novel explanation for the presence of non-ideal coiled coil structure in M1, a feature that is evolutionarily conserved among M proteins in general.The M1 protein contains three fibrinogen binding sites, one in the A region and two in the B repeats. Both binding sites in the B repeats are essential to establish a supramolecular complex capable of activating neutrophils. However the function of the binding site in the A region is still unknown. In addition to contributing to immune activation, the M1-fibrinogen interaction contributes to immune evasion. Surface recruitment of fibrinogen has been shown to protect against complement deposition and subsequent phagocytosis. We investigated the role of each fibrinogen binding site in conferring protection from phagocytosis.

Motivation Protein–protein interactions are central to all biological processes. One frequently observed mode of such interactions is the α-helical coiled coil (CC). Thus, an ability to extract, visualize and analyze CC interfaces quickly and without expert guidance would facilitate a wide range of biological research. In 2001, we reported Socket, which locates and characterizes CCs in protein structures based on the knobs-into-holes (KIH) packing between helices in CCs. Since then, studies of natural and de novo designed CCs have boomed, and the number of CCs in the RCSB PDB has increased rapidly. Therefore, we have updated Socket and made it accessible to expert and nonexpert users alike. Results The original Socket only classified CCs with up to six helices. Here, we report Socket2, which rectifies this oversight to identify CCs with any number of helices, and KIH interfaces with any of the 20 proteinogenic residues or incorporating nonnatural amino acids. In addition, we have developed a new and easy-to-use web server with additional features. These include the use of NGL Viewer for instantly visualizing CCs, and tabs for viewing the sequence repeats, helix-packing angles and core-packing geometries of CCs identified and calculated by Socket2. Availability and implementation Socket2 has been tested on all modern browsers. It can be accessed freely at http://coiledcoils.chm.bris.ac.uk/socket2/home.html. The source code is distributed using an MIT licence and available to download under the Downloads tab of the Socket2 home page.

De novo-designed protein domains are increasingly being applied in biotechnology, cell biology, and synthetic biology. Therefore, it is imperative that these proteins be robust to superficial changes; i.e., small changes to their amino acid sequences should not cause gross structural changes. In turn, this allows properties such as stability and solubility to be tuned without affecting structural attributes like tertiary fold and quaternary interactions. Reliably designed proteins with predictable behaviors may then be used as scaffolds to incorporate function, e.g., through the introduction of features for small-molecule, metal, or macromolecular binding, and enzyme-like active sites. Generally, achieving this requires the starting protein fold to be well understood. Herein, we focus on designing α-helical coiled coils, which are well studied, widespread, and often direct protein-protein interactions in natural systems. Our initial investigations reveal that a previously designed parallel, homotetrameric coiled coil, CC-Tet, is not robust to sequence changes that were anticipated to maintain its structure. Instead, the alterations switch the oligomeric state from tetramer to trimer. To improve the robustness of designed homotetramers, additional sequences based on CC-Tet were produced and characterized in solution and by X-ray crystallography. Of these updated sequences, one is robust to truncation and to changes in surface electrostatics; we call this CC-Tet*. Variants of the general CC-Tet* design provide a set of homotetrameric coiled coils with unfolding temperatures in the range from 40 to >95 °C. We anticipate that these will be of use in applications requiring robust and well-defined tetramerization domains.

The heterotrimeric kinesin-2 consists of two distinct motor subunits and an accessory protein, KAP, which binds to the coiled-coil stalk domains and one of the tail domains of the motor subunits. Genetic studies revealed that KAP is essential for the kinesin-2 functions in cilia, flagella, and axon. However, the structural significance of the KAP binding on kinesin-2 assembly and stability is not known. Here, using the Fluorescence Lifetime assay, we show that DmKAP binding selectively reduces the distance between the C-terminal ends of Drosophila kinesin-2 stalk heterodimer. Insertion of a missense mutation (E551K) in the Drosophila kinesin-2α stalk fragment, which was shown to reduce the structural dynamics of the stalk heterodimer earlier, also reduced the distances at both the N- and C-terminal ends of the stalk heterodimer independent of DmKAP. The zipping effect, particularly at the N-terminal end of the mutant stalk heterodimer, is further enhanced in the presence of DmKAP. Together, these results suggest that the KAP binding could alter the structural dynamics of kinesin-2 stalk heterodimer at the C-terminal end and stabilize the association between the stalk domains.

Protein-protein interactions of cellular importance are mediated by coiled coils (CCs), the ubiquitous structural motif formed by the association of two or more α-helices in a knobs into holes manner. Coronins, actin-associated multi-functional proteins that possess distinct cytoskeleton-dependent and independent functions, oligomerize through their C-terminal CC domain. The structure of the L. donovani coronin CC domain (LdCoroCC; PDB ID 5CX2) revealed, in addition to a novel topology and architecture, an inherent asymmetry, with one of the helices of the 4-helix bundle axially shifted (~2 turns). The structural analysis identified that steric hindrance by Ile 486, Leu 493 and Met 500 as the cause for this asymmetry. To experimentally validate this hypothesis and to better understand the sequence-structure relationship in CCs, these amino acids have been mutated (I486A, L493A, M500V and the double mutant I486A-L493A) and characterized. Thermal CD studies suggest that the I486A and M500V mutants have comparable Tm values to LdCoroCC, while the other mutants have lower melting temperatures. The mutant crystal structures (I486A, M500V and the double mutant) retain the 'ade' core packing as LdcoroCC. While the M500V structure is similar to LdCoroCC, the I486A and the I486A-L493A structures show an asymmetry to symmetry transition. This study reveals crucial role of residues at position 'a' in coiled-coil domain play an important role in stabilizing the asymmetry in LdCoroCC, which might be necessary pursue specific biological function(s) inside the Leishmania.

Golgins are an abundant class of peripheral membrane proteins of the Golgi. These very long (50–400 nm) rod-like proteins initially capture cognate transport vesicles, thus enabling subsequent SNARE-mediated membrane fusion. Here, we explore the hypothesis that in addition to serving as vesicle tethers, Golgins may also possess the capacity to phase separate and, thereby, contribute to the internal organization of the Golgi. GM130 is the most abundant Golgin at the cis Golgi. Remarkably, overexpressed GM130 forms liquid droplets in cells analogous to those described for numerous intrinsically disordered proteins with low complexity sequences, even though GM130 is neither low in complexity nor intrinsically disordered. Virtually pure recombinant GM130 also phase-separates into dynamic, liquid-like droplets in close to physiological buffers and at concentrations similar to its estimated local concentration at the cis Golgi.

C-di-GMP, bis-(3′-5′)-cyclic dimeric guanosine monophosphate, is a key signaling molecule that regulates many important physiological processes in bacteria. C-di-GMP is synthesized by diguanylate cyclase (DGC) containing the homodimeric GGDEF domain. There are many uncharacterized hypothetical proteins annotated as a putative DGC in bacteria including hyperthermophiles; however, their structures still remain unexplored. Here, we solved the crystal structure of the GGDEF-like domain of Tm0107 protein from Thermotoga maritima at a resolution of 2.1 A, which shares sequence similarities with DGC proteins in other bacteria. Tm0107 consists of an N-terminal coiled-coil and C-terminal GGDEF-like domain. We showed that the GGDEF-like domain of Tm0107 exists as monomer in solution and is structurally similar to other GGDEF domains. Two zinc ions are coordinated at the interface between two Tm0107 monomers. Based on our measurements of the Stokes radii of Tm0107 by analytical gel filtration, we propose a dimer model of Tm0107 containing both the N-terminal coiled coil and C-terminal GGDEF-like domains. Based on the model, Tm0107 forms a homodimer in a manner different compared to other structurally characterized DGC proteins. These results provide useful structural information about putative DGC proteins containing protein sequences similar to that of Tm0107, which is widely conserved in hyperthermophiles.

Short peptide-based inhibition of fusion remains an attractive goal in antihuman immunodeficiency virus (HIV) research based on its potential for the development of technically and economically desirable antiviral agents. Herein, we report the use of the dithiol bisalkylation reaction to generate a series of m-xylene thioether-stapled 22-residue α-helical peptides that have been identified as fusion inhibitors targeting HIV-1 glycoprotein 41 (gp41). The peptide sequence is based on the helix-zone binding domain of the gp41 C-terminal heptad repeat region. We found that one of these stapled peptides, named hCS6ERE, showed promising inhibitory potency against HIV-1 Env-mediated cell-cell fusion and viral replication at a level comparable to the clinically used 36-mer peptide T20. Furthermore, combining hCS6ERE with a fusion inhibitor having a different target site, such as HP23, produced synergistic anti-HIV-1 activity. Collectively, our study offers new insight into the design of anti-HIV peptides with short sequences.

How nitric oxide (NO) activates its primary receptor, α1/β1 soluble guanylyl cyclase (sGC or GC‐1), remains unknown. Likewise, how stimulatory compounds enhance sGC activity is poorly understood, hampering development of new treatments for cardiovascular disease. NO binding to ferrous heme near the N‐terminus in sGC activates cyclase activity near the C‐terminus, yielding cGMP production and physiological response. CO binding can also stimulate sGC, but only weakly in the absence of stimulatory small‐molecule compounds, which together lead to full activation. How ligand binding enhances catalysis, however, has yet to be discovered. Here, using a truncated version of sGC from Manduca sexta, we demonstrate that the central coiled‐coil domain, the most highly conserved region of the ~150,000 Da protein, not only provides stability to the heterodimer but is also conformationally active in signal transduction. Sequence conservation in the coiled coil includes the expected heptad‐repeating pattern for coiled‐coil motifs, but also invariant positions that disfavor coiled‐coil stability. Full‐length coiled coil dampens CO affinity for heme, while shortening of the coiled coil leads to enhanced CO binding. Introducing double mutation αE447L/βE377L, predicted to replace two destabilizing glutamates with leucines, lowers CO binding affinity while increasing overall protein stability. Likewise, introduction of a disulfide bond into the coiled coil results in reduced CO affinity. Taken together, we demonstrate that the heme domain is greatly influenced by coiled‐coil conformation, suggesting communication between heme and catalytic domains is through the coiled coil. Highly conserved structural imperfections in the coiled coil provide needed flexibility for signal transduction.

Lipid packing has a strong influence on the formation and structural dynamics of cell membranes. Techniques to modulate lipid packing may thus enable modification of cellular functions and events. An 18-residue amphiphilic helical peptide derived from the N-terminal segment of epsin-1 (EpN18) is reported to induce positive membrane curvature and to loosen lipid packing in the cell membrane. In this study, it is shown that EpN18, crosslinked to a leucine-zipper peptide K4, is recruited to the cell surface by interacting with a cell-surface-expressed E3 leucine-zipper segment. Cell-surface tethering markedly enhanced loosening of lipid packing, which led to the promotion of membrane translocation of octaarginine. The loosening of lipid packing by EpN18 was also confirmed by analyzing the generalized polarization value with a membrane-environment-sensitive dye, 2-hydroxy-3-{2-[(2-hydroxyethyl)dimethylamino]ethyl}-4-{2-[6-(dibutylamino)-2-naphthyl]ethenyl}pyridiniumdibromide (di-4-ANEPPDHQ). This approach thus shows promise for the control of lipid packing and related cellular events.

Mutations in the NPHP1 gene, coding for human nephrocystin-1 (NPHP1), cause the autosomal recessive disease nephronophthisis, the most common cause of end-stage renal disease in children and adolescents. The function and structure of NPHP1 are still poorly characterized. NPHP1 presents a modular structure well in keeping with its role as an adaptor protein: it harbors an SH3 domain flanked by two glutamic acid-rich regions and a conserved C-terminal nephrocystin homology domain (NHD). Similar to other NPHP protein family members, its N-terminus contains a putative coiled-coil domain (NPHP1CC) that is supposed to play an important role in NPHP1 self-association and/or protein-protein interactions. Structural studies proving its structure and its oligomerization state are still lacking. Here we demonstrate that NPHP1CC is monomeric in solution and unexpectedly folds into an autonomous domain forming a three-stranded antiparallel coiled coil suitable for protein-protein interactions. Notably, we found that the NPHP1CC shares remarkable structural similarities with the three-stranded coiled coil of the BAG domain protein family, which is known to mediate the anti-apoptotic function of these proteins, suggesting a possible similar role for NPHP1CC. In agreement with this hypothesis, we show that in the context of the full-length protein the NPHP1CC is fundamental to regulate resistance to apoptotic stimuli.

Tropomyosin is the major regulator of the thin filament. In striated muscle its function is to bind troponin complex and control the access of myosin heads to actin in a Ca2+-dependent manner. It also participates in the maintenance of thin filament length by regulation of tropomodulin and leiomodin, the pointed end-binding proteins. Because the size of the overlap between actin and myosin filaments affects the number of myosin heads which interact with actin, the filament length is one of the determinants of force development. Numerous point mutations in genes encoding tropomyosin lead to single amino acid substitutions along the entire length of the coiled coil that are associated with various types of cardiomyopathy and skeletal muscle disease. Specific regions of tropomyosin interact with different binding partners; therefore, the mutations affect diverse tropomyosin functions. In this review, results of studies on mutations in the genes TPM1 and TPM3, encoding Tpm1.1 and Tpm3.12, are described. The paper is particularly focused on mutation-dependent alterations in the mechanisms of actin-myosin interactions and dynamics of the thin filament at the pointed end.

Ezrin plays an important role in the development and progression of human esophageal squamous cell carcinoma (ESCC), providing a link between the cortical actin cytoskeleton and the plasma membrane to govern membrane structure and protrusions. However, the mechanism by which ezrin is activated still remains unknown in ESCC. Here, we identify a novel interaction between ezrin and heat shock protein family B (small) member 1 (HSPB1) in ESCC cells by mass spectroscopy and co-immunoprecipitation. HSPB1 only interacts with inactive ezrin and binds to the α-helical coiled coil region of ezrin. Knockdown of HSPB1 resulted to the decline of phosphorylation at ezrin Thr567, markedly suppressing the ability of ezrin to bind to the actin cytoskeleton and migration of ESCC cells. Furthermore, neither the constitutively active phosphomimetic ezrin T567D, nor inactivated ezrin T567A could restore cell migration following HSPB1 knockdown. Low HSPB1 expression was associated with favorable overall survival of ESCC patients. Taken together, HSPB1, as an important partner, participates in the activation of ezrin and merits further evaluation as a novel therapeutic target against human ESCC.

Laminins are large cell-adhesive glycoproteins that are required for the formation and function of basement membranes in all animals. Structural studies by electron microscopy in the early 1980s revealed a cross-shaped molecule, which subsequently was shown to consist of three distinct polypeptide chains. Crystallographic studies since the mid-1990s have added atomic detail to all parts of the laminin heterotrimer. The three short arms of the cross are made up of continuous arrays of disulphide-rich domains. The globular domains at the tips of the short arms mediate laminin polymerization; the surface regions involved in this process have been identified by structure-based mutagenesis. The long arm of the cross is an α-helical coiled coil of all three chains, terminating in a cell-adhesive globular region. The molecular basis of cell adhesion to laminins has been revealed by recent structures of heterotrimeric integrin-binding fragments and of a laminin fragment bound to the carbohydrate modification of dystroglycan. The structural characterization of the laminin molecule is essentially complete, but we still have to find ways of imaging native laminin polymers at molecular resolution.

In situ investigations in living cell membranes are important to elucidate the dynamic behaviors of membrane proteins in complex biomembrane environments. Protein-specific labeling is a key technique for the detection of a target protein by fluorescence imaging. The use of post-translational labeling methods using a genetically encodable tag and synthetic probes targeting the tag offer a smaller label size, labeling with synthetic fluorophores, and precise control of the labeling ratio in multicolor labeling compared with conventional genetic fusions with fluorescent proteins. This review focuses on tag-probe labeling studies for live-cell analysis of membrane proteins based on heterodimeric peptide pairs that form coiled-coil structures. The robust and simple peptide-peptide interaction enables not only labeling of membrane proteins by noncovalent interactions, but also covalent crosslinking and acyl transfer reactions guided by coiled-coil assembly. A number of studies have demonstrated that membrane protein behaviors in live cells, such as internalization of receptors and the oligomeric states of various membrane proteins (G-protein-coupled receptors, epidermal growth factor receptors, influenza A M2 channel, and glycopholin A), can be precisely analyzed using coiled-coil labeling, indicating the potential of this labeling method in membrane protein research.

Early Endosomal Antigen 1 (EEA1) is a key protein in endosomal trafficking and is implicated in both autoimmune and neurological diseases. The C-terminal FYVE domain of EEA1 binds endosomal membranes, which contain phosphatidylinositol-3-phosphate (PI(3)P). Although it is known that FYVE binds PI(3)P specifically, it has not previously been described of how FYVE attaches and binds to endosomal membranes. In this study, we employed both coarse-grained (CG) and atomistic (AT) molecular dynamics (MD) simulations to determine how FYVE binds to PI(3)P-containing membranes. CG-MD showed that the dominant membrane binding mode resembles the crystal structure of EEA1 FYVE domain in complex with inositol-1,3-diphospate (PDB ID 1JOC). FYVE, which is a homodimer, binds the membrane via a hinge mechanism, where the C-terminus of one monomer first attaches to the membrane, followed by the C-terminus of the other monomer. The estimated total binding energy is ~70 kJ/mol, of which 50–60 kJ/mol stems from specific PI(3)P-interactions. By AT-MD, we could partition the binding mode into two types: (i) adhesion by electrostatic FYVE-PI(3)P interaction, and (ii) insertion of amphipathic loops. The AT simulations also demonstrated flexibility within the FYVE homodimer between the C-terminal heads and coiled-coil stem. This leads to a dynamic model whereby the 200 nm long coiled coil attached to the FYVE domain dimer can amplify local hinge-bending motions such that the Rab5-binding domain at the other end of the coiled coil can explore an area of 0.1 μm2 in the search for a second endosome with which to interact., Author summary Peripheral proteins bind to the surfaces of specific membranes within eukaryotic cells and thereby coordinate dynamic interactions between sub-cellular compartments. The FYVE domain of EEA1 recognizes the membranes of endosomes by binding to the anionic headgroup of a specific lipid, phosphatidylinositol-3-phosphate (PI(3)P), which is present in those membranes. We use molecular dynamics simulations at two different resolutions (coarse-grained and all atom) to define the mechanism whereby a dimeric FYVE domain binds to model membranes containing PI(3)P. Hinge-bending flexibility between the bound FYVE domain and the coiled coil stalk to which it is attached is observed. In the intact EEA1 molecule the coiled coil is 200 nm long. This ‘amplifies’ the flexibility observed at the FYVE hinge end such that the other end of the EAA1 protein can sweep out an area of about 0.1 μm2 in its search for a second endosome with which to interact. In this way EAA1 acts as a long stalk with two ‘sticky’ ends to help draw together two specific membrane surfaces.

MicroRNAs (miRs) have been demonstrated to be associated with multiple processes in the development and progression of human malignancies. Previous studies have observed aberrant downregulation of miR‑144 in several types of cancer, including osteosarcoma. However, the function of miR‑144 and the underlying mechanism in osteosarcoma remain to be elucidated. The present study indicated that miR‑144 was markedly downregulated in osteosarcoma tissues and cell lines compared with that in the normal controls. Restoration of miR‑144 significantly inhibited cell proliferation, migration and invasion of MG‑63 osteosarcoma cells. In addition, Rho‑associated coiled‑coil containing protein kinase 1 (ROCK1) was identified as a novel target of miR‑144 in MG‑63 osteosarcoma cells. Furthermore, knockdown of ROCK1 suppressed the proliferation, migration and invasion of MG‑63 osteosarcoma cells to a similar extent to the effects of miR‑144 overexpression. In addition, the mRNA expression of ROCK1 was increased in osteosarcoma tissues and was negatively correlated with the expression of miR‑144. In conclusion, the results of the present study suggested that miR‑144 acts as a tumor suppressor by targeting ROCK1 in osteosarcoma.

Apolipoprotein L-I (APOL1) is a channel-forming effector of innate immunity. The common human APOL1 variant G0 provides protection against infection with certain Trypanosoma and Leishmania parasite species, but it cannot protect against the trypanosomes responsible for human African trypanosomiasis. Human APOL1 variants G1 and G2 protect against human-infective trypanosomes but also confer a higher risk of developing chronic kidney disease. Trypanosome-killing activity is dependent on the ability of APOL1 to insert into membranes at acidic pH and form pH-gated cation channels. We previously mapped the channel's pore-lining region to the C-terminal domain (residues 332–398) and identified a membrane-insertion domain (MID, residues 177–228) that facilitates acidic pH-dependent membrane insertion. In this article, we further investigate structural determinants of cation channel formation by APOL1. Using a combination of site-directed mutagenesis and targeted chemical modification, our data indicate that the C-terminal heptad-repeat sequence (residues 368–395) is a bona fide leucine zipper domain (ZIP) that is required for cation channel formation as well as lysis of trypanosomes and mammalian cells. Using protein-wide cysteine-scanning mutagenesis, coupled with the substituted cysteine accessibility method, we determined that, in the open channel state, both the N-terminal domain and the C-terminal ZIP domain are exposed on the intralumenal/extracellular side of the membrane and provide evidence that each APOL1 monomer contributes four transmembrane domains to the open cation channel conformation. Based on these data, we propose an oligomeric topology model in which the open APOL1 cation channel is assembled from the coiled-coil association of C-terminal ZIP domains.

The catalytically-active inclusion bodies (CatIBs) represent a promising strategy for immobilizing enzyme without additional carriers and chemicals, which has aroused great attention in academic and industrial communities. In this work, we discovered two natural parallel right-handed coiled-coil tetramer peptides from PDB database by a structural mining strategy. The two self-assembling peptides, NSPdoT from rotavirus and HVdoT from human Vasodilator-stimulated phosphoprotein, efficiently induced the CatIBs formation of a (R)-Hydroxynitrile lyase from Arabidopsis thaliana (AtHNL) in Escherichia coli cells. This is convenient to simultaneously purify and immobilize the target proteins as biocatalysts. As expected, HVdoT-AtHNL and NSPdoT-AtHNL possessed drastically increased tolerance toward lower pH values, which will be very critical to synthesize cyanohydrins under acidic condition for suppressing the non-enzymatic side reaction. In addition. AtHNL-CatIBs are produced at high yield in host cells as bioactive microparticles, which exhibited high thermal and pH stabilities. Therefore, the CatIBs method represent a promising application for the immobilization of enzymes in the biocatalysis field.

Templated chemistry offers the prospect of addressing specificity challenges occurring in bioconjugation reactions. Here, we show two peptide-templated amide-bond forming reactions that enable the concurrent labelling of two different membrane proteins with two different peptide nucleic acid (PNA) barcodes. The reaction system is based on the mutually selective coiled coil interaction between two thioester-linked PNA–peptide conjugates and two cysteine peptides serving as genetically encoded peptide tags. Orthogonal coiled coil templated covalent labelling is highly specific, quantitative and proceeds within a minute. To demonstrate the usefulness, we evaluated receptor internalisation of two membranous receptors EGFR (epidermal growth factor) and ErbB2 (epidermal growth factor receptor 2) by first staining PNA-tagged proteins with fluorophore–DNA conjugates and then erasing signals from non-internalized receptors via toehold-mediated strand displacement., A pair of orthogonal coiled coils templates highly specific live cell bioconjugation of two different proteins. PNA tagging and hybridisation with fluorophore–DNA reporters enables rapid dual receptor internalisation analysis of EGFR and ErbB2.

Funder: A.G. Levintis Foundation, PRK1 is a member of the protein kinase C-related kinase (PRK) family of serine/threonine kinases and a downstream effector of Rho GTPases. PRK1 has three N-terminal Homology Region 1 (HR1) domains (HR1a, HR1b and HR1c), which form antiparallel coiled coils that interact with Rho family GTPases. PRK1 also has a C2-like domain that targets it to the plasma membrane and a kinase domain, which is a member of the protein kinase C superfamily. PRK1 is involved in cytoskeletal regulation, cell adhesion, cell cycle progression and the immune response, and is implicated in cancer. There is currently no structural information for the HR1c domain. The 1H, 15N and 13C NMR backbone and sidechain resonance assignment of the HR1c domain presented here forms the basis for this domain's structural characterisation. This work will also enable studies of interactions between the three HR1 domains in an effort to obtain structural insight into the regulation of PRK1 activity.

Active DNA transposases like the Drosophila P element transposase (DmTNP) undergo oligomerization as a prerequisite for transposition. Human THAP9 (hTHAP9) is a catalytically active but functionally uncharacterized homologue of DmTNP. Here we report (using co-immunoprecipitation, pull down, colocalization, and proximity ligation assays) that both full length and truncated hTHAP9 (corresponding to amino-terminal DNA binding and predicted coiled coil domains) undergo homo-oligomerization, predominantly in the nuclei of HEK293T cells. Interestingly, the oligomerization is shown to be partially mediated by DNA. However, mutating the leucines (either individually or together) or deleting the predicted coiled coil region did not significantly affect oligomerization. Thus, we highlight the importance of DNA and the amino-terminal regions of hTHAP9 for their ability to form higher-order oligomeric states. We also report that Hcf-1, THAP1, THAP10, and THAP11 are possible protein interaction partners of hTHAP9. Elucidating the functional relevance of the different putative oligomeric state(s) of hTHAP9 would help answer questions about its interaction partners as well as its unknown physiological roles.

Fibrinogen is an abundant blood plasma protein that, inter alia, participates in blood coagulation. It polymerizes to form a fibrin clot that is among the major components of the thrombus. Fibrinogen reactions with various reactive metabolites may induce post-translational modifications (PTMs) into the protein structure that affect the architecture and properties of fibrin clots. We reviewed the previous literature to find the positions of PTMs of fibrinogen. For 7 out of 307 reported PTMs, we used molecular dynamics simulations to characterize their effect on the behavior of the fibrinogen coiled-coil domain. Interactions of the γ-coil with adjacent chains give rise to π-helices in Aα and Bβ chains of even unmodified fibrinogen. The examined PTMs suppress fluctuations of the γ-coil, which may affect the fibrinolysis and stiffness of the fibrin fibers. Citrullination of AαR104 and oxidations of γP70 and γP76 to glutamic semialdehyde unfold the α-helical structure of Aα and Bβ chains. Oxidation of γM78 to methionine sulfoxide induces the formation of an α-helix in the γ-coil region. Our findings suggest that certain PTMs alter the protein secondary structure. Thus, the altered protein structure may indicate the presence of PTMs in the molecule and consequently of certain metabolites within the system.

Biochemical studies require large quantities of proteins, which are typically obtained using bacterial overexpression. However, the folding machinery in bacteria is inadequate for expressing many mammalian proteins, which additionally undergo posttranslational modifications (PTMs) that bacteria, yeast, or insect cells cannot perform. Many proteins also require native N- and C-termini and cannot tolerate extra tag amino acids for proper function. Tropomyosin (Tpm), a coiled coil protein that decorates most actin filaments in cells, requires both native N- and C-termini and PTMs, specifically N-terminal acetylation (Nt-acetylation), to polymerize along actin filaments. Here, we describe a new method that combines native protein expression in human cells with an intein-based purification tag that can be precisely removed after purification. Using this method, we expressed several nonmuscle Tpm isoforms (Tpm1.6, Tpm1.7, Tpm2.1, Tpm3.1, Tpm3.2, and Tpm4.2) and the muscle isoform Tpm1.1. Proteomics analysis revealed that human-cell-expressed Tpms present various PTMs, including Nt-acetylation, Ser/Thr phosphorylation, Tyr phosphorylation, and Lys acetylation. Depending on the Tpm isoform (humans express up to 40 Tpm isoforms), Nt-acetylation occurs on either the initiator methionine or on the second residue after removal of the initiator methionine. Human-cell-expressed Tpms bind F-actin differently than their Escherichia coli-expressed counterparts, with or without N-terminal extensions intended to mimic Nt-acetylation, and they can form heterodimers in cells and in vitro. The expression method described here reveals previously unknown features of nonmuscle Tpms and can be used in future structural and biochemical studies with Tpms and other proteins, as shown here for α-synuclein.

The proteasome is a large protease complex that degrades many different cellular proteins. In eukaryotes, the 26S proteasome contains six different subunits of the ATPases associated with diverse cellular activities family, Rpt1–Rpt6, which form a hexameric ring as part of the base subcomplex that drives unfolding and translocation of substrates into the proteasome core. Archaeal proteasomes contain only a single Rpt-like ATPases associated with diverse cellular activities ATPase, the proteasome-activating nucleotidase, which forms a trimer of dimers. A key proteasome-activating nucleotidase proline residue (P91) forms cis- and trans-peptide bonds in successive subunits around the ring, allowing efficient dimerization through upstream coiled coils. However, the importance of the equivalent Rpt prolines for eukaryotic proteasome assembly was unknown. Here we showed that the equivalent proline is highly conserved in Rpt2, Rpt3, and Rpt5, and loosely conserved in Rpt1, in deeply divergent eukaryotes. Although in no case was a single Pro-to-Ala substitution in budding yeast strongly deleterious to growth, the rpt5–P76A mutation decreased levels of the protein and induced a mild proteasome assembly defect. Moreover, the rpt2–P103A, rpt3–P93A, and rpt5–P76A mutations all caused synthetic defects when combined with deletions of specific proteasome base assembly chaperones. The rpt2–P103A rpt5–P76A double mutant had uniquely strong growth defects attributable to defects in proteasome base formation. Several Rpt subunits in this mutant formed aggregates that were cleared, at least in part, by Hsp42 chaperone-mediated protein quality control. We propose that the conserved Rpt linker prolines promote efficient 26S proteasome base assembly by facilitating specific ATPase heterodimerization.

A promising strategy to neutralize HIV-1 is to target the gp41 spike subunit to block membrane fusion with the cell. We previously designed a series of single-chain proteins (named covNHR) that mimic the trimeric coiled-coil structure of the gp41 N-terminal heptad repeat (NHR) region and potently inhibit HIV-1 cell infection by avidly binding the complementary C-terminal heptad repeat (CHR) region. These proteins constitute excellent tools to understand the structural and thermodynamic features of this therapeutically important interaction. Gp41, as with many coiled-coil proteins, contains in core positions of the NHR trimer several highly conserved, buried polar residues, the role of which in gp41 structure and function is unclear. Here we produced three covNHR mutants by substituting each triad of polar residues for the canonical isoleucine. The mutants preserve their helical structure and show an extremely increased thermal stability. However, increased hydrophobicity enhances their self-association. Calorimetric analyses show a marked influence of mutations on the binding thermodynamics of CHR-derived peptides. The mutations do not affect however the in vitro HIV-1 inhibitory activity of the proteins. The results support a role of buried core polar residues in maintaining structural uniqueness and promoting an energetic coupling between conformational stability and NHR–CHR binding.

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.

Mimicking the extracellular matrix (ECM) continues to be a goal in the field of regenerative medicine. Herein, we report a modified trimeric GCN4 coiled-coil sequence containing three ligands for metal ions specifically positioned for crosslinked assembly (TriCross). In the presence of metal ions, TriCross assembles into a three-dimensional (3D) matrix with significant cavities to accommodate cells. The matrix was found to be stable in media with serum, and mild removal of the metal leads to disassembly. By assembling TriCross with a suspension of cells in media, the matrix encapsulates cells during the assembly process leading to high cell viability. Further disassembly under mild conditions allows for the release of cells from the scaffold. As such, this peptide-based material displays many of the characteristics necessary for successful 3D cell culture.