Coiled-coil Protein Research Articles

A Transgenic Mouse Model of Eccentric Left Ventricular Hypertrophy With Preserved Ejection Fraction Exhibits Alterations in the Autophagy-Lysosomal Pathway.

The ubiquitin-proteasome system (UPS) and the autophagy-lysosomal pathway (ALP) are the main proteolytic systems involved in cellular homeostasis. Since cardiomyocytes, as terminally differentiated cells, lack the ability to share damaged proteins with their daughter cells, they are especially reliant on these protein degradation systems for their proper function. Alterations of the UPS and ALP have been reported in a wide range of cardiac diseases, including cardiomyopathies. In this study, we determined whether the UPS and ALP are altered in a mouse model of eccentric left ventricular (LV) hypertrophy expressing both cyclin T1 and Gαq under the control of the cardiac-specific α-myosin heavy chain promoter (double transgenic; DTG). Compared to wild-type (WT) littermates, DTG mice showed higher end-diastolic (ED) LV wall thicknesses and diameter with preserved ejection fraction (EF). The cardiomyopathic phenotype was further confirmed by an upregulation of the fetal gene program and genes associated with fibrosis as well as a downregulation of genes involved in Ca2+ handling. Likewise, higher NT-proBNP levels were detected in DTG mice. Investigation of the UPS showed elevated steady-state levels of (poly)ubiquitinated proteins without alterations of all proteasomal activities in DTG mice. Evaluation of ALP key marker revealed a mixed pattern with higher protein levels of microtubule-associated protein 1 light chain 3 beta (LC3)-I and lysosomal-associated membrane protein-2, lower protein levels of beclin-1 and FYVE and coiled-coil domain-containing protein 1 (FYCO1) and unchanged protein levels of p62/SQSTM1 in DTG mice when compared to WT. At transcriptional level, a > 1.2-fold expression was observed for Erbb2, Hdac6, Lamp2, Nrg1, and Sqstm1, while a < 0.8-fold expression was revealed for Fyco1 in DTG mice. The results related to the ALP suggested overall a repression of the ALP during the initiation process, but an induction of the ALP at the level of autophagosome-lysosome fusion and the delivery of ubiquitinated cargo to the ALP for degradation.

BBLN-1 is essential for intermediate filament organization and apical membrane morphology

Epithelial tubes are essential components of metazoan organ systems that control the flow of fluids and the exchange of materials between body compartments and the outside environment. The size and shape of the central lumen confer important characteristics to tubular organs and need to be carefully controlled. Here, we identify the small coiled-coil protein BBLN-1 as a regulator of lumen morphology in the C.elegans intestine. Loss of BBLN-1 causes the formation of bubble-shaped invaginations of the apical membrane into the cytoplasm of intestinal cells and abnormal aggregation of the subapical intermediate filament (IF) network. BBLN-1 interacts with IF proteins and localizes to the IF network in an IF-dependent manner. The appearance of invaginations is a result of the abnormal IF aggregation, indicating a direct role for the IF network in maintaining lumen homeostasis. Finally, we identify bublin (BBLN) as the mammalian ortholog of BBLN-1. When expressed in the C.elegans intestine, BBLN recapitulates the localization pattern of BBLN-1 and can compensate for the loss of BBLN-1 in early larvae. In mouse intestinal organoids, BBLN localizes subapically, together with the IF protein keratin 8. Our results therefore may have implications for understanding the role of IFs in regulating epithelial tube morphology in mammals.

Extremely Thermostabilizing Core Mutations in Coiled-Coil Mimetic Proteins of HIV-1 gp41 Produce Diverse Effects on Target Binding but Do Not Affect Their Inhibitory Activity.

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.

Single-molecule mechanics of synthetic aromatic amide helices: Ultrafast and robust non-dissipative winding

Summary Because of proteins’ many degrees of conformational freedom, programming protein folding dynamics, overall elasticity, and motor functions remains an elusive objective. Instead, smaller and simpler objects, such as synthetic foldamers, may be amenable to design. However, little is known about their mechanical performance. Here, we show that reducing molecular size may not compromise mechanical properties. We report that helical aromatic oligoamides as small as 1 nm possess outstanding elasticity and outperform most natural helices. Using single-molecule force spectroscopy, we characterize their folding trajectories and intermediate states. We show that they cooperatively and reversibly unwind at high forces. They extend up to 3.8 times their original length and rewind against considerable forces on a timescale of 10 μs. Pulling and relaxing cycles follow the same trace up to a very high loading rate, indicating that the mechanical energy accumulated during the stretching does not dissipate and is immediately reusable.

TACC3 is a prognostic biomarker for kidney renal clear cell carcinoma and correlates with immune cell infiltration and T cell exhaustion.

Growing evidence has demonstrated that transforming acidic coiled-coil protein 3 (TACC3), a member of the TACC family, may be involved in regulating cell mitosis, transcription, and tumorigenesis. However, the role of TACC3 in kidney renal clear cell carcinoma (KIRC) remains unknown. In this study, multiple databases were used to determine the pattern of TACC3 in KIRC. We found that high TACC3 expression was associated with poor overall survival (OS) in stage I, II, IV and grade 3 KIRC patients. Univariate and multivariate Cox regression analyses showed that TACC3 was an independent risk factor for OS among KIRC patients. Moreover, TACC3 expression correlated with immune cell infiltration levels of B cells, T cells (CD8+, CD4+, follicular helper, regulatory and gamma delta), total and resting natural killer cells, total and activated dendritic cells, and resting mast cells. Furthermore, T cell exhaustion markers, such as PD1, CTLA4, LAG3 and TIM-3 were highly expressed in TACC3 overexpressing tissues. In addition, GSEA analysis revealed that the role of TACC3 in KIRC may be closely linked to immune-associated pathways. Therefore, our study reveals that TACC3 is a prognostic biomarker for OS among KIRC patients and may be associated with immune cell infiltration and T cell exhaustion.

Free-Standing Photocrosslinked Protein Polymer Hydrogels for Sustained Drug Release.

The fabrication of protein hydrogels consisting of different properties and functional motifs is critical in the development of protein-based materials for biomedical applications. Here, we report the design and characterization of a triblock protein polymer, CEC, composed of two different self-assembling domains derived from elastin protein (E) and coiled-coil protein (C), photopolymerized with a NHS-diazirine (D) crosslinker into a CEC-D hydrogel. The optimal photocrosslinker concentration and exposure time is determined to fabricate a free-standing hydrogel. Upon increasing the concentration of the CEC-D monomer and environmental temperature, the CEC-D hydrogel's conformation decreases in helical content from 58.0% to 44.8% and increases in β-content from 25.9% to 38.1%. These gels experience 55 ± 6% protein erosion from the free-standing gel in 13 days as the gel films gradually decrease in size. The swelling ratio of 12 ± 1% denotes that the gel has a swelling ability comparable to other protein hydrogels. These photocrosslinked CEC-D hydrogels can be employed for drug delivery with high encapsulation and 14 ± 2% release of curcumin into the supernatant in a week long study. Overall, the photocrosslinked CEC-D hydrogels exhibit stability, swelling ability, and sustained release of drug.

Epitope and Paratope Mapping by HDX-MS Combined with SPR Elucidates the Difference in Bactericidal Activity of Two Anti-NadA Monoclonal Antibodies.

Characterization of antigen-antibody interactions is crucial for understanding antibody-mediated protection against pathogens, biopharmaceutical development, as well as evaluation of the immune response post vaccination. Bexsero is a multicomponent vaccine against Neisseria meningitidis serogroup B in which one of the key vaccine antigens is Neisserial adhesin A (NadA), a trimeric coiled-coil protein. Two NadA-specific monoclonal antibodies (mAbs) isolated from Bexsero-vaccinated individuals have been shown to have similar binding affinity and appear to recognize a similar antigen region, yet only one of the mAbs is bactericidal. In this study, we use hydrogen/deuterium exchange mass spectrometry (HDX-MS) to perform an in-depth study of the interaction of the two mAbs with NadA antigen using a combined epitope and paratope mapping strategy. In addition, we use surface plasmon resonance (SPR) to investigate the stoichiometry of the binding of the two mAbs to NadA. While epitope mapping only identifies a clear binding impact of one of the mAbs on NadA, the paratope mapping analyses shows that both mAbs are binding to NadA through several complementarity determining regions spanning both heavy and light chains. Our results highlight the advantage of combined epitope and paratope mapping HDX-MS experiments and supporting biochemical experiments to characterize antigen-antibody interactions. Through this combined approach, we provide a rationale for how the binding stoichiometry of the two mAbs to the trimeric NadA antigen can explain the difference in bactericidal activity of the two mAbs.

The Golgin Protein RUD3 Regulates Fusarium graminearum Growth and Virulence.

Golgins are coiled-coil proteins that play prominent roles in maintaining the structure and function of the Golgi complex. However, the role of golgin proteins in phytopathogenic fungi remains poorly understood. In this study, we functionally characterized the Fusarium graminearum golgin protein RUD3, a homolog of ScRUD3/GMAP-210 in Saccharomyces cerevisiae and mammalian cells. Cellular localization observation revealed that RUD3 is located in the cis-Golgi. Deletion of RUD3 caused defects in vegetative growth, ascospore discharge, deoxynivalenol (DON) production, and virulence. Moreover, the Δrud3 mutant showed reduced expression of tri genes and impairment of the formation of toxisomes, both of which play essential roles in DON biosynthesis. We further used green fluorescent protein (GFP)-tagged SNARE protein SEC22 (SEC22-GFP) as a tool to study the transport between the endoplasmic reticulum (ER) and Golgi and observed that SEC22-GFP was retained in the cis-Golgi in the Δrud3 mutant. RUD3 contains the coiled coil (CC), GRAB-associated 2 (GA2), GRIP-related Arf binding (GRAB), and GRAB-associated 1 (GA1) domains, which except for GA1, are indispensable for normal localization and function of RUD3, whereas only CC is essential for normal RUD3-RUD3 interaction. Together, these results demonstrate how the golgin protein RUD3 mediates retrograde trafficking in the ER-to-Golgi pathway and is necessary for growth, ascospore discharge, DON biosynthesis, and pathogenicity in F. graminearumIMPORTANCEFusarium head blight (FHB) caused by the fungal pathogen Fusarium graminearum is an economically important disease of wheat and other small grain cereal crops worldwide, and limited effective control strategies are available. A better understanding of the regulation mechanisms of F. graminearum development, deoxynivalenol (DON) biosynthesis, and pathogenicity is therefore important for the development of effective control management of this disease. Golgins are attached via their extreme carboxy terminus to the Golgi membrane and are involved in vesicle trafficking and organelle maintenance in eukaryotic cells. In this study, we systematically characterized a highly conserved Golgin protein, RUD3, and found that it is required for vegetative growth, ascospore discharge, DON production, and pathogenicity in F. graminearum Our findings provide a comprehensive characterization of the golgin family protein RUD3 in plant-pathogenic fungus, which could help to identify a new potential target for effective control of this devastating disease.

Loss of Coiled-Coil Protein Cep55 Impairs Neural Stem Cell Abscission and Results in p53-Dependent Apoptosis in Developing Cortex.

To build the brain, embryonic neural stem cells (NSCs) tightly regulate their cell divisions, undergoing a polarized form of cytokinesis that is poorly understood. Cytokinetic abscission is mediated by the midbody to sever the daughter cells at the apical membrane. In cell lines, the coiled-coil protein Cep55 was reported to be required for abscission. Mutations of Cep55 in humans cause a variety of cortical malformations. However, its role in the specialized divisions of NSCs is unclear. Here, we elucidate the roles of Cep55 in abscission and brain development. KO of Cep55 in mice causes abscission defects in neural and non-neural cell types, and postnatal lethality. The brain is disproportionately affected, with severe microcephaly at birth. Quantitative analyses of abscission in fixed and live cortical NSCs show that Cep55 acts to increase the speed and success rate of abscission, by facilitating ESCRT recruitment and timely microtubule disassembly. However, most NSCs complete abscission successfully in the absence of Cep55 Those that fail show a tissue-specific response: binucleate NSCs and neurons elevate p53, but binucleate fibroblasts do not. This leads to massive apoptosis in the brain, but not other tissues. Double KO of both p53 and Cep55 blocks apoptosis but only partially rescues Cep55-/- brain size. This may be because of the persistent NSC cell division defects and p53-independent premature cell cycle exit. This work adds to emerging evidence that abscission regulation and error tolerance vary by cell type and are especially crucial in neural stem cells as they build the brain.SIGNIFICANCE STATEMENT During brain growth, embryonic neural stem cells (NSCs) must divide many times. In the last step of cell division, the daughter cell severs its connection to the mother stem cell, a process called abscission. The protein Cep55 is thought to be essential for recruiting proteins to the mother-daughter cell connection to complete abscission. We find that Cep55 mutants have very small brains with disturbed structure, but almost normal size bodies. NSC abscission can occur, but it is slower than normal, and failures are increased. Furthermore, NSCs that do fail abscission activate a signal for programmed cell death, whereas non-neural cells do not. Blocking this signal only partly restores brain growth, showing that regulation of abscission is crucial for brain development.

Study on CCDC69 interfering with the prognosis of patients with breast cancer through PPAR signal pathway.

Coiled-coil domain-containing protein 69 (CCDC69) is a novel gene and limited knowledge in known in breast cancer. In the present study, we aimed to explore the relationship between CCDC69 and breast cancer, demonstrate the clinicopathological significance and prognostic role of CCDC69 in breast cancer, and analyze the possible mechanism of CCDC69 affecting the prognosis of breast cancer. First, from GEO database, TIMER, GEPIA, and OncoLnc, we selected CCDC69 as the potential gene which closely involved in breast cancer progression. Next, by real-time PCR detection, the expression of CCDC69 in breast cancer tissue was notably lower than that in normal breast tissues (p=0.0002). In addition, our immunohistochemistry indicated that the positive expression rate of CCDC69 in the triple-negative breast cancer (TNBC) was lower than that in the non-TNBC (p=0.0362), and it was negatively correlated with the expression of Ki67 (p=0.001). Further enrichment analysis of CCDC69 and the similar genes performed on FunRich3.1.3 revealed that these genes were significantly associated with fat differentiation, and most of them were related to peroxisome proliferator-activated receptor (PPAR) signal pathway. Collectively, our findings suggest that CCDC69 is down regulated in breast cancer tissue especially in TNBC which has higher malignant grade and poorer clinical prognosis.

A specific basal body linker protein provides the connection function for basal body inheritance in trypanosomes

Centrioles and basal bodies (CBBs) are found in physically linked pairs, and in mammalian cells intercentriole connections (G1-G2 tether and S-M linker) regulate centriole duplication and function. In trypanosomes BBs are not associated with the spindle and function in flagellum/cilia nucleation with an additional role in mitochondrial genome (kinetoplast DNA [kDNA]) segregation. Here, we describe BBLP, a BB/pro-BB (pBB) linker protein in Trypanosoma brucei predicted to be a large coiled-coil protein conserved in the kinetoplastida. Colocalization with the centriole marker SAS6 showed that BBLP localizes between the BB/pBB pair, throughout the cell cycle, with a stronger signal in the old flagellum BB/pBB pair. Importantly, RNA interference (RNAi) depletion of BBLP leads to a conspicuous splitting of the BB/pBB pair associated only with the new flagellum. BBLP RNAi is lethal in the bloodstream form of the parasite and perturbs mitochondrial kDNA inheritance. Immunogold labeling confirmed that BBLP is localized to a cytoskeletal component of the BB/pBB linker, and tagged protein induction showed that BBLP is incorporated de novo in both new and old flagella BB pairs of dividing cells. We show that the two aspects of CBB disengagement-loss of orthogonal orientation and ability to separate and move apart-are consistent but separable events in evolutionarily diverse cells and we provide a unifying model explaining centriole/BB linkage differences between such cells.

Nrf1 Knock-Down in the Hypothalamic Paraventricular Nucleus Alleviates Hypertension Through Intervention of Superoxide Production-Removal Balance and Mitochondrial Function.

Oxidative stress in the hypothalamic paraventricular nucleus (PVN) contributes greatly to the development of hypertension. The recombinant nuclear respiratory factor 1 (Nrf1) regulates the transcription of several genes related to mitochondrial respiratory chain function or antioxidant expression, and thus may be involved in the pathogenesis of hypertension. Here we show that in the two-kidney, one-clip (2K1C) hypertensive rats the transcription level of Nrf1 was elevated comparing to the normotensive controls. Knocking down of Nrf1 in the PVN of 2K1C rats can significantly reduce their blood pressure and level of plasma norepinephrine (NE). Analysis revealed significant reduction of superoxide production level in both whole cell and mitochondria, along with up-regulation of superoxide dismutase 1 (Cu/Zn-SOD), NAD(P)H: quinone oxidoreductase 1 (NQO1), thioredoxin-dependent peroxiredoxin 3 (Prdx3), cytochrome c (Cyt-c) and glutathione synthesis rate-limiting enzyme (glutamyl-cysteine ligase catalytic subunit (Gclc) and modifier subunit (Gclm)), and down-regulation of cytochrome c oxidase subunit VI c (Cox6c) transcription after Nrf1 knock-down. In addition, the reduced ATP production and elevated mitochondrial membrane potential in the PVN of 2K1C rats were reinstated with Nrf1 knock-down, together with restored expression of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α), mitochondrial transcription factor A (Tfam), coiled-coil myosin-like BCL2-interacting protein (Beclin1), and Mitofusin 1 (Mfn1), which are related to the mitochondrial biogenesis, fusion, and autophagy. Together, the results indicate that the PVN Nrf1 is associated with the development of 2K1C-induced hypertension, and Nrf1 knock-down in the PVN can alleviate hypertension through intervention of mitochondrial function and restorement of the production-removal balance of superoxide.

Self-assembly and regulation of protein cages from pre-organised coiled-coil modules

Coiled-coil protein origami (CCPO) is a modular strategy for the de novo design of polypeptide nanostructures. CCPO folds are defined by the sequential order of concatenated orthogonal coiled-coil (CC) dimer-forming peptides, where a single-chain protein is programmed to fold into a polyhedral cage. Self-assembly of CC-based nanostructures from several chains, similarly as in DNA nanotechnology, could facilitate the design of more complex assemblies and the introduction of functionalities. Here, we show the design of a de novo triangular bipyramid fold comprising 18 CC-forming segments and define the strategy for the two-chain self-assembly of the bipyramidal cage from asymmetric and pseudo-symmetric pre-organised structural modules. In addition, by introducing a protease cleavage site and masking the interfacial CC-forming segments in the two-chain bipyramidal cage, we devise a proteolysis-mediated conformational switch. This strategy could be extended to other modular protein folds, facilitating the construction of dynamic multi-chain CC-based complexes.

Designed folding pathway of modular coiled-coil-based proteins

Natural proteins are characterised by a complex folding pathway defined uniquely for each fold. Designed coiled-coil protein origami (CCPO) cages are distinct from natural compact proteins, since their fold is prescribed by discrete long-range interactions between orthogonal pairwise-interacting coiled-coil (CC) modules within a single polypeptide chain. Here, we demonstrate that CCPO proteins fold in a stepwise sequential pathway. Molecular dynamics simulations and stopped-flow Förster resonance energy transfer (FRET) measurements reveal that CCPO folding is dominated by the effective intra-chain distance between CC modules in the primary sequence and subsequent folding intermediates, allowing identical CC modules to be employed for multiple cage edges and thus relaxing CCPO cage design requirements. The number of orthogonal modules required for constructing a CCPO tetrahedron can be reduced from six to as little as three different CC modules. The stepwise modular nature of the folding pathway offers insights into the folding of tandem repeat proteins and can be exploited for the design of modular protein structures based on a given set of orthogonal modules.

Overexpression of GSE1 Related to Trastuzumab Resistance in Gastric Cancer Cells.

Gastric cancer is one of the most prevalent human cancers with poor prognosis. Trastuzumab is a well-used targeted drug for gastric cancer with HER2 amplification. Trastuzumab resistance restrains the clinical use of trastuzumab. In this study, we reported human Gse1 coiled-coil protein (GSE1) promoted trastuzumab resistance in HER2-positive gastric cancer cells. Acquired trastuzumab-resistant gastric cancer cells overexpressed GSE1, and depletion of GSE1 decreased the trastuzumab resistance of trastuzumab-resistant gastric cancer cells. BCL-2 was a downstream gene positively regulated by GSE1 and also performed promoting the role of trastuzumab resistance in HER2-positive gastric cancer cells. A high level of GSE1 was associated with a high risk of tumor lymph node metastasis and higher clinical stage in HER2-positive gastric cancer patients. GSE1 was a potential target that could be used for HER2-positive gastric cancer therapy.

Biochemical Role of Phosphorylation of Serine-283 of Mammalian Tropomyosin Tpm1.1 (Alpha)

Tropomyosin is an alpha helical coiled-coil protein (284 amino acids per chain) having a regulatory function in the thin filament of the sarcomere. The major isoform of tropomyosin, Tpm 1.1 (alpha), in the skeletal and cardiac muscles of mammals and birds is phosphorylated at serine-283. The degree of phosphorylation is age-related; a high level is found in the mammalian fetal heart of approximately 0.7 mole per mole that drops to 0.3 in the adult. The current study is aimed at understanding the relationship between this particular covalent modification and the proteolytic susceptibility of tropomyosin and interaction with F-actin.

Giardia intestinalis coiled-coil cytolinker protein 259 interacts with actin and tubulin.

Giardia intestinalis is a human parasite that causes a diarrheal disease in developing countries. G. intestinalis has a cytoskeleton (CSK) composed of microtubules and microfilaments, and the Giardia genome does not code for the canonical CSK-binding proteins described in other eukaryotic cells. To identify candidate actin and tubulin cross-linking proteins, we performed a BLAST analysis of the Giardia genome using a spectraplakins consensus sequence as a query. Based on the highest BLAST score, we selected a 259-kDa sequence designated as a cytoskeleton linker protein (CLP259). The sequence was cloned in three fragments and characterized by immunoprecipitation, confocal microscopy, and mass spectrometry (MS). CLP259 was located in the cytoplasm in the form of clusters of thick rods and colocalized with actin at numerous sites and with tubulin in the median body. Immunoprecipitation followed by mass spectrometry revealed that CLP259 interacts with structural proteins such as giardins, SALP-1, axonemal, and eight coiled-coils. The vesicular traffic proteins detected were Mu adaptin, Vacuolar ATP synthase subunit B, Bip, Sec61 alpha, NSF, AP complex subunit beta, and dynamin. These results indicate that CLP259 in trophozoites is a CSK linker protein for actin and tubulin and could act as a scaffold protein driving vesicular traffic.

The Small GTPase Arf6 Functions as a Membrane Tether in a Chemically-Defined Reconstitution System.

Arf-family small GTPases are essential protein components for membrane trafficking in all eukaryotic endomembrane systems, particularly during the formation of membrane-bound, coat protein complex-coated transport carriers. In addition to their roles in the transport carrier formation, a number of Arf-family GTPases have been reported to physically associate with coiled-coil tethering proteins and multisubunit tethering complexes, which are responsible for membrane tethering, a process of the initial contact between transport carriers and their target subcellular compartments. Nevertheless, whether and how indeed Arf GTPases are involved in the tethering process remain unclear. Here, using a chemically-defined reconstitution approach with purified proteins of two representative Arf isoforms in humans (Arf1, Arf6) and synthetic liposomes for model membranes, we discovered that Arf6 can function as a bona fide membrane tether, directly and physically linking two distinct lipid bilayers even in the absence of any other tethering factors, whereas Arf1 retained little potency to trigger membrane tethering under the current experimental conditions. Arf6-mediated membrane tethering reactions require trans-assembly of membrane-anchored Arf6 proteins and can be reversibly controlled by the membrane attachment and detachment cycle of Arf6. The intrinsic membrane tethering activity of Arf6 was further found to be significantly inhibited by the presence of membrane-anchored Arf1, suggesting that the tethering-competent Arf6-Arf6 assembly in trans can be prevented by the heterotypic Arf1-Arf6 association in a cis configuration. Taken together, these findings lead us to postulate that self-assemblies of Arf-family small GTPases on lipid bilayers contribute to driving and regulating the tethering events of intracellular membrane trafficking.

Revealing biophysical properties of KfrA-type proteins as a novel class of cytoskeletal, coiled-coil plasmid-encoded proteins

BackgroundDNA binding KfrA-type proteins of broad-host-range bacterial plasmids belonging to IncP-1 and IncU incompatibility groups are characterized by globular N-terminal head domains and long alpha-helical coiled-coil tails. They have been shown to act as transcriptional auto-regulators.ResultsThis study was focused on two members of the growing family of KfrA-type proteins encoded by the broad-host-range plasmids, R751 of IncP-1β and RA3 of IncU groups. Comparative in vitro and in silico studies on KfrAR751 and KfrARA3 confirmed their similar biophysical properties despite low conservation of the amino acid sequences. They form a wide range of oligomeric forms in vitro and, in the presence of their cognate DNA binding sites, they polymerize into the higher order filaments visualized as “threads” by negative staining electron microscopy. The studies revealed also temperature-dependent changes in the coiled-coil segment of KfrA proteins that is involved in the stabilization of dimers required for DNA interactions.ConclusionKfrAR751 and KfrARA3 are structural homologues. We postulate that KfrA type proteins have moonlighting activity. They not only act as transcriptional auto-regulators but form cytoskeletal structures, which might facilitate plasmid DNA delivery and positioning in the cells before cell division, involving thermal energy.

Triangular in Vivo Self-Assembling Coiled-Coil Protein Origami.

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.