Regulation of polysome assembly on the endoplasmic reticulum by a coiled-coil protein, p180

A coiled-coil microtubule-bundling protein, p180, was originally identified as one of the ribosome receptor candidates on the rough endoplasmic reticulum (ER) and is highly expressed in secretory tissues. Recently, we reported that p180 plays crucial roles in upregulating collagen biosynthesis, mainly by facilitating ribosome association on the ER. Here, we provide evidence that p180 is required to form translationally active polysome/translocon complexes on the ER. Assembly of highly-developed polysomes on the ER was severely perturbed upon loss of p180. p180 associates with polysome/translocon complexes through multiple contact sites: it was coimmunoprecipitated with the translocon complex independently of ribosomes, while it can also bind to ribosomal large subunit specifically. The responsible domain of p180 for membrane polysome assembly was identified in the C-terminal coiled-coil region. The degree of ribosome occupation of collagen and fibronectin mRNAs was regulated in response to increased traffic demands. This effect appears to be exerted in a manner specific for a specified set of mRNAs. Collectively, our data suggest that p180 is required to form translationally active polysome/translocon complexes on the ER membrane, and plays a pivotal role in highly efficient biosynthesis on the ER membrane through facilitating polysome formation in professional secretory cells.     

Open-and-shut cases in coiled-coil assembly: alpha-sheets and alpha-cylinders

The coiled coil is a ubiquitous protein-folding motif. It generally is accepted that coiled coils are characterized by sequence patterns known as heptad repeats. Such patterns direct the formation and assembly of amphipathic alpha-helices, the hydrophobic faces of which interface in a specific manner first proposed by Crick and termed "knobs-into-holes packing". We developed software, SOCKET, to recognize this packing in protein structures. As expected, in a trawl of the protein data bank, we found examples of canonical coiled coils with a single contiguous heptad repeat. In addition, we identified structures with multiple, overlapping heptad repeats. This observation extends Crick's original postulate: Multiple, offset heptad repeats help explain assemblies with more than two helices. Indeed, we have found that the sequence offset of the multiple heptad repeats is related to the coiled-coil oligomer state. Here we focus on one particular sequence motif in which two heptad repeats are offset by two residues. This offset sets up two hydrophobic faces separated by approximately 150 degrees -160 degrees around the alpha-helix. In turn, two different combinations of these faces are possible. Either similar or opposite faces can interface, which leads to open or closed multihelix assemblies. Accordingly, we refer to these two forms as alpha-sheets and alpha-cylinders. We illustrate these structures with our own predictions and by reference to natural variants on these designs that have recently come to light.     

Real-time observation of coiled-coil domains and subunit assembly in intermediate filaments

We have utilized electron paramagnetic resonance spectroscopy to study secondary structure, subunit interaction, and molecular orientation of vimentin molecules within intact intermediate filaments and assembly intermediates. Spectroscopy data prove alpha-helical coiled-coil structures at individual amino acids 316-336 located in rod 2B. Analysis of positions 305, 309, and 312 identify this region as conforming to the helical pattern identified within 316-336 and thus demonstrates that, contrary to some previous predictions, this region is in an alpha-helical conformation. We show that by varying the position of the spin label, we can identify both intra- and inter-dimer interactions. With a label attached to the outside of the alpha-helix, we have been able to measure interactions between positions 348 of separate dimers as they align together in intact filaments, identifying the exact point of overlap. By mixing different spin-labeled proteins, we demonstrate that the interaction at position 348 is the result of an anti-parallel arrangement of dimers. This approach provides high resolution structural information (<2 nm resolution), can be used to identify molecular arrangements between subunits in an intact intermediate filament, and should be applicable to other noncrystallizable filamentous systems as well as to the study of protein fibrils.     

Laminin chain assembly by triple and double stranded coiled-coil structures.

We have previously provided evidence that laminin assembly occurs by the specific interaction of the alpha-helical domains of the A, B1, and B2 chains, located within the long arm of the molecule (Hunter, I., Schulthess, T., Bruch, M., Beck, K., and Engel, J. (1990) Eur. J. Biochem. 188, 205-211). Recent evidence for noncoordinate synthesis of the laminin chains, and in particular, the absence of the 400-kDa A chain from laminins produced by a number of cell types, has led us to examine the molecular mechanism of laminin assembly using the isolated A and B1-B2 chains of laminin fragment E8. E8A shows little tendency to self-associate, and when renatured from urea forms globular structures with little detectable alpha-helix. In contrast, E8B1-B2 renatures to form rod-like molecules, 30 nm in length. The rod-like structure, high alpha-helix content, and sharp thermal transition indicate that they are double stranded coiled coils. When mixed in equimolar amounts, E8A and E8B1-B2 renature to form molecules which are biochemically and ultrastructurally indistinguishable from native E8. If E8A and E8B1-B2 are renatured separately and mixed at a 1:1 molar ratio, they also form E8 molecules. These results suggest a mechanism of laminin assembly which involves the formation of a double coiled-coil B1-B2 intermediate with which the A chain subsequently interacts to form a triple coiled-coil laminin molecule. In addition, our results indicate that isoforms consisting of the B1 and B2 chains only would form stable "laminin-like" structures.     

Role of a novel coiled-coil domain-containing protein CCDC69 in regulating central spindle assembly

The formation of the central spindle (or the spindle midzone) is essential for cytokinesis in animal cells. In this study, we report that coiled-coil domain-containing protein 69 (CCDC69) is implicated in controlling the assembly of central spindles and the recruitment of midzone components. Exogenous expression of CCDC69 in HeLa cells interfered with microtubule polymerization and disrupted the formation of bipolar mitotic spindles. Endogenous CCDC69 proteins were localized to the central spindle during anaphase. RNA interference (RNAi)-mediated knockdown of CCDC69 led to the formation of aberrant central spindles and disrupted the localization of midzone components such as aurora B kinase, protein regulator of cytokinesis 1 (PRC1), MgcRacGAP/HsCYK-4, and polo-like kinase 1 (Plk1) at the central spindle. Aurora B kinase was found to bind to CCDC69 and this binding depended on the coiled-coil domains at the C-terminus of CCDC69. Further, disruption of aurora B function in HeLa cells by treatment with a small chemical inhibitor led to the mislocalization of CCDC69 at the central spindle. Our results indicate that CCDC69 acts as a scaffold to regulate the recruitment of midzone components and the assembly of central spindles.     

Regulation of alpha-helical coiled-coil dimerization in chicken skeletal muscle light meromyosin.

The dimerization specificity of the light meromyosin (LMM) domain of chicken neonatal and adult myosin isoforms was analyzed by metal chelation chromatography. Our results show that neonatal and adult LMMs associate preferentially, although not exclusively, as homodimeric coiled-coils. Using chimeric LMM constructs combining neonatal and adult sequences, we observed that a stretch of 183 amino acids of sequence identity at the N terminus of the LMM was sufficient to allow the adult LMM to dimerize in a non-selective manner. In contrast, sequence identity in the remaining C-terminal 465 amino acids had only a modest effect on the dimerization selectivity of the adult isoform. Sequence identity at the N terminus also promoted dimerization of the neonatal LMM to a greater degree than sequence identity at the C terminus. However, the N terminus had only a partial effect on the dimerization specificity of the neonatal sequence, and residues distributed throughout the LMM were capable of affecting dimerization selectivity of this isoform. These results indicated that dimerization preference of the neonatal and adult isoforms was affected to a different extent by sequence identity at a given region of the LMM.     

Role of a novel coiled-coil domain-containing protein CCDC69 in regulating central spindle assembly

The formation of the central spindle (or the spindle midzone) is essential for cytokinesis in animal cells. In this study, we report that coiled-coil domain-containing protein 69 (CCDC69) is implicated in controlling the assembly of central spindles and the recruitment of midzone components. Exogenous expression of CCDC69 in HeLa cells interfered with microtubule polymerization and disrupted the formation of bipolar mitotic spindles. Endogenous CCDC69 proteins were localized to the central spindle during anaphase. RNA interference (RNAi)-mediated knockdown of CCDC69 led to the formation of aberrant central spindles and disrupted the localization of midzone components such as aurora B kinase, protein regulator of cytokinesis 1 (PRC1), MgcRacGAP/HsCYK-4, and polo-like kinase 1 (Plk1) at the central spindle. Aurora B kinase was found to bind to CCDC69 and this binding depended on the coiled-coil domains at the C-terminus of CCDC69. Further, disruption of aurora B function in HeLa cells by treatment with a small chemical inhibitor led to the mislocalization of CCDC69 at the central spindle. Our results indicate that CCDC69 acts as a scaffold to regulate the recruitment of midzone components and the assembly of central spindles.Key words: CCDC69, aurora B, Plk1, central spindles, midzone components, cytokinesis     

X-ray crystal structure of a TRPM assembly domain reveals an antiparallel four-stranded coiled-coil

Transient receptor potential (TRP) channels comprise a large family of tetrameric cation-selective ion channels that respond to diverse forms of sensory input. Earlier studies showed that members of the TRPM subclass possess a self-assembling tetrameric C-terminal cytoplasmic coiled-coil domain that underlies channel assembly and trafficking. Here, we present the high-resolution crystal structure of the coiled-coil domain of the channel enzyme TRPM7. The crystal structure, together with biochemical experiments, reveals an unexpected four-stranded antiparallel coiled-coil architecture that bears unique features relative to other antiparallel coiled-coils. Structural analysis indicates that a limited set of interactions encode assembly specificity determinants and uncovers a previously unnoticed segregation of TRPM assembly domains into two families that correspond with the phylogenetic divisions seen for the complete subunits. Together, the data provide a framework for understanding the mechanism of TRPM channel assembly and highlight the diversity of forms found in the coiled-coil fold.     

Dynein light chain LC8 promotes assembly of the coiled-coil domain of swallow protein

LC8 is a highly conserved light-chain subunit of cytoplasmic dynein that is thought to play a fundamental role in both the assembly of the motor complex and the recruitment of cargo. An interaction between LC8 and the Drosophila protein swallow has been previously characterized and supports a role for dynein in the localization of maternal morphogens during oogenesis. Swallow is required for the proper localization of bicoid mRNA, the anterior determinant that plays a critical role in establishment of the Drosophila embryonic axis. In this work, we prepared constructs of swallow, each containing a predicted coiled-coil domain and variable surrounding segments that lie within the domain proposed to interact with LC8. The interaction between LC8 and swallow domains was characterized by glutathione S-transferase (GST) pull-down assays, limited proteolysis followed by mass spectrometry, and circular dichroic spectroscopy. Hydrodynamic measurements, covalent cross-linking, and circular dichroic spectroscopy show that this domain of swallow is an unstable dimeric coiled-coil. Upon LC8 binding, however, the coiled-coil becomes significantly more stable. A possible general role for LC8 in macromolecular assembly is discussed.     

Anti-adhesive properties of fish tropomyosins

Aims:  We have recently found that preconditioning of stainless steel surfaces with an aqueous fish muscle extract can significantly impede bacterial adhesion. The purpose of this study was to identify and characterize the primary components associated with this bacteria-repelling effect.
Methods and Results:  The anti-adhesive activity was assayed against Escherchia coli K-12, and bacterial adhesion was quantified by crystal violet staining and sonication methods. Proteolytic digestion, elution and fractionation experiments revealed that the anti-adhesive activity of the extract was linked to the formation of a proteinaceous conditioning film composed primarily of fish tropomyosins. These fibrous proteins formed a considerable anti-adhesive conditioning layer on and reduced bacterial adhesion to several different materials including polystyrene, vinyl plastic, stainless steel and glass. The protein adsorption profiles obtained from the various materials did not differ significantly, but elution was often incomplete making minor qualitative/quantitative differences indiscernible.
Conclusions:  The data highlights the significance of protein conditioning films on bacterial adhesion and emphasizes the importance of substratum's physiochemical properties and exposure time with regards to protein adsorption/elution efficiency and subsequent bacterial adhesion.
Significance and Impact of the Study:  Fish tropomyosin-coatings could potentially offer a nontoxic and relatively inexpensive measure of reducing bacterial colonization of inert surfaces.     

Novel coiled-coil cell division factor ZapB stimulates Z ring assembly and cell division

Formation of the Z ring is the first known event in bacterial cell division. However, it is not yet known how the assembly and contraction of the Z ring are regulated. Here, we identify a novel cell division factor ZapB in Escherichia coli that simultaneously stimulates Z ring assembly and cell division. Deletion of zapB resulted in delayed cell division and the formation of ectopic Z rings and spirals, whereas overexpression of ZapB resulted in nucleoid condensation and aberrant cell divisions. Localization of ZapB to the divisome depended on FtsZ but not FtsA, ZipA or FtsI, and ZapB interacted with FtsZ in a bacterial two-hybrid analysis. The simultaneous inactivation of FtsA and ZipA prevented Z ring assembly and ZapB localization. Time lapse microscopy showed that ZapB–GFP is present at mid-cell in a pattern very similar to that of FtsZ. Cells carrying a zapB deletion and the ftsZ84 ^ts allele exhibited a synthetic sick phenotype and aberrant cell divisions. The crystal structure showed that ZapB exists as a dimer that is 100% coiled-coil. In vitro , ZapB self-assembled into long filaments and bundles. These results raise the possibility that ZapB stimulates Z ring formation directly via its capacity to self-assemble into larger structures.     

Intracellular coiled-coil domain engaged in subunit interaction and assembly of melastatin-related transient receptor potential channel 2

TRPM2 channels, activated by adenosine diphosphoribose and related molecules, are assembled as oligomers and most likely tetramers. However, the molecular determinants driving the subunit interaction and assembly of the TRPM2 channels are not well defined. Here we examined, using site-directed mutagenesis in conjunction with co-immunoprecipitation and patch clamp recording, the role of a coiled-coil domain in the intracellular C terminus of TRPM2 subunit in subunit interaction and channel assembly. Deletion of the coiled-coil domain resulted in severe disruption of the subunit interaction and substantial loss of the adenosine diphosphoribose-evoked channel currents. Individual or combined mutations to glutamine of the hydrophobic residues at positions a and d of the abcdef heptad repeat, key residues for protein-protein interaction, significantly reduced the subunit interaction and channel currents; the mutational effects on the subunit interaction and channel currents were clearly correlated. Furthermore, deletion of the coiled-coil domain in a pore mutant subunit abolished its dominant negative phenotypic functional suppression. These results provide strong evidence that the coiled-coil domain is critically engaged in the TRPM2 subunit interaction and such interaction is required for assembly of functional TRPM2 channel. The coiled-coil domain, which is highly conserved within the TRPM subfamily, may serve as a general structural element governing the assembly of TRPM channels.     

Regulation of c-Fes tyrosine kinase and biological activities by N-terminal coiled-coil oligomerization domains

The cytoplasmic protein-tyrosine kinase Fes has been implicated in cytokine signal transduction, hematopoiesis, and embryonic development. Previous work from our laboratory has shown that active Fes exists as a large oligomeric complex in vitro. However, when Fes is expressed in mammalian cells, its kinase activity is tightly repressed. The Fes unique N-terminal sequence has two regions with strong homology to coiled-coil-forming domains often found in oligomeric proteins. Here we show that disruption or deletion of the first coiled-coil domain upregulates Fes tyrosine kinase and transforming activities in Rat-2 fibroblasts and enhances Fes differentiation-inducing activity in myeloid leukemia cells. Conversely, expression of a Fes truncation mutant consisting only of the unique N-terminal domain interfered with Rat-2 fibroblast transformation by an activated Fes mutant, suggesting that oligomerization is essential for Fes activation in vivo. Coexpression with the Fes N-terminal region did not affect the transforming activity of v-Src in Rat-2 cells, arguing against a nonspecific suppressive effect. Taken together, these findings suggest a model in which Fes activation may involve coiled-coil-mediated interconversion of monomeric and oligomeric forms of the kinase. Mutation of the first coiled-coil domain may activate Fes by disturbing intramolecular coiled-coil interaction, allowing for oligomerization via the second coiled-coil domain. Deletion of the second coiled-coil domain blocks fibroblast transformation by an activated form of c-Fes, consistent with this model. These results provide the first evidence for regulation of a nonreceptor protein-tyrosine kinase by coiled-coil domains.