Characterization of the matrilin coiled-coil domains reveals seven novel isoforms

Matrilins constitute a family of four oligomeric extracellular proteins that are involved in the development and homeostasis of cartilage and bone. To reveal their homo- and heterotypic oligomerization propensities, we analyzed the four human matrilin coiled-coil domains by biochemical and biophysical methods. These studies not only confirmed the homo- and heterotypic oligomerization states reported for the full-length proteins but revealed seven novel matrilin isoforms. Specific heterotrimeric interactions of variable chain stoichiometries were observed between matrilin-1 and matrilin-2, matrilin-1 and matrilin-4, and matrilin-2 and matrilin-4. In addition, matrilin-1 formed two different specific heterotetramers with matrilin-3. Interestingly, a distinct heterotrimer consisting of three different chains was formed between matrilin-1, matrilin-2, and matrilin-4. No interactions, however, were observed between matrilin-2 and matrilin-3 or between matrilin-3 and matrilin-4. Both homo- and heterotypic oligomers folded into parallel disulfide-linked structures, although coiled-coil formation was not dependent on disulfide bridge formation. Our results indicate that the heterotypic preferences seen for the matrilin coiled-coil domains are the result of the packing of the hydrophobic core rather than ionic interactions. Mass spectrometry revealed that the concentrations of the individual chains statistically determined the stoichiometry of the heteromers, suggesting that formation of the different matrillin chain combinations is controlled by expression levels.     

Site-specific modification of de novo designed coiled-coil polypeptides with inorganic redox complexes.

A stepwise procedure for preparing of site-specific binuclear metallopeptides is described. The modification procedure involves the alkylation of a cysteine side chain by reaction with [Ru(bpy)(2)(phen-ClA)](2+), where bpy = 2,2'-bipyridine and phen-ClA = 5-chloroacetamido-1,10-phenanthroline, followed by the coordination of a ruthenium pentammine complex to a histidine residue located elsewhere along the sequence. The apo and metalated versions of the peptides C10H21(30-mer) and H10C21(30-mer) display circular dichroism spectra having minima at 208 and 222 nm, with theta(222)/theta(208) = 1.04 to indicate that these peptides exist as alpha-helical coiled-coils in aqueous solution. When the ruthenium polypyridyl complex is attached to C10H21(30-mer), the Delta-l and Lambda-l diastereomers of the resulting metallopeptide can be readily separated from each other by reversed-phase HPLC. However, in the case of the related H10C21(30-mer) metallopeptide, the two diastereomers cannot be chromatographically resolved. These results indicate how the subtle interplay between peptide conformation/sequence and metal complex geometry may alter some of the physical characteristics of metallopeptides.     

Characterization of a new four-chain coiled-coil: influence of chain length on stability.

Limited information is available on inherent stabilities of four-chain-coils. We have developed a model system to study this folding motif using synthetic peptides derived from sequences contained in the tetramerization domain of Lac repressor. These peptides are tetrameric as judged by both gel filtration and sedimentation equilibrium and the tetramers are fully helical as determined by CD. The four-chain coiled-coils are well folded as judged by the cooperativity of thermal unfolding and by the extent of dispersion in aliphatic chemical shifts seen in NMR spectra. In addition, we measured the chain length dependence of this four-chain coiled-coil. To this end, we developed a general procedure for nonlinear curve fitting of denaturation data in oligomeric systems. The dissociation constants for bundles that contain alpha-helical chains 21, 28, and 35 amino acids in length are 3.1 x 10(-12), 6.7 x 10(-23), and 1.0 x 10(-38) M3, respectively. This corresponds to tetramer stabilities (in terms of the peptide monomer concentration) of 180 microM, 51 nM, and 280 fM, respectively. Finally, we discuss the rules governing coiled-coil formation in light of the work presented here.     

Cohesin and related coiled-coil domain-containing complexes physically and functionally connect the dots across the genome

Interactions between genetic regions located across the genome maintain its three-dimensional organization and function. Recent studies point to key roles for a set of coiled-coil domain-containing complexes (cohibin, cohesin, condensin and monopolin) and related factors in the regulation of DNA-DNA connections across the genome. These connections are critical to replication, recombination, gene expression as well as chromosome segregation.     

Characterization of three new chlorophyll-protein complexes

Three new chlorophyll-proteins with electrophoretic mobilities intermediate between those of the P₇₀₀ chl a-protein and the light-harvesting chl a,b-protein complexes are reported and their absorption spectra and polypeptide composition are characterized. Two of these chlorophyll-proteins, bands II_b and II_a, contain approximately equal amounts of chl a and b, have polypeptide compositions similar to that of the light-harvesting chl a,b-protein and probably represent oligomers of the latter complex. The third new chlorophyll-protein contains only chl a and its major polypeptide(s) is in the 42 kd region. Indirect evidence indicates this chlorophyll-protein is associated with the reaction-center of photosystem II.     

The use of coiled-coil interactions for the analysis and micropreparative isolation of adenomatous polyposis coli protein complexes.

The coiled coil is a common structural motif found both as the dominant structure in fibrous proteins and as an oligomerization domain in a variety of cytoskeletal and extracellular matrix proteins. Coiled-coils typically consist of two to four helices that are supercoiled around one another in either parallel or antiparallel orientations. In the past few years our knowledge of the structure and specificity of coiled coil interactions has increased, allowing the de novo design and preparation of coiled-coils with well-defined structure and specificity. Indeed, the design and synthesis of a peptide that binds specifically to a single coiled-coil-containing protein, adenomatous polyposis coli (APC) has been reported. We have optimized solid-phase synthesis techniques to produce a modified form of the anti-APC peptide that contains a biotin moiety specifically placed so as to allow selective orientation onto the surface of a biosensor or affinity support. These peptide surfaces have been used to both monitor and purify APC and APC complexes from cellular extracts.     

Characterization of Rab45/RASEF containing EF-hand domain and a coiled-coil motif as a self-associating GTPase

Rab-family GTPases function as key regulators for membrane traffic. Among them, Rab45/RASEF is an atypical GTPase in that it contains a coiled-coil motif at the mid region and a distinct N-terminal EF-hand domain with C-terminal Rab-homology domain. Here, we provide the initial biochemical characterization and intracellular localization of human Rab45. Rab45 bound guanine nucleotide tri- and di-phosphates through the C-terminal Rab domain. Rab45 was capable of self-interacting, and the self-interaction required the mid region containing the coiled-coil motif. Rab45 expressed in HeLa cells was localized in a small patch in the perinuclear area of the cell, and the localization was regulated by the guanine nucleotide-bound states of Rab45. Interestingly, the mid region, together with Rab domain, appeared to be essential for the characteristic perinuclear localization of Rab45, indicating that the self-interaction may be involved in the intracellular localization of Rab45.     

Comparative analysis of coiled-coil prediction methods

In this study we compare commonly used coiled-coil prediction methods against a database derived from proteins of known structure. We find that the two older programs COILS and PairCoil/MultiCoil are significantly outperformed by two recent developments: Marcoil, a program built on hidden Markov models, and PCOILS, a new COILS version that uses profiles as inputs; and to a lesser extent by a PairCoil update, PairCoil2. Overall Marcoil provides a slightly better performance over the reference database than PCOILS and is considerably faster, but it is sensitive to highly charged false positives, whereas the weighting option of PCOILS allows the identification of such sequences.     

Characterization of mRNA-protein complexes from mammalian cells.

In a previous report we described the use of oligo(dT)-cellulose for the isolation of mRNA-protein complexes from EDTA-dissociated polysomes extracted from normally growing or adenovirus infected KB-cells (I). Experiments presented here provide evidence that proteins involved in these complexes bind specifically to mRNA since: a) the proteins and mRNA cosediment through sucrose gradients, b) they adsorb and elute from oligo(dT)-cellulose together, and c) analysis of the products from ribonuclease digestion experiments show that the poly (A) end and a separate small fraction of the mRNA are resistant to the enzymes and attached to protein.     

Characterization of dodecylphosphocholine/myelin basic protein complexes

The stoichiometry of myelin basic protein (MBP)/dodecylphosphocholine (DPC) complexes and the location of protein segments in the micelle have been investigated by electron paramagnetic resonance (EPR), ultracentrifugation, photon correlation light scattering, 31P, 13C, and 1H nuclear magnetic resonance (NMR), and electron microscopy. Ultracentrifugation measurements indicate that MBP forms stoichiometrically well-defined complexes consisting of 1 protein molecule and approximately 140 detergent molecules. The spin-labels 5-, 12-, and 16-doxylstearate have been incorporated into DPC/MBP aggregates. EPR spectral parameters and 13C and 1H NMR relaxation times indicate that the addition of MBP does not affect the environment and location of the labels or the organization of the micelles except for a slight increase in size. Previous results indicating that the protein lies primarily near the surface of the micelle have been confirmed by comparing 13C NMR spectra of the detergent with and without protein with spectra of protein/detergent aggregates containing spin-labels. Electron micrographs of the complexes taken by using the freeze-fracture technique confirm the estimated size obtained by light-scattering measurements. Overall, these results indicate that mixtures of MBP and DPC can form highly porous particles with well-defined protein and lipid stoichiometry. The structural integrity of these particles appears to be based on protein-lipid interactions. In addition, electron micrographs of aqueous DPC/MBP suspensions show the formation of a small amount of material consisting of large arrays of detergent micelles, suggesting that MBP is capable of inducing large changes in the overall organization of the detergent.     

Regulation of coiled-coil assembly in tropomyosins

Tropomyosins (TMs) are a family of actin filament-binding proteins. They consist of nearly 100% alpha-helix and assemble into parallel coiled-coil dimers. In vertebrates, TMs are encoded by four genes that give rise to at least 17 distinct isoforms through the use of alternative RNA splicing and alternative promoters. We have studied various aspects of the coiled-coil interactions among muscle and nonmuscle isoforms by the use of transfection of epitope-tagged constructs, followed by immunoprecipitation, SDS-PAGE, and Western blot analyses. For coiled-coil interactions between high-molecular-weight isoforms (284 amino acids), the information for homo- versus heterodimerization is contained in large part within the alternatively spliced exons of nonmuscle and muscle (skeletal and smooth) isoforms. Furthermore, sequences located in alternatively spliced exons encoding amino acids 39-80 (exons 2a/2b), amino acids 189-213 (exons 6a/6b), and amino acids 258-284 (exons 9a/9d) are critical for the selective formation of homo- versus heterodimers. Among low-molecular-weight isoforms (248 amino acids), TM-4 and TM-5 can form either homodimers or heterodimers. The trigger sequence (amino acids 190-202) is required for homodimerization of TM-4, but not heterodimerization of TM-4 with TM-5. How the dimeric state of TMs might play a role in their cellular localization and function is discussed.     

Characterization of cells comprising murine thymocyte multicellular complexes

Characterization of complexes between thymocytes and thymic stromal cells was facilitated in the present study by demonstrating that complexes would reform if cells were incubated for 1.5 to 2 h in vitro at ambient temperature. Several immunologic approaches were used to determine the phenotype of complexed cells. Bound T cells were 97% double-positive (CD4 and CD8), 3% double-negative and greater than 99% CD3 positive by using immunoperoxidase immunohistology on cytospins. Five percent expressed the TCR beta-chain and 1 to 2% were IL-2R positive. The percentages were the same whether complexes were preformed in vivo or formed in vitro. Despite the apparent absence of single positive cells in complexes, when isolated CD4 or CD8 positive cells were tested for their ability to bind to adherent thymic macrophages, each subpopulation contained some cells which were capable of complex formation. When thymocytes were fractionated by density, steroid sensitivity or peanut agglutinin positivity then allowed to form complexes, cells with an immature phenotype had a greater propensity for complex formation. Central stromal cells all were class II MHC gene product (I-A and I-E) positive, expressed macrophage-associated Ag (B23.1 and MAC-1), were negative for cytokeratin but positive for vimentin, were reactive with a polyclonal antimacrophage serum, but did not express dendritic cell Ag (33D1). The data demonstrate that immature thymocytes bind exclusively to class II MHC gene product positive thymic macrophages. This binding step may play a role in the acquisition of T cell function in the thymus.     

Characterization of Cep135, a novel coiled-coil centrosomal protein involved in microtubule organization in mammalian cells.

By using monoclonal antibodies raised against isolated clam centrosomes, we have identified a novel 135-kD centrosomal protein (Cep135), present in a wide range of organisms. Cep135 is located at the centrosome throughout the cell cycle, and localization is independent of the microtubule network. It distributes throughout the centrosomal area in association with the electron-dense material surrounding centrioles. Sequence analysis of cDNA isolated from CHO cells predicted a protein of 1,145-amino acid residues with extensive alpha-helical domains. Expression of a series of deletion constructs revealed the presence of three independent centrosome-targeting domains. Overexpression of Cep135 resulted in the accumulation of unique whorl-like particles in both the centrosome and the cytoplasm. Although their size, shape, and number varied according to the level of protein expression, these whorls were composed of parallel dense lines arranged in a 6-nm space. Altered levels of Cep135 by protein overexpression and/or suppression of endogenous Cep135 by RNA interference caused disorganization of interphase and mitotic spindle microtubules. Thus, Cep135 may play an important role in the centrosomal function of organizing microtubules in mammalian cells.     

Identification of targets for calcium signaling through the copine family of proteins. Characterization of a coiled-coil copine-binding motif

We provide evidence that copines, members of a ubiquitous family of calcium-dependent, membrane-binding proteins, may represent a universal transduction pathway for calcium signaling because we find copines are capable of interacting with a wide variety of "target" proteins including MEK1, protein phosphatase 5, and the CDC42-regulated kinase, that are themselves components of intracellular signaling pathways. The copine target proteins were identified by yeast two-hybrid screening and the interactions were verified in vitro using purified proteins. In the majority of cases the copine binds to a domain of the target protein that is predicted to form a characteristic coiled-coil. A consensus sequence for the coiled-coil copine-binding site was derived and found to have predictive value for identifying new copine targets. We also show that interaction with copines may result in recruitment of target proteins to membrane surfaces and regulation of the enzymatic activities of target proteins.     

Thermodynamic analysis of self-assembly in coiled-coil biomaterials

Coiled-coil protein structural motifs have proven amenable to the design of structurally well-defined biomaterials. Mesoscale structural properties can be fairly well predicted based on rules governing the chemical interactions between the helices that define this structural motif. We explore the role of the hydrophobic core residues on the self-assembly of a coiled-coil polymer through a mutational analysis coupled with a salting-out procedure. Because the resultant polymers remain in solution, a thermodynamic approach is applied to characterize the polymer assembly using conventional equations from polymer theory to extract nucleation and elongation parameters. The stabilities and lengths of the polymers are measured using circular dichroism spectropolarimetry, sizing methods including dynamic light scattering and analytical ultracentrifugation, and atomic force microscopy to assess mesoscale morphology. Upon mutating isoleucines at two core positions to serines, we find that polymer stability is decreased while the degree of polymerization is about the same. Differences in results from circular dichroism and dynamic light scattering experiments suggest the presence of a stable intermediate state, and a scheme is proposed for how this intermediate might relate to the monomer and polymer states.     

Solution structural characterization of coiled-coil peptide-polymer side-conjugates

Detailed structural characterization of protein-polymer conjugates and understanding of the interactions between covalently attached polymers and biomolecules will build a foundation to design and synthesize hybrid biomaterials. Conjugates based on simple protein structures are ideal model system to achieve these ends. Here we present a systematic structural study of coiled-coil peptide-poly(ethylene glycol) (PEG) side-conjugates in solution, using circular dichroism, dynamic light scattering, and small-angle X-ray scattering, to determine the conformation of conjugated PEG chains. The overall size and shape of side-conjugates were determined using a cylindrical form factor model. Detailed structural information of the covalently attached PEG chains was extracted using a newly developed model where each peptide-PEG conjugate was modeled as a Gaussian chain attached to a cylinder, which was further arranged in a bundle-like configuration of three or four cylinders. The peptide-polymer side-conjugates were found to retain helix bundle structure, with the polymers slightly compressed in comparison with the conformation of free polymers in solution. Such detailed structural characterization of the peptide-polymer conjugates, which elucidates the conformation of conjugated PEG around the peptide and assesses the effect of PEG on peptide structure, will contribute to the rational design of this new family of soft materials.     

Characterization of the DNA binding specificity of Shelterin complexes

The Shelterin complex associates with telomeres and plays an essential role in telomere protection and telomerase regulation. In its most abundant form, the complex is composed of six core components: TRF1, TRF2, POT1, TIN2, TPP1 and RAP1. Of these subunits, three can interact directly with either single-stranded (POT1) or double-stranded (TRF1, TRF2) telomeric DNA. In this report, we have developed assays to measure the DNA binding activity of Shelterin complexes in human cell extracts. With these assays, we have characterized the composition and DNA binding specificity of two Shelterin complexes: a 6-member complex that contains all six core components and a second complex that lacks TRF1. Our results show that both of these complexes bind with high affinity (K(D) = 1.3-1.5 × 10(-9) M) and selectively to ds/ss-DNA junctions that carry both a binding site for POT1 (ss-TTAGGGTTAG) and a binding site for the SANT/Myb domain of TRF1 or TRF2 (ds-TTAGGGTTA). This DNA binding specificity suggests the preferential recruitment of these complexes to areas of the telomere where ss- and ds-DNA are in close proximity, such as the 3'-telomeric overhang, telomeric DNA bubbles and the D-loop at the base of T-loops.