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1.
J Cell Biol ; 135(6 Pt 1): 1583-92, 1996 Dec.
Article in English | MEDLINE | ID: mdl-8978824

ABSTRACT

After denervation of muscle, motor axons reinnervate original synaptic sites. A recombinant fragment of the synapse specific laminin beta 2 chain (s-laminin) was reported to inhibit motor axon growth. Consequently, a specific sequence (leucine-arginine-glutamate, LRE) of the laminin beta 2 chain was proposed to act as a stop signal and to mediate specific reinnervation at the neuromuscular junction (Porter, B.E., J. Weis, and J.R. Sanes. 1995. Neuron. 14:549-559). We demonstrate here that native chick laminin-4, which contains the beta 2 chain and is present in the synaptic basement membrane, does not inhibit but rather promotes motor axon growth. In native heterotrimeric laminin, the LRE sequence of the beta 2 chain is found in a triple coiled-coil region that is formed by all three subunits. We show here that the effect of LRE depends on the structural context. Whereas a recombinant randomly coiled LRE peptide indeed inhibited outgrowth by chick motoneurons, a small recombinant triple coiled-coil protein containing this sequence did not.


Subject(s)
Axons/physiology , Laminin/genetics , Motor Neurons/physiology , Promoter Regions, Genetic , Amino Acid Sequence , Animals , Cell Division/genetics , Cell Division/physiology , Cells, Cultured , Chick Embryo , Culture Media , Laminin/chemistry , Laminin/physiology , Mice , Molecular Sequence Data , Neurites/physiology , Protein Conformation
2.
J Cell Biol ; 153(6): 1287-300, 2001 Jun 11.
Article in English | MEDLINE | ID: mdl-11402071

ABSTRACT

The microtubule-binding integral 63 kD cytoskeleton-linking membrane protein (CLIMP-63; former name, p63) of the rough endoplasmic reticulum (ER) is excluded from the nuclear envelope. We studied the mechanism underlying this ER subdomain-specific localization by mutagenesis and structural analysis. Deleting the luminal but not cytosolic segment of CLIMP-63 abrogated subdomain-specific localization, as visualized by confocal microscopy in living cells and by immunoelectron microscopy using ultrathin cryosections. Photobleaching/recovery analysis revealed that the luminal segment determines restricted diffusion and immobility of the protein. The recombinant full-length luminal segment of CLIMP-63 formed alpha-helical 91-nm long rod-like structures as evident by circular dichroism spectroscopy and electron microscopy. In the analytical ultracentrifuge, the luminal segment sedimented at 25.7 S, indicating large complexes. The complexes most likely arose by electrostatic interactions of individual highly charged coiled coils. The findings indicate that the luminal segment of CLIMP-63 is necessary and sufficient for oligomerization into alpha-helical complexes that prevent nuclear envelope localization. Concentration of CLIMP-63 into patches may enhance microtubule binding on the cytosolic side and contribute to ER morphology by the formation of a protein scaffold in the lumen of the ER.


Subject(s)
Endoplasmic Reticulum/metabolism , Membrane Proteins , Phosphoproteins/metabolism , Trans-Activators , Animals , Binding Sites , COS Cells , Chlorocebus aethiops , Nuclear Envelope/metabolism , Phosphoproteins/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism
3.
Science ; 274(5288): 761-5, 1996 Nov 01.
Article in English | MEDLINE | ID: mdl-8864111

ABSTRACT

Oligomerization by the formation of alpha-helical bundles is common in many proteins. The crystal structure of a parallel pentameric coiled coil, constituting the oligomerization domain in the cartilage oligomeric matrix protein (COMP), was determined at 2.05 angstroms resolution. The same structure probably occurs in two other extracellular matrix proteins, thrombospondins 3 and 4. Complementary hydrophobic interactions and conserved disulfide bridges between the alpha helices result in a thermostable structure with unusual properties. The long hydrophobic axial pore is filled with water molecules but can also accommodate small apolar groups. An "ion trap" is formed inside the pore by a ring of conserved glutamines, which binds chloride and probably other monatomic anions. The oligomerization domain of COMP has marked similarities with proposed models of the pentameric transmembrane ion channels in phospholamban and the acetylcholine receptor.


Subject(s)
Extracellular Matrix Proteins/chemistry , Glycoproteins/chemistry , Ion Channels/chemistry , Protein Conformation , Protein Structure, Secondary , Amino Acid Sequence , Animals , Cartilage Oligomeric Matrix Protein , Chloride Channels/chemistry , Chlorides/chemistry , Crystallography, X-Ray , Disulfides/chemistry , Glutamine/chemistry , Humans , Hydrogen Bonding , Matrilin Proteins , Models, Molecular , Molecular Sequence Data , Sequence Alignment
4.
Structure ; 8(3): 223-30, 2000 Mar 15.
Article in English | MEDLINE | ID: mdl-10745004

ABSTRACT

BACKGROUND: The parallel two-stranded alpha-helical coiled coil is the most frequently encountered subunit-oligomerization motif in proteins. The simplicity and regularity of this motif have made it an attractive system to explore some of the fundamental principles of protein folding and stability and to test the principles of de novo design. RESULTS: The X-ray crystal structure of the 18-heptad-repeat alpha-helical coiled-coil domain of the actin-bundling protein cortexillin I from Dictyostelium discoideum is a tightly packed parallel two-stranded alpha-helical coiled coil. It harbors a distinct 14-residue sequence motif that is essential for coiled-coil formation, and is a prerequisite for the assembly of cortexillin I. The atomic structure reveals novel types of ionic coiled-coil interactions. In particular, the structure shows that a characteristic interhelical and intrahelical salt-bridge pattern, in combination with the hydrophobic interactions occurring at the dimer interface, is the key structural feature of its coiled-coil trigger site. CONCLUSIONS: The knowledge gained from the structure could be used in the de novo design of alpha-helical coiled coils for applications such as two-stage drug targeting and delivery systems, and in the design of coiled coils as templates for combinatorial helical libraries in drug discovery and as synthetic carrier molecules.


Subject(s)
Microfilament Proteins/chemistry , Crystallography, X-Ray , Leucine Zippers , Models, Molecular , Protein Conformation , Protozoan Proteins , Salts/chemistry
5.
J Mol Biol ; 250(1): 74-9, 1995 Jun 30.
Article in English | MEDLINE | ID: mdl-7602598

ABSTRACT

The long arm of laminin in which three polypeptide chains alpha, beta, and gamma are assembled in an alpha-helical coiled-coil structure is stabilized by non-covalent interactions and disulfide bridges. The stabilizing role of the disulfide linkage between the beta and gamma-chains at the C-terminal region of the assembly domain was investigated with about 100-residue long recombinant fragments. Circular dichroism spectra and electron micrographs were identical for linked and non-linked species and indicated two-stranded coiled-coil structures with about 100% alpha-helicity at 20 degrees C. Thermal transition profiles revealed an increase of the melting temperature from 42 degrees C to 60.4 degrees C upon disulfide formation at a chain concentration of 25 microM. The enthalpy of interaction was identical for the two species but the negative entropy involved in joining the two chains was reduced by the disulfide bonds. At chain concentrations of 10 microM the Gibbs free energy delta G was by 17.5 kJ/mol more negative for the disulfide-linked than for the unlinked chains. Because of the concentration dependence of the entropy of the non-linked chains, this difference decreased with increasing concentration and, by extrapolation at chain concentrations of 10 mM, the stability of both structures would be the same. As a competing reaction, beta-chains associated to four-stranded bundles which probably consist of pairs of two-stranded coiled-coils. After disulfide formation a biphasic transition curve was observed which indicated two different ways of connecting the chains in the bundle.


Subject(s)
Disulfides/chemistry , Laminin/chemistry , Protein Structure, Secondary , Circular Dichroism , Hot Temperature , Laminin/ultrastructure , Peptide Fragments/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/ultrastructure , Thermodynamics
6.
J Mol Biol ; 250(1): 64-73, 1995 Jun 30.
Article in English | MEDLINE | ID: mdl-7602597

ABSTRACT

Recombinant fragments alpha, beta, and gamma were prepared comprising about 100 C-terminal residues of the corresponding polypeptide chains in the three-stranded alpha-helical coiled-coil domain of laminin. Circular dichroism spectra, thermal transition profiles, non-denaturing gels, analytical ultracentrifugation, and calorimetry indicated alpha-helicity and high thermal stabilities for the beta gamma heterodimer and homoassociates of beta. Very little or no coiled-coil formation was found for alpha and gamma. The thermal melting profiles and their concentration dependencies were quantitatively described by a two-state mechanism in which two unfolded chains combine to a fully alpha-helical dimer. For the beta gamma dimer the melting temperature was Tm = 42 degrees C at a chain concentration of 25 microM in 5 mM sodium phosphate buffer (pH 7.4). Addition of 100 mM NaCl decreased the Tm slightly but the relative stability of beta gamma and beta beta coiled-coils was not significantly changed, indicating that electrostatic interactions alone are not responsible for chain selection. Upon addition of 1 M urea the Tm value dropped by about 10 degrees C. The enthalpy changes for the formation of the coiled-coil were delta H degrees = -304(+/- 30) kJ/mol for the beta gamma heterodimer and -198(+/- 20) kJ/mol for the beta-homoassociates. Gibbs free energies and entropies amounted to delta G degrees = -42.8 kJ and delta S degrees = -876 J/mol K for the heteroassembly and -37.8 kJ/mol and -537 J/mol K for the homoassembly of beta. This low preference for heteroassociation of the fragment is smaller than the chain selectivity observed for larger fragments and intact laminin. Deletion of ten residues from the C-terminal region of the gamma-fragment which were recently reported as an essential assembly-site was not sufficient to abolish heteroassociation. Interaction of alpha-fragment with double-stranded beta gamma coiled-coils reflected the formation of a three-stranded coiled-coil in laminin but for the small recombinant fragments association between alpha and beta-homoassociates was also observed. The C-terminal 100 residues in the coiled-coil domain are therefore not alone responsible for the high specificity of chain selection in laminin.


Subject(s)
Laminin/chemistry , Protein Conformation , Protein Structure, Secondary , Amino Acid Sequence , Animals , Base Sequence , Calorimetry , Kinetics , Mice , Molecular Sequence Data , Molecular Weight , Peptide Fragments/chemistry , Recombinant Proteins/chemistry , Sequence Deletion/physiology , Thermodynamics
7.
J Mol Biol ; 308(5): 1081-9, 2001 May 18.
Article in English | MEDLINE | ID: mdl-11352592

ABSTRACT

Recombinant expression of collagens and fragments of collagens is often difficult, as their biosynthesis requires specific post-translational enzymes, in particular prolyl 4-hydroxylase. Although the use of hydroxyproline-deficient variants offers one possibility to overcome this difficulty, these proteins usually differ markedly in stability when compared with the hydroxyproline-containing analogs. Here, we report a method to stabilize collagen-like peptides by fusing them to the N terminus of the bacteriophage T4 fibritin foldon domain. The isolated foldon domain and the chimeric protein (GlyProPro)(10)foldon were expressed in a soluble form in Escherichia coli. The recombinant proteins and the synthetic (ProProGly)(10) peptide were characterized by circular dichroism (CD) spectroscopy, differential scanning calorimetry, and analytical ultracentrifugation. We show that the foldon domain, which comprises only 27 amino acid residues, forms an obligatory trimer with a high degree of thermal stability. The CD thermal unfolding profiles recorded from foldon are monophasic and completely reversible upon cooling. Similar Van't Hoff and calorimertic enthalpy values of trimer formation indicated a cooperative all-or-none transition. As reported previously, (ProProGly)(10) peptides form collagen triple helices of only moderate stability. When fused to the foldon domain, however, triple helix formation of (GlyProPro)(10) is concentration independent, and the midpoint temperature of the triple helix unfolding is significantly increased. The stabilizing function of the trimeric foldon domain is explained by the close vicinity of its N termini, which induce a high local concentration in the range of 1 M for the C termini of the collagen-like-peptide. Collagen-foldon fusion proteins should be potentially useful to study receptor-collagen interactions.


Subject(s)
Bacteriophage T4/chemistry , Collagen/chemistry , Collagen/metabolism , Protein Engineering , Recombinant Fusion Proteins/chemistry , Viral Proteins/chemistry , Viral Proteins/metabolism , Amino Acid Sequence , Biopolymers/chemistry , Biopolymers/genetics , Biopolymers/metabolism , Calorimetry, Differential Scanning , Circular Dichroism , Collagen/chemical synthesis , Collagen/genetics , Escherichia coli/genetics , Molecular Sequence Data , Peptides/chemical synthesis , Peptides/chemistry , Peptides/genetics , Peptides/metabolism , Protein Structure, Quaternary , Protein Structure, Secondary , Protein Structure, Tertiary , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Solubility , Temperature , Thermodynamics , Ultracentrifugation , Viral Proteins/genetics
8.
J Mol Biol ; 279(1): 189-99, 1998 May 29.
Article in English | MEDLINE | ID: mdl-9636709

ABSTRACT

Recombinant rat liver GTP cyclohydrolase I has been prepared by heterologous gene expression in Escherichia coli and characterized by biochemical and biophysical methods. Correlation averaged electron micrograph images of preferentially oriented enzyme particles revealed a fivefold rotational symmetry of the doughnut-shaped views with an average particle diameter of 10 nm. Analytical ultracentrifugation and quantitative scanning transmission electron microscopy yielded average molecular masses of 270 kDa and 275 kDa, respectively. Like the Escherichia coli homolog, these findings suggest that the active enzyme forms a homodecameric protein complex consisting of two fivefold symmetric pentameric rings associated face-to-face. Examination of the amino acid sequence combined with calcium-binding experiments and mutational analysis revealed a high-affinity, EF-hand-like calcium-binding loop motif in eukaryotic enzyme species, which is absent in bacteria. Intrinsic fluorescence measurements yielded an approximate dissociation constant of 10 nM for calcium and no significant binding of magnesium. Interestingly, a loss of calcium-binding capacity observed for two rationally designed mutations within the presumed calcium-binding loop of the rat GTP cyclohydrolase I yielded a 45% decrease in enzyme activity. This finding suggests that failure of calcium binding may be the consequence of a mutation recently identified in the causative GTP cyclohydrolase I gene of patients suffering from dopa responsive dystonia.


Subject(s)
Calcium/metabolism , GTP Cyclohydrolase/chemistry , Protein Conformation , Animals , Binding Sites , Circular Dichroism , GTP Cyclohydrolase/genetics , GTP Cyclohydrolase/ultrastructure , Genetic Engineering , Liver/enzymology , Microscopy, Electron , Rats
9.
Protein Sci ; 7(2): 389-402, 1998 Feb.
Article in English | MEDLINE | ID: mdl-9521116

ABSTRACT

Backbone 15N relaxation parameters (R1, R2, 1H-15N NOE) have been measured for a 22-residue recombinant variant of the S-peptide in its free and S-protein bound forms. NMR relaxation data were analyzed using the "model-free" approach (Lipari & Szabo, 1982). Order parameters obtained from "model-free" simulations were used to calculate 1H-15N bond vector entropies using a recently described method (Yang & Kay, 1996), in which the form of the probability density function for bond vector fluctuations is derived from a diffusion-in-a-cone motional model. The average change in 1H-15N bond vector entropies for residues T3-S15, which become ordered upon binding of the S-peptide to the S-protein, is -12.6+/-1.4 J/mol.residue.K. 15N relaxation data suggest a gradient of decreasing entropy values moving from the termini toward the center of the free peptide. The difference between the entropies of the terminal and central residues is about -12 J/mol residue K, a value comparable to that of the average entropy change per residue upon complex formation. Similar entropy gradients are evident in NMR relaxation studies of other denatured proteins. Taken together, these observations suggest denatured proteins may contain entropic contributions from non-local interactions. Consequently, calculations that model the entropy of a residue in a denatured protein as that of a residue in a di- or tri-peptide, might over-estimate the magnitude of entropy changes upon folding.


Subject(s)
Entropy , Peptide Fragments/chemistry , Protein Folding , Ribonuclease, Pancreatic/chemistry , Magnetic Resonance Spectroscopy , Models, Chemical , Nitrogen Isotopes , Protein Binding , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Ribonuclease, Pancreatic/metabolism
10.
Protein Sci ; 6(8): 1734-45, 1997 Aug.
Article in English | MEDLINE | ID: mdl-9260286

ABSTRACT

The C-terminal oligomerization domain of chicken cartilage matrix protein is a trimeric coiled coil comprised of three identical 43-residue chains. NMR spectra of the protein show equivalent magnetic environments for each monomer, indicating a parallel coiled coil structure with complete threefold symmetry. Sequence-specific assignments for 1H-, 15N-, and 13C-NMR resonances have been obtained from 2D 1H NOESY and TOCSY spectra, and from 3D HNCA, 15N NOESY-HSQC, and HCCH-TOCSY spectra. A stretch of alpha-helix encompassing five heptad repeats (35 residues) has been identified from intra-chain HN-HN and HN-H alpha NOE connectivities. 3JHNH alpha coupling constants, and chemical shift indices. The alpha-helix begins immediately downstream of inter-chain disulfide bonds between residues Cys 5 and Cys 7, and extends to near the C-terminus of the molecule. The threefold symmetry of the molecule is maintained when the inter-chain disulfide bonds that flank the N-terminus of the coiled coil are reduced. Residues Ile 21 through Glu 36 show conserved chemical shifts and NOE connectivities, as well as strong protection from solvent exchange in the oxidized and reduced forms of the protein. By contrast, residues Ile 10 through Val 17 show pronounced chemical shift differences between the oxidized and reduced protein. Strong chemical exchange NOEs between HN resonances and water indicate solvent exchange on time scales faster than 10 s, and suggests a dynamic fraying of the N-terminus of the coiled coil upon reduction of the disulfide bonds. Possible roles for the disulfide crosslinks of the oligomerization domain in the function of cartilage matrix protein are proposed.


Subject(s)
Extracellular Matrix Proteins , Glycoproteins/chemistry , Amino Acid Sequence , Animals , Biopolymers , Cartilage , Chickens , Magnetic Resonance Spectroscopy , Matrilin Proteins , Molecular Sequence Data , Oxidation-Reduction , Protein Structure, Secondary , Recombinant Proteins/chemistry
11.
Matrix Biol ; 15(8-9): 555-65; discussion 567-8, 1997 Mar.
Article in English | MEDLINE | ID: mdl-9138288

ABSTRACT

Subunit oligomerization of many proteins is mediated by alpha-helical coiled-coil domains. 3,4-Hydrophobic heptad repeat sequences, the characteristic feature of the coiled-coil protein folding motif, have been found in a wide variety of gene products including cytoskeletal, nuclear, muscle, cell surface, extracellular, plasma, bacterial, and viral proteins. Whereas the majority of coiled-coil structures is represented by intracellular alpha-helical bundles that contain two polypeptide chains, examples of extracellular coiled-coil proteins are fewer in number. Most proteins located in the extracellular space form three-stranded alpha-helical assemblies. Recently, five-stranded coiled coils have been identified in thrombospondins 3 and 4 and in cartilage oligomeric matrix protein, and the formation of a heterotetramer has been observed in in vitro studies with the recombinant asialoglycoprotein receptor oligomerization domain. Coiled-coil domains in laminins and probably also in tenascins and thrombospondins are responsible for the formation of tissue-specific isoforms by selective oligomerization of different polypeptide chains.


Subject(s)
Extracellular Matrix Proteins/chemistry , Protein Structure, Secondary , Amino Acid Sequence , Animals , Asialoglycoprotein Receptor , Cartilage , Crystallography, X-Ray , Extracellular Space , Macromolecular Substances , Membrane Glycoproteins/chemistry , Models, Structural , Molecular Sequence Data , Receptors, Cell Surface/chemistry , Recombinant Proteins/chemistry , Thrombospondins
12.
Matrix Biol ; 19(4): 283-8, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10963988

ABSTRACT

Collagen triple helices, coiled coils and other oligomerization domains mediate the subunit assembly of a large number of proteins. Oligomerization leads to functional advantages of multivalency and high binding strength, increased structure stabilization and combined functions of different domains. These features seen in naturally occurring proteins can be engineered by protein design by combining oligomerization domains with functional domains.


Subject(s)
Collagen/chemistry , Oligopeptides/chemistry , Animals , Cross-Linking Reagents , Humans , Protein Engineering , Protein Structure, Tertiary
13.
FEBS Lett ; 446(1): 75-80, 1999 Mar 05.
Article in English | MEDLINE | ID: mdl-10100618

ABSTRACT

The pKa values of eight glutamic acid residues in the homotrimeric coiled coil domain of chicken matrilin-1 have been determined from 2D H(CA)CO NMR spectra recorded as a function of the solution pH. The pKa values span a range between 4.0 and 4.7, close to or above those for glutamic acid residues in unstructured polypeptides. These results suggest only small favorable contributions to the stability of the coiled coil from the ionization of its acidic residues.


Subject(s)
Extracellular Matrix Proteins/chemistry , Glycoproteins/chemistry , Protein Conformation , Animals , Escherichia coli , Matrilin Proteins
14.
J Neurochem ; 71(6): 2615-25, 1998 Dec.
Article in English | MEDLINE | ID: mdl-9832163

ABSTRACT

Previous experiments suggested that the human cell adhesion molecule L1 interacts with different integrins via its sixth immunoglobulin-like domain in an RGD-dependent manner. Here we have described the expression of this domain from early postnatal mouse brain, analyzed the structure of the recombinant protein by circular dichroism and fluorescence spectroscopy, and performed solid-phase binding studies to alpha(v)beta3, alpha(IIb)beta3, and alpha5beta1 integrins. The domain was found to have the expected beta-sheet organization, which was lost in the presence of guanidine hydrochloride. The midpoint of the single-step transition occurred at 1.5 M guanidine hydrochloride. The sixth immunoglobulin-like domain of mouse brain L1 contains two RGD motifs and was found to bind in a concentration-dependent and saturable way to alpha(v)beta3, alpha(IIb)beta3, and alpha5beta1 integrins, suggesting specific interactions with these ligands. However, only the interaction to alpha(v)beta3 could be inhibited in a concentration-dependent manner by an RGD-containing peptide, and the IC50 was determined to be approximately 20 nM. Mutants of the domain, which lack either one or both of the RGD sites, demonstrated that the RGD site comprising residues 562-564 is involved in the interaction to alpha(v)beta3. Our findings indicate an RGD-independent mechanism for the interactions to alpha(IIb)beta3 and alpha5beta1, as no involvement of any RGD motif could be demonstrated.


Subject(s)
Immunoglobulins/genetics , Neural Cell Adhesion Molecules/genetics , Neural Cell Adhesion Molecules/physiology , Platelet Glycoprotein GPIIb-IIIa Complex/physiology , Receptors, Fibronectin/physiology , Receptors, Vitronectin/physiology , Amino Acid Sequence , Animals , Drug Interactions , Mice , Mice, Inbred Strains , Neural Cell Adhesion Molecules/metabolism , Oligopeptides/genetics , Oligopeptides/physiology , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Receptors, Fibronectin/metabolism , Receptors, Vitronectin/metabolism , Recombinant Proteins
15.
J Biol Chem ; 271(38): 23558-65, 1996 Sep 20.
Article in English | MEDLINE | ID: mdl-8798565

ABSTRACT

Dissociated sponge cells quickly reaggregate in a species-specific manner, differentiate, and reconstruct tissue, providing a very handy system to investigate the molecular basis of more complex intercellular recognition processes. Species-specific cell adhesion in the marine sponge Microciona prolifera is mediated by a supramolecular complex with a Mr = 2 x 10(7), termed aggregation factor. Guanidinium hydrochloride/cesium chloride dissociative gradients and rhodamine B isothiocyanate staining indicated the presence of several proteins with different degrees of glycosylation. Hyaluronate has been found to be associated with the aggregation factor. Chemical deglycosylation revealed a main component accounting for nearly 90% of the total protein. The cDNA-deduced amino acid sequence predicts a 35-kDa protein (MAFp3), the first sponge aggregation factor core protein ever described. The open reading frame is uninterrupted upstream from the amino terminus of the mature protein, and the deduced amino acid sequence for this region has been found to contain a long stretch sharing homology with the Na+-Ca2+ exchanger protein. A putative hyaluronic acid binding domain and several putative N- and O-glycosylation signals are present in MAFp3, as well as eight cysteines, some of them involved in intermolecular disulfide bridges. Northern blot data suggest variable expression, and Southern blot analysis reveals the presence of other related gene sequences. According to the respective molecular masses, one aggregation factor molecule would contain about 300 MAFp3 units, suggesting that sponge cell adhesion might be based on the assembly of multiple small glycosylated protein subunits.


Subject(s)
Cell Adhesion Molecules/chemistry , Cell Adhesion/physiology , Glycoproteins/chemistry , Porifera/chemistry , Amino Acid Sequence , Amino Acids/analysis , Animals , Base Sequence , Blotting, Northern , Blotting, Southern , Cell Adhesion Molecules/genetics , DNA, Complementary/genetics , Gene Library , Glycoproteins/genetics , Hyaluronic Acid/analysis , Molecular Sequence Data , Porifera/genetics , Protein Conformation , Protein Denaturation , Sequence Analysis, DNA , Sequence Homology, Amino Acid
16.
J Biol Chem ; 275(16): 11672-7, 2000 Apr 21.
Article in English | MEDLINE | ID: mdl-10766786

ABSTRACT

We have recently identified a distinct 13-residue sequence pattern that occurs with limited sequence variations in many two-stranded coiled coils but not in trimers, tetramers, or pentamers. This coiled-coil trigger pattern was demonstrated to be indispensable for the assembly of the oligomerization domain of the actin-bundling protein cortexillin I from Dictyostelium discoideum and the leucine zipper domain of the yeast transcriptional activator GCN4. With the aim to extend our knowledge on trigger sequences we have investigated the human macrophage scavenger receptor type A oligomerization domain as a representative of three-stranded coiled coils. We prepared a variety of recombinant N- and C-terminal deletion mutants from the full-length oligomerization domain by heterologous gene expression in Escherichia coli and assessed their ability to form trimeric coiled-coil structures by circular dichroism spectroscopy and analytical ultracentrifugation. Deletion mapping identified a distinct seven-residue sequence that was absolutely required for proper coiled-coil formation, supporting our previous results that heptad repeats alone are not sufficient for oligomerization. The finding that all fragments containing this particular sequence exhibited similar thermal stabilities indicates primarily a stabilizing function of the coiled-coil trigger. Based on sequence similarity, we suggest that functionally related sites are present in other three-stranded coiled-coil proteins.


Subject(s)
CD36 Antigens/chemistry , Macrophages/metabolism , Membrane Proteins , Receptors, Immunologic/chemistry , Receptors, Lipoprotein , Amino Acid Sequence , Animals , Cattle , Circular Dichroism , Humans , Mice , Molecular Sequence Data , Protein Conformation , Protein Structure, Secondary , Rabbits , Receptors, Scavenger , Scavenger Receptors, Class A , Scavenger Receptors, Class B , Structure-Activity Relationship , Ultracentrifugation
17.
J Biol Chem ; 271(50): 31996-2001, 1996 Dec 13.
Article in English | MEDLINE | ID: mdl-8943247

ABSTRACT

The human hepatic asialoglycoprotein receptor is a noncovalent hetero-oligomer composed of two homologous subunits, H1 and H2, with an as yet unknown stoichiometry. Ligand specificity and binding affinity depend on the arrangement of the subunits in the complex. An 80-amino acid segment connecting the transmembrane and the carbohydrate binding domains contains heptad repeats characteristic of alpha-helical coiled coil structure. We expressed and purified corresponding peptides, H1S and H2S, and confirmed by circular dichroism spectroscopy that they can assume alpha-helical conformation. Oxidative cross-linking of amino-terminal cysteines generated specific covalent oligomers, indicating that separately H1S forms trimers and H2S tetramers. Upon mixing, covalent heterotetramers were formed with a preferred stoichiometry of 2 H1S and 2 H2S peptides. These results suggest that the stalk segments of the receptor subunits oligomerize to constitute an alpha-helical coiled coil stalk on top of which the carbohydrate binding domains are exposed for ligand binding. We propose that the functional asialoglycoprotein receptor is a 2:2 heterotetramer.


Subject(s)
Asialoglycoproteins/metabolism , Receptors, Cell Surface/chemistry , Amino Acid Sequence , Asialoglycoprotein Receptor , Circular Dichroism , Fluorescent Antibody Technique , Humans , Liver/chemistry , Molecular Sequence Data , Protein Conformation , Receptors, Cell Surface/metabolism
18.
J Biol Chem ; 273(17): 10602-8, 1998 Apr 24.
Article in English | MEDLINE | ID: mdl-9553121

ABSTRACT

We have investigated the oligomerization process of tenascin-C using a variety of recombinant wild-type and mutant polypeptide chain fragments produced by heterologous gene expression in Escherichia coli. Biochemical and biophysical analyses of the structures and assemblies of these fragments indicated a sequential two-step oligomerization mechanism of tenascin-C involving the concerted interaction of two distinct domains and cysteines 64, 111, and 113. First, the sequence between alanine 114 and glutamine 139 initiates hexabrachion formation via a parallel three-stranded coiled coil. Subsequently, the tenascin assembly domain, which is unique to the tenascins, is responsible for the connection of two triplets to a hexamer. The oligomerization of the tenascin assembly domains by the three-stranded coiled coil increases their homophilic binding affinity and is an important prerequisite for tenascin-C hexamerization. Although formation of the characteristic hexabrachion structure involves the covalent linkage of the six subunits by cysteine residues, mutational analysis indicates that hexamer formation is not dependent on intermolecular disulfide bonds. Most interestingly, substitution of glutamate 130 within the coiled-coil domain by leucine or alanine resulted in the formation of parallel four-stranded helix structures, which further associated to dodecamers. Aside from supporting a sequential process of tenascin-C assembly, this finding provides experimental evidence that non-core residues can have profound effects on the oligomerization states of coiled coils.


Subject(s)
Protein Folding , Tenascin/chemistry , Amino Acid Sequence , Amino Acid Substitution , Animals , Biopolymers , Humans , Microscopy, Electron , Molecular Sequence Data , Protein Conformation , Recombinant Proteins/chemistry , Sequence Homology, Amino Acid
19.
J Cell Sci ; 112 ( Pt 21): 3627-39, 1999 Nov.
Article in English | MEDLINE | ID: mdl-10523499

ABSTRACT

The human (beta)-cytoplasmic actin differs by only 15 amino acids from Act88F actin which is the only actin expressed in the indirect flight muscle (IFM) of Drosophila melanogaster. To test the structural and functional significance of this difference, we ectopically expressed (beta)-cytoplasmic actin in the IFM of Drosophila that lack endogenous Act88F. When expression of the heterologous actin was regulated by approximately 1.5 kb of the 5' promoter region of the Act88F gene, little (beta)-cytoplasmic actin accumulated in the IFM of the flightless transformants. Including Act88F-specific 5' and 3' untranslated regions (UTRs) yielded transformants that expressed wild-type amounts of (beta)-cytoplasmic actin. Despite the assembly of (beta)-cytoplasmic actin containing thin filaments to which endogenous myosin crossbridges attached, sarcomere organization was deficient, leaving the transformants flightless. Rather than affecting primarily actin-myosin interactions, our findings suggest that the (beta)-cytoplasmic actin isoform is not competent to interact with other actin-binding proteins in the IFM that are involved in the organization of functional myofibrils.


Subject(s)
Actins/genetics , Cytoplasm/genetics , Drosophila melanogaster/physiology , Flight, Animal/physiology , Muscle, Skeletal/chemistry , Animals , Cytoplasm/chemistry , Gene Expression Regulation/genetics , Humans , Microscopy, Electron , Muscle, Skeletal/physiology , Muscle, Skeletal/ultrastructure , Mutagenesis/genetics , Promoter Regions, Genetic/genetics , Protein Isoforms/genetics , Transgenes/genetics
20.
Biochemistry ; 38(40): 13263-9, 1999 Oct 05.
Article in English | MEDLINE | ID: mdl-10529199

ABSTRACT

A detailed understanding of GABAB receptor assembly is an important issue in view of its role as attractive target for treatment of epilepsy, anxiety, depression, cognitive defects, and nociceptive disorders. Heteromerization of GABAB-R1 and GABAB-R2 subunits is a prerequisite for the formation of a functional GABAB receptor. Each individual subunit contains one stretch of approximately 30 amino acid residues within its intracellular C-terminal domain that mediates heteromer formation. To investigate the mechanism of the GABAB-R1/GABAB-R2 interaction and to assess the subunit stoichiometry of the complex, recombinant polypeptide chain fragments containing the heteromerization site were produced by heterologous gene expression in Escherichia coli. When mixed in equimolar amounts, these peptides preferentially formed parallel coiled-coil heterodimers under physiological buffer conditions. This demonstrates that the short C-terminal regions are sufficient to determine the specificity of interaction between GABAB receptor subunits. In contrast, isolated GABAB-R1 peptides folded into relatively unstable homodimers, whereas GABAB-R2 peptides were largely unstructured. Together with the data reported in the literature, the results presented here indicate that the functional GABAB receptor is a heterodimer assembled by parallel coiled-coil alpha-helices.


Subject(s)
Receptors, GABA-B/metabolism , Adult , Amino Acid Sequence , Circular Dichroism , Dimerization , Humans , Molecular Sequence Data , Peptide Fragments/chemical synthesis , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Structure, Secondary , Receptors, GABA-B/chemistry , Receptors, GABA-B/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Repetitive Sequences, Amino Acid , Sequence Alignment , Static Electricity
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