The laminin-binding domain of agrin is structurally related to N-TIMP-1.

Agrin is the key organizer of postsynaptic differentiation at the neuromuscular junction. This organization activity requires the binding of agrin to the synaptic basal lamina. Binding is conferred by the N-terminal agrin (NtA) domain, which mediates a high-affinity interaction with the coiled coil domain of laminins. Here, we report the crystal structure of chicken NtA at 1.6 A resolution. The structure reveals that NtA harbors an oligosaccharide/oligonucleotide-binding fold with several possible sites for the interaction with different ligands. A high structural similarity of NtA with the protease inhibition domain in tissue inhibitor of metalloproteinases-1 (TIMP-1) supports the idea of additional functions of agrin besides synaptogenic activity.

Subdomain-specific localization of CLIMP-63 (p63) in the endoplasmic reticulum is mediated by its luminal alpha-helical segment.

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.

Stabilization of short collagen-like triple helices by protein engineering.

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.

An intrahelical salt bridge within the trigger site stabilizes the GCN4 leucine zipper.

We previously reported that a helical trigger segment within the GCN4 leucine zipper monomer is indispensable for the formation of its parallel two-stranded coiled coil. Here, we demonstrate that the intrinsic secondary structure of the trigger site is largely stabilized by an intrahelical salt bridge. Removal of this surface salt bridge by a single amino acid mutation induced only minor changes in the backbone structure of the GCN4 leucine zipper dimer as verified by nuclear magnetic resonance. The mutation, however, substantially destabilized the dimeric structure. These findings support the proposed hierarchic folding mechanism of the GCN4 coiled coil in which local helix formation within the trigger segment precedes dimerization.

What are oligomerization domains good for?

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.

Crystal structure of a naturally occurring parallel right-handed coiled coil tetramer.

The crystal structure of a polypeptide chain fragment from the surface layer protein tetrabrachion from Staphylothermus marinus has been determined at 1.8 A resolution. As proposed on the basis of the presence of 11-residue repeats, the polypeptide chain fragment forms a parallel right-handed coiled coil structure. Complementary hydrophobic interactions and complex networks of surface salt bridges result in an extremely thermostable tetrameric structure with remarkable properties. In marked contrast to left-handed coiled coil tetramers, the right-handed coiled coil reveals large hydrophobic cavities that are filled with water molecules. As a consequence, the packing of the hydrophobic core differs markedly from that of a right-handed parallel coiled coil tetramer that was designed on the basis of left-handed coiled coil structures.

Toward a high-resolution structure of phospholamban: design of soluble transmembrane domain mutants.

Determination of a high-resolution structure of the phospholamban (PLB) transmembrane domain by X-ray crystallography or NMR is handicapped by the hydrophobic nature of the peptide. Interestingly, the crystal structure of the five-stranded parallel coiled-coil oligomerization domain from cartilage oligomeric matrix protein (COMPcc) shows marked similarities to a model proposed for the pentameric transmembrane domain of PLB. Contrary to the putative coiled-coil domain of PLB, COMPcc contains mostly hydrophilic amino acids on the surface, resulting in a soluble molecule. Here, we report the design of soluble PLB transmembrane domain variants by combining the surface residues of COMPcc and the hydrophobic interior of the transmembrane domain of PLB. The soluble PLB variants formed pentameric structures as revealed by analytical ultracentrifugation. After redox shuffling, they showed unspecific disulfide bridge patterns similar to that of the chemically synthesized wild-type PLB transmembrane domain. These results suggest a structural homology between the soluble PLB mutants and the wild-type PLB transmembrane domain. Together with the data reported in the literature, they furthermore indicate that residues Leu37, Ile40, Leu44, and Ile47 of the PLB sequence specify pentamer formation. In contrast, a designed recombinant COMPcc mutant, COMP-ARCC, which was engineered to contain the two PLB cysteines that potentially could form an interchain disulfide bridge, formed a specific disulfide bond pattern. This finding indicates structural differences between the transmembrane domain of PLB and COMPcc. The soluble PLB variants may be used to determine a high-resolution structure of the PLB pentamer by X-ray crystallography.

A distinct seven-residue trigger sequence is indispensable for proper coiled-coil formation of the human macrophage scavenger receptor oligomerization domain.

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.

The coiled-coil trigger site of the rod domain of cortexillin I unveils a distinct network of interhelical and intrahelical salt bridges.

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.

Op18/stathmin caps a kinked protofilament-like tubulin tetramer.

Oncoprotein 18/stathmin (Op18), a regulator of microtubule dynamics, was recombinantly expressed and its structure and function analysed. We report that Op18 by itself can fold into a flexible and extended alpha-helix, which is in equilibrium with a less ordered structure. In complex with tubulin, however, all except the last seven C-terminal residues of Op18 are tightly bound to tubulin. Digital image analysis of Op18:tubulin electron micrographs revealed that the complex consists of two longitudinally aligned alpha/beta-tubulin heterodimers. The appearance of the complex was that of a kinked protofilament-like structure with a flat and a ribbed side. Deletion mapping of Op18 further demonstrated that (i) the function of the N-terminal part of the molecule is to 'cap' tubulin subunits to ensure the specificity of the complex and (ii) the complete C-terminal alpha-helical domain of Op18 is necessary and sufficient for stable Op18:tubulin complex formation. Together, our results suggest that besides sequestering tubulin, the structural features of Op18 enable the protein specifically to recognize microtubule ends to trigger catastrophes.

Interaction of agrin with laminin requires a coiled-coil conformation of the agrin-binding site within the laminin gamma1 chain.

Coiled-coil domains are found in a wide variety of proteins, where they typically specify subunit oligomerization. Recently, we have demonstrated that agrin, a multidomain heparan sulfate proteoglycan with a crucial role in the development of the nerve-muscle synapse, binds to the three-stranded coiled-coil domain of laminin-1. The interaction with laminin mediates the integration of agrin into basement membranes. Here we characterize the binding site within the laminin-1 coiled coil in detail. Binding assays with individual laminin-1 full-length chains and fragments revealed that agrin specifically interacts with the gamma1 subunit of laminin-1, whereas no binding to alpha1 and beta1 chains was detected. By using recombinant gamma1 chain fragments, we mapped the binding site to a sequence of 20 residues. Furthermore, we demonstrate that a coiled-coil conformation of this binding site is required for its interaction with agrin. The finding that recombinant gamma1 fragments bound at least 10-fold less than native laminin-1 indicates that the structure of the three-stranded coiled-coil domain of laminin is required for high-affinity agrin binding. Interestingly, no binding to a chimeric gamma2 fragment was observed, indicating that the interaction of agrin with laminin is isoform specific.

Substitution of flight muscle-specific actin by human (beta)-cytoplasmic actin in the indirect flight muscle of Drosophila.

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.

Domain analysis of cortexillin I: actin-bundling, PIP(2)-binding and the rescue of cytokinesis.

Cortexillins are actin-bundling proteins that form a parallel two-stranded coiled-coil rod. Actin-binding domains of the alpha-actinin/spectrin type are located N-terminal to the rod and unique sequence elements are found in the C-terminal region. Domain analysis in vitro revealed that the N-terminal domains are not responsible for the strong actin-filament bundling activity of cortexillin I. The strongest activity resides in the C-terminal region. Phosphatidylinositol 4,5-bisphosphate (PIP(2)) suppresses this bundling activity by binding to a C-terminal nonapeptide sequence. These data define a new PIP(2)-regulated actin-bundling site. In vivo the PIP(2)-binding motif enhances localization of a C-terminal cortexillin I fragment to the cell cortex and improves the rescue of cytokinesis. This motif is not required, however, for translocation to the cleavage furrow. A model is presented proposing that cortexillin translocation is based on a mitotic cycle of polar actin polymerization and midzone depolymerization.

Heterodimerization of a functional GABAB receptor is mediated by parallel coiled-coil alpha-helices.

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.

Contributions of the ionization states of acidic residues to the stability of the coiled coil domain of matrilin-1.

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.

Structural analysis of the sixth immunoglobulin-like domain of mouse neural cell adhesion molecule L1 and its interactions with alpha(v)beta3, alpha(IIb)beta3, and alpha5beta1 integrins.

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.

An autonomous folding unit mediates the assembly of two-stranded coiled coils.

Subunit oligomerization of many proteins is mediated by coiled-coil domains. Although the basic features contributing to the thermodynamic stability of coiled coils are well understood, the mechanistic details of their assembly have not yet been dissected. Here we report a 13-residue sequence pattern that occurs with limited sequence variations in many two-stranded coiled coils and that is absolutely required for the assembly of the Dictyostelium discoideum actin-bundling protein cortexillin I and the yeast transcriptional activator GCN4. The functional relationship between coiled-coil "trigger" sequences was manifested by replacing the intrinsic trigger motif of GCN4 with the related sequence from cortexillin I. We demonstrate that these trigger sequences represent autonomous helical folding units that, in contrast to arbitrarily chosen heptad repeats, can mediate coiled-coil formation. Aside from being of general interest for protein folding, trigger motifs should be of particular importance in the protein de novo design.

All-trans retinol, vitamin D and other hydrophobic compounds bind in the axial pore of the five-stranded coiled-coil domain of cartilage oligomeric matrix protein.

The potential storage and delivery function of cartilage oligomeric matrix protein (COMP) for cell signaling molecules was explored by binding hydrophobic compounds to the recombinant five-stranded coiled-coil domain of COMP. Complex formation with benzene, cyclohexane, vitamin D3 and elaidic acid was demonstrated through increases in denaturation temperatures of 2-10 degreesC. For all-trans retinol and all-trans retinoic acid, an equilibrium dissociation constant KD = 0.6 microM was evaluated by fluorescence titration. Binding of benzene and all-trans retinol into the hydrophobic axial pore of the COMP coiled-coil domain was proven by the X-ray crystal structures of the corresponding complexes at 0.25 and 0.27 nm resolution, respectively. Benzene binds with its plane perpendicular to the pore axis. The binding site is between the two internal rings formed by Leu37 and Thr40 pointing into the pore of the COMP coiled-coil domain. The retinol beta-ionone ring is positioned in a hydrophobic environment near Thr40, and the 1.1 nm long isoprene tail follows a completely hydrophobic region of the pore. Its terminal hydroxyl group complexes with a ring of the five side chains of Gln54. A mutant in which Gln54 is replaced by Ile binds all-trans retinol with affinity similar to the wild-type, demonstrating that hydrophobic interactions are predominant.

NMR structure of a parallel homotrimeric coiled coil.

The solution structure of the oligomerization domain of cartilage matrix protein (also known as matrilin-1) has been determined by heteronuclear NMR spectroscopy. The domain folds into a parallel, disulfide-linked, three-stranded, alpha-helical coiled coil, spanning five heptad repeats in the amino acid sequence. The sequence of the first two heptad repeats shows some deviations from the consensus of hydrophobic and hydrophilic residue preferences. While the corresponding region of the coiled coil has a higher intrinsic flexibility, backbone alpha-helix and superhelix parameters are consistent with a regular coiled coil structure.