Therese Schulthess

The Crystal Structure of a Five-Stranded Coiled Coil in COMP: A Prototype Ion Channel?

Oligomerization by the formation of α-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 α 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.

Two Adjacent Trimeric Fas Ligands Are Required for Fas Signaling and Formation of a Death-Inducing Signaling Complex

ABSTRACT The membrane-bound form of Fas ligand (FasL) signals apoptosis in target cells through engagement of the death receptor Fas, whereas the proteolytically processed, soluble form of FasL does not induce cell death. However, soluble FasL can be rendered active upon cross-linking. Since the minimal extent of oligomerization of FasL that exerts cytotoxicity is unknown, we engineered hexameric proteins containing two trimers of FasL within the same molecule. This was achieved by fusing FasL to the Fc portion of immunoglobulin G1 or to the collagen domain of ACRP30/adiponectin. Trimeric FasL and hexameric FasL both bound to Fas, but only the hexameric forms were highly cytotoxic and competent to signal apoptosis via formation of a death-inducing signaling complex. Three sequential early events in Fas-mediated apoptosis could be dissected, namely, receptor binding, receptor activation, and recruitment of intracellular signaling molecules, each of which occurred independently of the subsequent one. These results demonstrate that the limited oligomerization of FasL, and most likely of some other tumor necrosis factor family ligands such as CD40L, is required for triggering of the signaling pathways.

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 Å 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.

Electron microscopic evidence for a mucin-like region in chick muscle α-dystroglycan

α‐Dystroglycan has been isolated from chicken cardiac muscle and its molecular weight was estimated to be ≈135 kDa. The avian protein interacts with murine Engelbreth‐Holm‐Swarm (EHS) tumor laminin via interaction with the C‐terminal LG4 and LG5 domains (fragment E3) of the laminin α‐chain. This laminin binding is calcium‐dependent and can be competed by heparin. Electron microscopy investigation on the shape of α‐dystroglycan suggests that the core protein consists of two roughly globular domains connected by a segment which most likely corresponds to a mucin‐like central region also predicted by sequence analysis on mammalian isoforms. This segment may act as a spacer in the dystrophin‐associated glycoproteins complex exposing the N‐terminal domain of α‐dystroglycan to laminin in the extracellular space.

A distinct 14 residue site triggers coiled‐coil formation in cortexillin I

We have investigated the process of the assembly of the Dictyostelium discoideum cortexillin I oligomerization domain (Ir) into a tightly packed, two‐stranded, parallel coiled‐coil structure using a variety of recombinant polypeptide chain fragments. The structures of these Ir fragments were analyzed by circular dichroism spectroscopy, analytical ultracentrifugation and electron microscopy. Deletion mapping identified a distinct 14 residue site within the Ir coiled coil, Arg311–Asp324, which was absolutely necessary for dimer formation, indicating that heptad repeats alone are not sufficient for stable coiled‐coil formation. Moreover, deletion of the six N‐terminal heptad repeats of Ir led to the formation of a four‐ rather than a two‐helix structure, suggesting that the full‐length cortexillin I coiled‐coil domain behaves as a cooperative folding unit. Most interestingly, a 16 residue peptide containing the distinct coiled‐coil ‘trigger’ site Arg311–Asp324 yielded ∼30% helix formation as monomer, in aqueous solution. pH titration and NaCl screening experiments revealed that the peptides helicity depends strongly on pH and ionic strength, indicating that electrostatic interactions by charged side chains within the peptide are critical in stabilizing its monomer helix. Taken together, these findings demonstrate that Arg311–Asp324 behaves as an autonomous helical folding unit and that this distinct Ir segment controls the process of coiled‐coil formation of cortexillin I.

TENASCIN-C HEXABRACHION ASSEMBLY IS A SEQUENTIAL TWO-STEP PROCESS INITIATED BY COILED-COIL ALPHA -HELICES

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 inEscherichia 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.

Electron microscopic structure of agrin and mapping of its binding site in laminin‐1

Agrin is a large, multidomain heparan sulfate proteoglycan that is associated with basement membranes of several tissues. Particular splice variants of agrin are essential for the formation of synaptic structures at the neuromuscular junction. The binding of agrin to laminin appears to be required for its localization to synaptic basal lamina and other basement membranes. Here, electron microscopy was used to determine the structure of agrin and to localize its binding site in laminin‐1. Agrin appears as an ∼95 nm long particle that consists of a globular, N‐terminal laminin‐binding domain, a central rod predominantly formed by the follistatin‐like domains and three globular, C‐terminal laminin G‐like domains. In a few cases, heparan sulfate glycosaminoglycan chains were seen emerging from the central portion of the core protein. Moreover, we show that agrin binds to the central region of the three‐stranded, coiled‐coil oligomerization domain in the long arm of laminin‐1, which mediates subunit assembly of the native laminin molecule. In summary, our data show for the first time a protein–protein interaction of the extracellular matrix that involves a coiled‐coil domain, and they assign a novel role to this domain of laminin‐1. Based on this, we propose that agrin associates with basal lamina in a polarized way.

Thermodynamics of Melittin Binding to Lipid Bilayers. Aggregation and Pore Formation

Lipid membranes act as catalysts for protein folding. Both alpha-helical and beta-sheet structures can be induced by the interaction of peptides or proteins with lipid surfaces. Melittin, the main component of bee venom, is a particularly well-studied example for the membrane-induced random coil-to-alpha-helix transition. Melittin in water adopts essentially a random coil conformation. The cationic amphipathic molecule has a high affinity for neutral and anionic lipid membranes and exhibits approximately 50-65% alpha-helix conformation in the membrane-bound state. At higher melittin concentrations, the peptide forms aggregates or pores in the membrane. In spite of the long-standing interest in melittin-lipid interactions, no systematic thermodynamic study is available. This is probably caused by the complexity of the binding process. Melittin binding to lipid vesicles is fast and occurs within milliseconds, but the binding process involves at least four steps, namely, (i) the electrostatic attraction of the cationic peptide to an anionic membrane surface, (ii) the hydrophobic insertion into the lipid membrane, (iii) the conformational change from random coil to alpha-helix, and (iv) peptide aggregation in the lipid phase. We have combined microelectrophoresis (measurement of the zeta potential), isothermal titration calorimetry, and circular dichroism spectroscopy to provide a thermodynamic analysis of the individual binding steps. We have compared melittin with a synthetic analogue, [D]-V(5,8),I(17),K(21)-melittin, for which alpha-helix formation is suppressed and replaced by beta-structure formation. The comparison reveals that the thermodynamic parameters for the membrane-induced alpha-helix formation of melittin are identical to those observed earlier for other peptides with an enthalpy h(helix) of -0.7 kcal/mol and a free energy g(helix) of -0.2 kcal/mol per peptide residue. These thermodynamic parameters hence appear to be of general validity for lipid-induced membrane folding. As g(helix) is negative, it further follows that helix formation leads to an enhanced membrane binding for the peptides or proteins involved. In this study, melittin binds by approximately 2 orders of magnitude better to the lipid membrane than [D]-V(5,8),I(17),K(21)-melittin which cannot form an alpha-helix. We also found conditions under which the isothermal titration experiment reports only the aggregation process. Melittin aggregation is an entropy-driven process with an endothermic heat of reaction (DeltaH(agg)) of approximately 2 kcal/mol and an aggregation constant of 20-40 M(-1).

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°C. For all‐trans retinol and all‐trans retinoic acid, an equilibrium dissociation constant KD = 0.6 μM 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 β‐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.

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 γ1 subunit of laminin‐1, whereas no binding to α1 and β1 chains was detected. By using recombinant γ1 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 γ1 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 γ2 fragment was observed, indicating that the interaction of agrin with laminin is isoform specific.