Ruth Landwehr

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.

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.

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.

Collagen stabilization at atomic level: crystal structure of designed (GlyProPro)10foldon.

In a designed fusion protein the trimeric domain foldon from bacteriophage T4 fibritin was connected to the C terminus of the collagen model peptide (GlyProPro)(10) by a short Gly-Ser linker to facilitate formation of the three-stranded collagen triple helix. Crystal structure analysis at 2.6 A resolution revealed conformational changes within the interface of both domains compared with the structure of the isolated molecules. A striking feature is an angle of 62.5 degrees between the symmetry axis of the foldon trimer and the axis of the triple helix. The melting temperature of (GlyProPro)(10) in the designed fusion protein (GlyProPro)(10)foldon is higher than that of isolated (GlyProPro)(10,) which suggests an entropic stabilization compensating for the destabilization at the interface.

Binding of Ca2+ influences susceptibility of laminin to proteolytic digestion and interactions between domain‐specific laminin fragments

tRNA (m5U54)-methyltransferase (EC 2.1.1.35) catalyzes the transfer of methyl groups from S-adenosyl-L-methionine to transfer ribonucleic acid (tRNA) and thereby forming 5-methyluridine (m5U, ribosylthymine) in position 54 of tRNA. This enzyme, which is involved in the biosynthesis of all tRNA chains in Escherichia coli, was purified 5800-fold. A hybrid plasmid carrying trmA, the structural gene for tRNA (m5U54)-methyltransferase was used to amplify genetically the production of this enzyme 40-fold. The purest fraction contained three polypeptides of 42 kDa, 41 kDa and 32 kDa and a heterogeneous 48-57-kDa RNA-protein complex. All the polypeptides seem to be related to the 42/41-kDa polypeptides previously identified as the tRNA (m5U54)-methyltransferase. RNA comprises about 50% (by mass) of the complex. The RNA seems not to be essential for the methylation activity, but may increase the activity of the enzyme. The amino acid composition is presented and the N-terminal sequence of the 42-kDa polypeptide was found to be: Met-Thr-Pro-Glu-His-Leu-Pro-Thr-Glu-Gln-Tyr-Glu-Ala-Gln-Leu-Ala-Glu-Lys- . The tRNA (m5U54)-methyltransferase has a pI of 4.7 and a pH optimum of 8.0. The enzyme does not require added cations but is stimulated by Mg2+. The apparent Km for tRNA and S-adenosyl-L-methionine are 80 nM and 17 microM, respectively.

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 Å 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.

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.

A Fragment of Laminin Comprising the Entire Long Arm

Laminin is one of the major noncollagenous protein of the extracellular matrix. It consists of three nonidentical polypeptide chains (designated A, B1 and B2) which are arranged into the structure of an elongated cross with three short arms and one long arm. The organization and insertion of the laminin molecule in the basement membrane may involve fixation of the divalent cation calcium, since laminin can be extracted from basement membrane containing tissues by calcium sequestrating reagents (Paulsson et al. 1987). In vitro studies have shown that laminin aggregates in the presence of calcium (Yurchenco et al. 1985).