Jörg Stetefeld

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.

Proteolytic E‐cadherin activation followed by solution NMR and X‐ray crystallography

Cellular adhesion by classical cadherins depends critically on the exact proteolytic removal of their N‐terminal prosequences. In this combined solution NMR and X‐ray crystallographic study, the consequences of propeptide cleavage of an epithelial cadherin construct (domains 1 and 2) were followed at atomic level. At low protein concentration, the N‐terminal processing induces docking of the tryptophan‐2 side‐chain into a binding pocket on the same molecule. At high concentration, cleavage induces dimerization (KD=0.72 mM, koff=0.7 s−1) and concomitant intermolecular exchange of the βA‐strands and the tryptophan‐2 side‐chains. Thus, the cleavage represents the switch from a nonadhesive to the functional form of cadherin.

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.

Storage function of cartilage oligomeric matrix protein: the crystal structure of the coiled-coil domain in complex with vitamin D3

The five‐stranded coiled‐coil domain of cartilage oligomeric matrix protein (COMPcc) forms a continuous axial pore with binding capacities for hydrophobic compounds, including prominent cell signalling molecules. Here, we report the X‐ray structure of the COMPcc domain in complex with vitamin D3 at 1.7 Å resolution. The COMPcc pentamer harbours two molecules of the steroid hormone precursor in a planar s‐trans conformation of the conjugated triene, with the aliphatic tails lying along the molecule axis. A hydrophilic ring of five Gln54 side chains divides the channel into two hydrophobic compartments in which the bound vitamin D3 pair is fixed in a head‐to‐head orientation. Vitamin D3 binding induces a volumetric increase of the cavities of ∼30% while the main chain distances of the pentamer are retained. This adaptation to the bulky ring systems of the ligands is accomplished by a rotamer re‐orientation of β‐branched side chains that form the knobs into holes of the coiled‐coil structure. Compared with binding of vitamin D and retinoic acid by their classical receptors, COMP exerts a distinct mechanism of interaction mainly defined by the pattern of hydrophobic core residues.

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.

Mapping of the laminin-binding site of the N-terminal agrin domain (NtA)

Agrin is a key organizer of acetylcholine receptor (AChR) clustering at the neuromuscular junction. The binding of agrin to laminin is required for its localization to synaptic basal lamina and other basement membranes. The high‐affinity interaction with the coiled‐coil domain of laminin is mediated by the N‐terminal domain of agrin. We have adopted a structurally guided site‐directed mutagenesis approach to map the laminin‐binding site of NtA. Mutations of L117 and V124 in the C‐terminal helix 3 showed that they are crucial for binding. Both residues are located in helix 3 and face the groove between the β‐barrel and the C‐terminal helical segment of NtA. Remark ably, the distance between both residues matches a heptad repeat distance of two aliphatic residues which are solvent exposed in the coiled‐coil domain of laminin. A lower but significant contribution originates from R43 and a charged cluster (E23, E24 and R40) at the open face of the β‐barrel structure. We propose that surface‐exposed, conserved residues of the laminin γ1 chain interact with NtA via hydrophobic and ionic interactions.

Structure/function analysis of spinalin, a spine protein of Hydra nematocysts

The nematocyst capsules of the cnidarians are specialized explosive organelles that withstand high osmotic pressures of ≈ 15 MPa (150 bar). A tight disulfide network involving cysteine‐rich capsule wall proteins, like minicollagens and nematocyst outer wall antigen, characterizes their molecular composition. Nematocyst discharge leads to the expulsion of a long inverted tubule that was coiled inside the capsule matrix before activation. Spinalin has been characterized as a glycine‐rich, histidine‐rich protein associated with spine structures on the surface of everted tubules. Here, we show that full‐length Hydra spinalin can be expressed recombinantly in HEK293 cells and has the property to form disulfide‐linked oligomers, reflecting its state in mature capsules. Furthermore, spinalin showed a high tendency to associate into dimers in vitro and in vivo. Our data, which show incomplete disulfide connectivity in recombinant spinalin, suggest a possible mechanism by which the spine structure may be linked to the overall capsule polymer.