The pentameric channel of COMPcc in complex with different fatty acids.

BACKGROUND: COMPcc forms a pentameric left-handed coiled coil that is known to bind hydrophilic signaling molecules such as vitamin D(3), and vitamin A. PRINCIPAL FINDINGS: In an integrated approach we reveal the unique binding properties of COMPcc for saturated and unsaturated fatty acids. Our observations suggest that residues Met33 (gating pore), Thr40/Asn41 (water chamber) and Gln54 (electrostatic trap) are key elements for the binding of fatty acids by COMPcc. In addition, this work characterizes the binding of various fatty acids to COMPcc using fluorescence spectroscopy. Our findings reveal a binding trend within the hydrophobic channel of COMPcc, namely, that is driven by length of the methylene tail and incorporation of unsaturation. CONCLUSION/SIGNIFICANCE: The unique binding properties imply that COMPcc may be involved in signalling functions in which hydrophilic ligands are involved. The pentameric channel is a unique carrier for lipophilic compounds. This opens the exciting possibility that COMPcc could be developed as a targeted drug delivery system.

Site specific cleavage mediated by MMPs regulates function of agrin.

BACKGROUND: Agrin is the key inducer of postsynaptic differentiations at the neuromuscular junction. The multidomain heparan sulfate proteoglycan is mediating via its N-terminal segment the interaction with laminin, whereas the C-terminal portion is responsible for Dystroglycan binding and clustering of the Acetylcholine receptor. Matrix metalloproteinases (MMP) are known to play essential roles in matrix remodeling, degradation and regulation of extracellular signaling networks. PRINCIPAL FINDINGS: Site-specific processing of Agrin provides key insight into regulatory effects of Matrix metalloproteinases (MMPs). Here, we present a detailed study of agrin processing by different MMPs together with a molecular understanding of binding and cleavage at both terminal fragments. The data suggest for a regulatory effect of MMP cleavage at particularly important functional sites of agrin. Cleave of agrin abolishes the agrin-laminin complex formation and the Acetylcholine receptor clustering at the neuromuscular junction. CONCLUSION/SIGNIFICANCE: Agrin is a target of specific MMP processing resulting in agrin subfragments with different regulatory activities. MMP processing is a powerful tool to regulate extracellular signaling networks.

The use of coiled-coil proteins in drug delivery systems.

The coiled-coil motif is found in approximately 10% of all protein sequences and is responsible for the oligomerization of proteins in a highly specific manner. Coiled-coil proteins exhibit a large diversity of function (e.g. gene regulation, cell division, membrane fusion, drug extrusion) thus demonstrating the significance of oligomerization in biological systems. The classical coiled-coil domain comprises a series of consecutive heptad repeats in the protein sequence that are readily identifiable by the location of hydrophobic residues at the 'a' and 'd' positions. This gives rise to an alpha-helical structure in which between 2 and 7 helices are wound around each other in the form of a left-handed supercoil. More recently, structures of coiled-coil domains have been solved that have an 11 residue (undecad) or a 15 residue (pentadecad) repeat, which show the formation of a right-handed coiled-coil structure. The high stability of coiled coils, together with the presence of large internal cavities in the pentameric coiled-coil domain of cartilage oligomerization matrix protein (COMPcc) and the tetrameric right-handed coiled coil of Staphylothermus marinus (RHCC) has led us and others to look for therapeutic applications. In this review, we present evidence in support of a vitamin A and vitamin D(3) binding activity for the pentameric COMPcc molecule. In addition, we will discuss exciting new developments which show that the RHCC tetramer is capable of binding the major anticancer drug cisplatin and the ability to fuse it to an antigenic epitope for the development of a new generation of vaccines.

An interdomain disulfide bridge links the NtA and first FS domain in agrin.

Agrin is a multidomain heparan sulfate proteoglycan involved in postsynaptic differentiation at the neuromuscular junction. Binding of agrin to synaptic basal lamina is mediated by the N-terminal agrin (NtA) domain. The NtA domain of agrin is followed by a tandem of nine follistatin-like (FS) domains forming a rod-like spacer to the laminin G-like domains of the molecule. Here we report that the most C-terminal cysteine residue of NtA (Cys123) forms an interdomain disulfide bond with the FOLN subdomain of the FS module. Remarkably, this single cysteine is flanked by Leu117 and Val124, which are two essential beta-branched amino acids forming the heterocomplex of NtA with the gamma 1 chain of laminin. Moreover, we show that this covalent linkage compensates for the seven amino acid residue splice insert at the very C-terminal helix H3 and causes a rigid interface between NtA and FS independent of the alternative mRNA splice event. These results suggest that the interdomain disulfide bond between the NtA and the first FS domain might be important for the proper folding of agrin.