Resveratrol (3,4,5-trihydroxy-trans-stilbene) is a natural phytoalexin found in grapes and wine, which shows antioxidant and antiproliferative activities. In this study we have investigated whether these properties are dependent on similar or different structural determinants of the molecule. To this purpose, resveratrol derivatives, in which all or each single hydroxylic function were selectively substituted with methyl groups, were synthesized. Analogues with the stilbenic double bond reduced or with the stereoisomery modified were also investigated. The antioxidant activity of these compounds was evaluated by measuring the inhibition of citronellal thermo-oxidation, or the reduction of 2,2-diphenyl-1-picrylhydrazyl radical. In addition, the protection against lipid peroxidation was determined in rat liver microsomes, and in human primary cell cultures. The antiproliferative activity was evaluated by a clonogenic assay, and by analysis of cell cycle progression and DNA synthesis. The results showed that the hydroxyl group in 4 position is not the sole determinant for antioxidant activity. In contrast, the presence of 4-OH together with stereoisomery in the trans-conformation (4-hydroxystyryl moiety) was absolutely required for inhibition of cell proliferation. Enzymatic assays in vitro demonstrated that inhibition of DNA synthesis was induced by a direct interaction of resveratrol with DNA polymerases ␣ and ␦.

Collagenases of the matrix metalloproteinase (MMP) family play major roles in morphogenesis, tissue repair, and human diseases, but how they recognize and cleave the collagen triple helix is not fully understood. Here, we report temperature-dependent binding of a catalytically inactive MMP-1 mutant (E200A) to collagen through the cooperative action of its catalytic and hemopexin domains. Contact between the two molecules was mapped by screening the Collagen Toolkit peptide library and by hydrogen/deuterium exchange. The crystal structure of MMP-1(E200A) bound to a triplehelical collagen peptide revealed extensive interactions of the 115-Å-long triple helix with both MMP-1 domains. An exosite in the hemopexin domain, which binds the leucine 10 residues C-terminal to the scissile bond, is critical for collagenolysis and represents a unique target for inhibitor development. The scissile bond is not correctly positioned for hydrolysis in the crystallized complex. A productive binding mode is readily modeled, without altering the MMP-1 structure or the exosite interactions, by axial rotation of the collagen homotrimer. Interdomain flexing of the enzyme and a localized excursion of the collagen chain closest to the active site, facilitated by thermal loosening of the substrate, may lead to the first transition state of collagenolysis. extracellular matrix | X-ray crystallography | protease T he interstitial collagens I, II, and III are the major structural proteins in connective tissues such as skin, bone, cartilage, tendon, and blood vessels (1). They consist of three α chains with repeating Gly-X-Y triplets (X and Y are often proline and hydroxyproline, respectively) that intertwine each other to form a triple helix of ∼300 nm in length (2). Interstitial collagens are resistant to most proteolytic enzymes, but vertebrate collagenases cleave them at a single site approximately three-quarters of the way from the N terminus of the triple helix, thus initiating collagenolysis (3). Owing to this unique activity, collagenases play important roles in embryo development, morphogenesis, tissue remodeling, wound healing, and human diseases, such as arthritis, cancer, and atherosclerosis (4, 5).Matrix metalloproteinase 1 (MMP-1) is a typical vertebrate collagenase (3). It consists of an N-terminal catalytic (Cat) domain containing an active-site zinc ion and a C-terminal hemopexin (Hpx) domain comprised of a four-bladed β-propeller, which are connected by a linker region (6, 7). Although the Cat domain can cleave a number of noncollagenous proteins including heat-denatured collagen (gelatin), its activity on native triplehelical collagen is negligible. The combination of the Cat and Hpx domains is required for MMP-1 to be able to degrade native collagen, and the same is true for all other collagenolytic MMPs, namely MMP-2, MMP-8, MMP-13, and MMP-14 (3). How collagenases interact with collagen and how the Hpx domain endows these enzymes with collagenolytic activity is not clearly understood. Another enigma of collagenolysis bec...

SummaryThe discoidin domain receptors, DDR1 and DDR2, are widely expressed receptor tyrosine kinases that are activated by triple-helical collagen. They control important aspects of cell behavior and are dysregulated in several human diseases. The major DDR2-binding site in collagens I–III is a GVMGFO motif (O is hydroxyproline) that also binds the matricellular protein SPARC. We have determined the crystal structure of the discoidin domain of human DDR2 bound to a triple-helical collagen peptide. The GVMGFO motifs of two collagen chains are recognized by an amphiphilic pocket delimited by a functionally critical tryptophan residue and a buried salt bridge. Collagen binding results in structural changes of DDR2 surface loops that may be linked to the process of receptor activation. A comparison of the GVMGFO-binding sites of DDR2 and SPARC reveals a striking case of convergent evolution in collagen recognition.

Receptor tyrosine kinases of the discoidin domain family, DDR1 and DDR2, are activated by different types of collagen and play important roles in cell adhesion, migration, proliferation, and matrix remodeling. In a previous study, we found that collagen binding by the discoidin domain receptors (DDRs) requires dimerization of their extracellular domains (Leitinger, B. (2003) J. Biol. Chem. 278, 16761-16769), indicating that the paradigm of ligand-induced receptor dimerization may not apply to the DDRs. Using chemical cross-linking and co-immunoprecipitation of differently tagged DDRs, we now show that the DDRs form ligand-independent dimers in the biosynthetic pathway and on the cell surface. We further show that both the extracellular and the cytoplasmic domains are individually dispensable for DDR1 dimerization. The DDR1 transmembrane domain contains two putative dimerization motifs, a leucine zipper and a GXXXG motif. Mutations disrupting the leucine zipper strongly impaired collagen-induced transmembrane signaling, although the mutant DDR1 proteins were still able to dimerize, whereas mutation of the GXXXG motif had no effect. A bacterial reporter assay (named TOXCAT) showed that the DDR1 transmembrane domain has a strong potential for selfassociation in a biological membrane and that this interaction occurs via the leucine zipper and not the GXXXG motif. Our results demonstrate that the DDRs exist as stable dimers in the absence of ligand and that receptor activation requires specific interactions made by the transmembrane leucine zipper. Receptor tyrosine kinases (RTKs)5 control many fundamental cellular processes, such as cell proliferation, differentiation, migration, and metabolism. RTK activity normally is under tight control, and dysregulated RTK activation is associated with most cancers, making RTKs important targets for cancer therapy (1). RTKs allow the cell to respond to external cues; ligand binding to the extracellular domain (ECD) of RTKs results in transphosphorylation of their cytoplasmic domains, which in turn leads to downstream signaling. The prevailing model of RTK activation states that receptors are monomeric in the absence of ligand but become dimerized upon ligand binding; dimerization brings the cytoplasmic domains in close proximity, favoring transphosphorylation (2). However, some studies have found dimerized RTKs in the absence of ligand, suggesting that activation may involve ligand-induced conformational changes within a dimeric receptor (e.g. Refs. 3-5).The discoidin domain receptor (DDR) family of RTKs consists of two members, DDR1 and DDR2, that are characterized by the presence of an extracellular discoidin homology (DS) domain. Uniquely among RTKs, the DDRs are activated by a major extracellular matrix component, triple-helical collagen (6, 7). Several collagen types bind to and activate the DDRs, with the two receptors displaying different specificities toward certain collagen types (6 -8). The DDRs are widely expressed in normal and malignant tissues and control developm...

SummaryThe discoidin domain receptors, DDR1 and DDR2, are constitutively dimeric receptor tyrosine kinases that are activated by triple-helical collagen. Aberrant DDR signaling contributes to several human pathologies, including many cancers. We have generated monoclonal antibodies (mAbs) that inhibit DDR1 signaling without interfering with collagen binding. The crystal structure of the monomeric DDR1 extracellular region bound to the Fab fragment of mAb 3E3 reveals that the collagen-binding discoidin (DS) domain is tightly associated with the following DS-like domain, which contains the epitopes of all mAbs. A conserved surface patch in the DS domain outside the collagen-binding site is shown to be required for signaling. Thus, the active conformation of the DDR1 dimer involves collagen-induced contacts between the DS domains, in addition to the previously identified association of transmembrane helices. The mAbs likely inhibit signaling by sterically blocking the extracellular association of DDR1 subunits.