Glycosylation is a key mechanism for orchestrating the bioactivity, metabolism and location of small molecules in living cells. In plants, a large multigene family of glycosyltransferases is involved in these processes, conjugating hormones, secondary metabolites, biotic and abiotic environmental toxins, to impact directly on cellular homeostasis. The red grape enzyme UDP-glucose:flavonoid 3-Oglycosyltransferase (VvGT1) is responsible for the formation of anthocyanins, the health-promoting compounds which, in planta, function as colourants determining flower and fruit colour and are precursors for the formation of pigmented polymers in red wine. We show that VvGT1 is active, in vitro, on a range of flavonoids. VvGT1 is somewhat promiscuous with respect to donor sugar specificity as dissected through full kinetics on a panel of nine sugar donors. The three-dimensional structure of VvGT1 has also been determined, both in its 'Michaelis' complex with a UDP-glucose-derived donor and the acceptor kaempferol and in complex with UDP and quercetin. These structures, in tandem with kinetic dissection of activity, provide the foundation for understanding the mechanism of these enzymes in small molecule homeostasis.

Glycosylation of macrolide antibiotics confers host cell immunity from endogenous and exogenous agents. The Streptomyces antibioticus glycosyltransferases, OleI and OleD, glycosylate and inactivate oleandomycin and diverse macrolides including erythromycin, respectively. The structure of these enzyme–ligand complexes, in tandem with kinetic analysis of site-directed variants, provide insight into the interaction of macrolides with their synthetic apparatus. Erythromycin binds to OleD and the 23S RNA of its target ribosome in the same conformation and, although the antibiotic contains a large number of polar groups, its interaction with these macromolecules is primarily through hydrophobic contacts. Erythromycin and oleandomycin, when bound to OleD and OleI, respectively, adopt different conformations, reflecting a subtle effect on sugar positioning by virtue of a single change in the macrolide backbone. The data reported here provide structural insight into the mechanism of resistance to both endogenous and exogenous antibiotics, and will provide a platform for the future redesign of these catalysts for antibiotic remodelling.

Overexpression of the epidermal growth factor (EGF) receptor (EGFR) in cancer cells correlates with tumor malignancy and poor prognosis for cancer patients. For this reason, the EGFR has become one of the main targets of anticancer therapies. Structural data obtained in the last few years have revealed the molecular mechanism for ligand-induced EGFR dimerization and subsequent signal transduction, and also how this signal is blocked by either monoclonal antibodies or small molecules. Nimotuzumab (also known as h-R3) is a humanized antibody that targets the EGFR and has been successful in the clinics. In this work, we report the crystal structure of the Fab fragment of Nimotuzumab, revealing some unique structural features in the heavy variable domain. Furthermore, competition assays show that Nimotuzumab binds to domain III of the extracellular region of the EGFR, within an area that overlaps with both the surface patch recognized by Cetuximab (another anti-EGFR antibody) and the binding site for EGF. A computer model of the Nimotuzumab-EGFR complex, constructed by docking and molecular dynamics simulations and supported by mutagenesis studies, unveils a novel mechanism of action, with Nimotuzumab blocking EGF binding while still allowing the receptor to adopt its active conformation, hence warranting a basal level of signaling. [Cancer Res 2009;69(14):5851-9]

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The plant cell wall degrading apparatus of anaerobic bacteria includes a large multienzyme complex termed the "cellulosome." The complex assembles through the interaction of enzyme-derived dockerin modules with the multiple cohesin modules of the noncatalytic scaffolding protein. Here we report the crystal structure of the Clostridium cellulolyticum cohesindockerin complex in two distinct orientations. The data show that the dockerin displays structural symmetry reflected by the presence of two essentially identical cohesin binding surfaces. In one binding mode, visualized through the A16S/L17T dockerin mutant, the C-terminal helix makes extensive interactions with its cohesin partner. In the other binding mode observed through the A47S/F48T dockerin variant, the dockerin is reoriented by 180°and interacts with the cohesin primarily through the N-terminal helix. Apolar interactions dominate cohesin-dockerin recognition that is centered around a hydrophobic pocket on the surface of the cohesin, formed by Leu-87 and Leu-89, which is occupied, in the two binding modes, by the dockerin residues Phe-19 and Leu-50, respectively. Despite the structural similarity between the C. cellulolyticum and Clostridium thermocellum cohesins and dockerins, there is no cross-specificity between the protein partners from the two organisms. The crystal structure of the C. cellulolyticum complex shows that organism-specific recognition between the protomers is dictated by apolar interactions primarily between only two residues, Leu-17 in the dockerin and the cohesin amino acid Ala-129. The biological significance of the plasticity in dockerin-cohesin recognition, observed here in C. cellulolyticum and reported previously in C. thermocellum, is discussed.