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Final Activity Report Summary - GLYCO NMR (NMR aided design of inhibitors for human glycosyltransferases)

During this project, protocols for the efficient recombinant expression of the enzyme human blood group B galactosyltransferase (GTB) and mutants for use in NMR spectroscopy have been set up and optimised. It has also included the preparation of uniformly 15N and 15N, 2H isotope labelled, as well as amino acid selective 15N/1H uniformly 2H labeled enzyme. NMR experiments revealing information about the molecular recognition of both, donor and acceptor substrates, have been carried out. Transferred NOE and STD NMR experiments allowed to study the binding processes looking at ligand signals, whereas preliminary TROSY HSQC experiments pave the way for future binding studies looking at protein signals.

Experimental data has been quantitatively analysed by using a complete matrix relaxation approach that, in combination with modelling calculations (docking), has allowed the refinement of the bound conformations of donor ligands UDP-Gal and UDP-Glc in the binding pocket of GTB, as well as obtaining kinetic rates for dissociation. In addition SPR experiments have delivered dissociation constants for some of the ligands. Finally, the role of the divalent cation has also been clarified. The results show that the bioactive conformer of UDP-Gal is a folded conformation showing close contacts between the galactose and the ribose sugars. This result agrees with the crystal structure of GTB:UDP complex, and determines the orientation of the galactose in the binding pocket, that could not be obtained by X-ray diffraction. The recognition of the donor by GTB involves a selection of minor conformers of the free ligand in solution. Mathematical analysis of the STD curves, yielded the dissociation rate for the binding of UDP-Gal (koff=10 Hz). Similar analysis for the H-antigen acceptor showed faster dissociation, with off-rates at least a factor of 10 higher. This can be important for the mechanism, as differences in residence times for substrates can be used by GTB for keeping the unstable donor safe from degradation and other side reactions, while different binding events occur in the adjacent binding pocket until a proper acceptor binds.

Studies with UDP-Glc showed that this ligand is recognized by the enzyme with a similar bound conformation (folded conformer). Nevertheless, virtually no transfer reaction to acceptor substrates is observed. From the bound conformations of both nucleotides we hypothesized that two key side chains (D302 and E303, conserved in retaining galactosyltransferases) act as tweezers that favour a further conformational transition of the hexopyranose towards the transition state, triggered upon binding of the acceptor. This necessarily involves an optimal distance between the OH-4 and the carboxylate that is possible only for the axial orientation of OH-4 in the galactose ring. On the other hand NMR experiments in the presence and in the absence of Mg2+ demonstrated that the ion cofactor is not a strict requirement for binding, but it is necessary for optimizing the orientation of the galactose ring in the binding pocket of GTB for an efficient transfer reaction. Thus, in the absence of Mg2+ ions, UDP-Gal and UDP-Glc bind in an extended conformation, presumably the most populated in solution, in which characteristically only the UDP part is recognized by the enzyme.

The addition of Mg2+ leads to the folded bound conformations, in which all, uracil, ribose, and Gal or Glc, present short contacts with the protein surface. The effect of the proper orientation of the hexose driven by the divalent cation on the catalytic efficiency is clear from activity assays that showed that, in the absence of Mg2+, the enzyme still processes the substrate, although with a notably reduced rate. In parallel, uniformly 2H and 15N labelled GTB, as well as 15N labelled GTB, were subjected to TROSY HSQC experiments at 700 MHz. The enzyme gives well resolved spectra only at high temperatures of above 50 C. Below this temperature the spectra indicate reversible dimerisation of the protein. Thus, it will be necessary to find experimental conditions where aggregation is prevented at physiological temperatures without loss of enzyme activity.

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