Background: Enzymes have evolved to recognise their target substrates with exquisite selectivity and specificity. Whether fragments of the substrate--perhaps never available to the evolving enzyme--are bound in the same manner as the parent substrate addresses the fundamental basis of specificity. An understanding of the relative contributions of individual portions of ligand molecules to the enzyme-binding interaction may offer considerable insight into the principles of substrate recognition.
Results: We report 12 crystal structures of Escherichia coli thymidylate synthase in complexes with available fragments of the substrate (dUMP), both with and without the presence of a cofactor analogue. The structures display considerable fidelity of binding mode and interactions. These complexes reveal several interesting features: the cofactor analogue enhances the localisation of substrate and substrate fragments near the reactive thiol; the ribose moiety reduces local disorder through additional specific enzyme-ligand interactions; the pyrimidine has multiple roles, ranging from stereospecificity to mechanistic competence; and the glycosidic linkage has an important role in the formation of a covalent attachment between substrate and enzyme.
Conclusions: The requirements of ligand-protein binding can be understood in terms of the binding of separate fragments of the ligand. Fragments which are subsystems of the natural substrate for the enzyme confer specific contributions to the binding affinity, orientation or electrostatics of the enzymatic mechanism. This ligand-binding analysis provides a complementary method to the more prevalent approaches utilising site-directed mutagenesis. In addition, these observations suggest a modular approach for rational drug design utilising chemical fragments.