Approaches to solving the rigid receptor problem by identifying a minimal set of flexible residues during ligand docking.

BACKGROUND: Using fixed receptor sites derived from high-resolution crystal structures in structure-based drug design does not properly account for ligand-induced enzyme conformational change and imparts a bias into the discovery and design of novel ligands. We sought to facilitate the design of improved drug leads by defining residues most likely to change conformation, and then defining a minimal manifold of possible conformations of a target site for drug design based on a small number of identified flexible residues. RESULTS: The crystal structure of thymidylate synthase from an important pathogenic target Pneumocystis carinii (PcTS) bound to its substrate and the inhibitor, BW1843U89, is reported here and reveals a new conformation with respect to the structure of PcTS bound to substrate and the more conventional antifolate inhibitor, CB3717. We developed an algorithm for determining which residues provide 'soft spots' in the protein, regions where conformational adaptation suggests possible modifications for a drug lead that may yield higher affinity. Remodeling the active site of thymidylate synthase with new conformations for only three residues that were identified with this algorithm yields scores for ligands that are compatible with experimental kinetic data. CONCLUSIONS: Based on the examination of many protein/ligand complexes, we develop an algorithm (SOFTSPOTS) for identifying regions of a protein target that are more likely to accommodate plastically to regions of a drug molecule. Using these indicators we develop a second algorithm (PLASTIC) that provides a minimal manifold of possible conformations of a protein target for drug design, reducing the bias in structure-based drug design imparted by structures of enzymes co-crystallized with inhibitors.

The structural mechanism for half-the-sites reactivity in an enzyme, thymidylate synthase, involves a relay of changes between subunits.

Thymidylate synthase (TS), a half-the-sites reactive enzyme, catalyzes the final step in the de novo biosynthesis of deoxythymidine monophosphate, dTMP, required for DNA replication. The cocrystal structure of TS from Pneumocystis carinii (PcTS), a new drug target for an important pathogen, with its substrate, deoxyuridine monophosphate (dUMP), and a cofactor mimic, CB3717, was determined. The structure, solved at 2.6 A resolution, shows an asymmetric dimer with two molecules of the substrate dUMP bound yet only one molecule of cofactor analogue bound. The structural evidence reveals that upon binding cofactor analogue and forming a covalent bond from the nucleophilic cysteine to the substrate, dUMP, at one active site, PcTS undergoes a conformational change that renders the opposite monomer incapable of forming a covalent bond or binding a molecule of cofactor analogue. The communication pathway between the two active sites is evident, allowing a structural definition of the basis of half-the-sites reactivity for thymidylate synthase and providing an example of such a mechanism for other half-the-sites reactive enzymes.

Crystal structure of a brominated RNA helix with four mismatched base pairs: An investigation into RNA conformational variability.

The X-ray crystal structure of a brominated RNA helix with four mismatched base pairs and sequence r(UG(Br)C(Br)CAGUUCGCUGGC)(2) was determined to 2.1 A using the methods of multiwavelength anomalous diffraction (MAD) applied to the bromine K-absorption edge. There are three molecules in the asymmetric unit with unique crystal-packing environments, revealing true conformational variability at high resolution for this sequence. The structure shows that the sequence itself does not define a consistent pattern of solvent molecules, with the exception of the mismatched base pairs, implying that specific RNA-protein interactions would occur only with the nucleotides. There are a number of significant tertiary interactions, some of which are a result of the brominated base pairs and others that are directly mediated by the RNA 2' hydroxyl groups. The mismatched base pairs exhibit a solvent network as well as a stacking pattern with their nearest neighbors that validate previous thermodynamic analysis.

Femtosecond studies of hematoporphyrin derivative in solution: effect of pH and solvent on the excited state dynamics.

We report direct femtosecond measurements of the excited state dynamics of hematoporphyrin derivative (HpD) in solution. The dynamics are found to be very sensitive to the solvent and pH of aqueous solutions. The decay of the excited singlet states is much faster in acidic and pH 7 buffer aqueous solutions (< 230 ps) than in basic aqueous solutions or organic solvents (> 10 ns). The dynamical results show strong correlation with static fluorescence measurements: weaker fluorescence in acidic and pH 7 buffer solutions corresponding to shorter-lived excited states. A new fast decay component with a time constant around 5 ps is identified both in acidic aqueous solutions and in organic solvents such as acetone and attributed to internal conversion from the second to the first excited singlet state of aggregates or certain oligomers in HpD, in accord with the observation that the fast decay component is larger at a higher concentration. Oxygen is found to have no effect on the dynamics on the time scale investigated, 1 ns, indicating that oxygen quenching of the singlet excited states is insignificant on this time scale. The sensitive solvent and pH dependence of the excited state dynamics has important clinical implications in the use of HpD as a photosensitizing agent.