Simone Brunie

Structure—activity relationships of methionyl-trna synthetase: graphics modelling and genetic engineering

Abstract The 3D structure of the methionyl-tRNA synthetase from E. coli has been investigated using X-ray analysis 1,2 at a resolution 1.8A. 90% of the molecule is now well defined and the zinc atom has been identified in a buried region of the molecule, close to the active site. At the same time, the refinement of the complex ATP-MetRS at 2.5 A has been carried out. The crystallographic R factor has been assigned a value of 25% at 2.5A with an overall temperature factor of 9A 2 and 22% when the individual temperature factors are refined. A Fourier difference map clearly reveals the electron density of the bound ATP, showing the phosphate groups deeply plunging into the active site. In parallel, the synthetase gene has been used to probe some of the enzyme structure-activity relationships. A series of 60 modified enzymes truncated at the C-terminus have been constructed in vitro and assayed for activity. In agreement with the graphics model, the results show that a minimum of 534 residues is necessary to sustain the aminoacylation reaction. A programme of site-directed mutagenesis is in progress: residues thought to be important for the catalytic activity, the metal coordination and tRNA interaction are being modified. Preliminary results are discussed in the light of the crystallographic model.

GEOMETRY OF INTERCALATION OF PSORALENS IN DNA APPROACHED BY MOLECULAR MECHANICS

The results of molecular mechanical calculations on intercalation complexes of 3‐car‐bethoxypsoralen, 5‐methoxypsoralen, 8‐methoxypsoralen, 7‐methylpyrido[3,4‐c]psoralen (MepyPs) and 7‐methylpyrido[4,3‐c]psoralen (2N‐MePyPs) with the double stranded duodecanucleotide d(CGCGATATCGCG)2 are presented. In the energy‐minimized structures, the psoralens are intercalated with their plane orthogonal to the helix axis. Stacking interactions between the furan ring of the psoralen and the adjacent bases are maximized in most derivatives studied, whereas the effect of the various substituents of the psoralen ring is to specifically push part of the molecule towards either the minor or the major groove, preventing a symmetrical intercalation (with respect to the two strands of the DNA). The relative position of the psoralen ring and of the adjacent thymine foreshadows the formation of furan‐side monoadducts in 3‐CPs, MePyPs and 2N‐MePyPs, whereas the formation of a pyrone‐side monoadduct appears as geometrically more favourable in 5‐MOP and both furan‐ and pyrone‐side monoadducts can be geometrically envisaged in 8‐MOP. A good correlation therefore exists between the more or less favourable equilibrium geometries and the experimentally observed photoreactions. The present study is the first attempt to characterize the geometrical parameters as part of a complex set of geometrical, dynamical and excited state parameters governing the overall DNA‐psoralen photoreaction.

Molecular mechanics and dynamics of DNA—furocoumarin complexes: Effect of the aromatization of the pyrone ring on the intercalation geometry☆

Results of molecular mechanics and dynamics calculations on intercalation complexes of DNA with various furocoumarins (psoralen, angelicin, 7-methylpyrido[3,4-c]psoralen and 7-methylpyrido[4,3-c]psoralen) and their corresponding aromatized derivatives are presented. These calculations were undertaken with the aim to elucidate the roles of the pyrone and pyridine moieties in the interactions which tend to orient the furocoumarins and pyridopsoralens between DNA base pairs. It appears that the intercalation geometries are very similar for the furocoumarins and related aromatized compounds. Therefore the oxygen and nitrogen atoms of the pyrone and pyridine moieties are not important in the orientation of the drug within the oligonucleotide.

A unique structural feature of a phospholipase A 2 is probed by molecular dynamics

The unusual catalytic network, revealed by the crystal structure of one of the two phospholipases A2 (PLA2) from the venom of the crotalid A.p.piscivorus has been probed using molecular dynamics. The catalytic network has been remodeled to a conformation similar to that found in all other PLA2, and the modeled structure has been submitted to energy minimization and molecular dynamics simulation, to explore the conformational space of the network. The calculations have yielded a large reorganization of the catalytic network, which gets a conformation close to that of the crystal structure. These results suggest that the unusual catalytic network observed in the studied PLA2 is a structural feature of the protein and not a crystal artifact.