Utilisation de multicouches planes et courbees pour l'analyse d'emissions X caracteristiques et d'effets d'anomalie en reflexion speculaire de rayonnement synchrotron

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In-plate protein crystallization, in situ ligand soaking and X-ray diffraction.

International audienc

X-ray structure reveals a new class and provides insight into evolution of alkaline phosphatases.

The alkaline phosphatase (AP) is a bi-metalloenzyme of potential applications in biotechnology and bioremediation, in which phosphate monoesters are nonspecifically hydrolysed under alkaline conditions to yield inorganic phosphate. The hydrolysis occurs through an enzyme intermediate in which the catalytic residue is phosphorylated. The reaction, which also requires a third metal ion, is proposed to proceed through a mechanism of in-line displacement involving a trigonal bipyramidal transition state. Stabilizing the transition state by bidentate hydrogen bonding has been suggested to be the reason for conservation of an arginine residue in the active site. We report here the first crystal structure of alkaline phosphatase purified from the bacterium Sphingomonas. sp. Strain BSAR-1 (SPAP). The crystal structure reveals many differences from other APs: 1) the catalytic residue is a threonine instead of serine, 2) there is no third metal ion binding pocket, and 3) the arginine residue forming bidentate hydrogen bonding is deleted in SPAP. A lysine and an aspargine residue, recruited together for the first time into the active site, bind the substrate phosphoryl group in a manner not observed before in any other AP. These and other structural features suggest that SPAP represents a new class of APs. Because of its direct contact with the substrate phosphoryl group, the lysine residue is proposed to play a significant role in catalysis. The structure is consistent with a mechanism of in-line displacement via a trigonal bipyramidal transition state. The structure provides important insights into evolutionary relationships between members of AP superfamily

Combining 'dry' co-crystallization and in situ diffraction to facilitate ligand screening by X-ray crystallography.

International audienceX-ray crystallography is an established technique for ligand screening in fragment-based drug-design projects, but the required manual handling steps - soaking crystals with ligand and the subsequent harvesting - are tedious and limit the throughput of the process. Here, an alternative approach is reported: crystallization plates are pre-coated with potential binders prior to protein crystallization and X-ray diffraction is performed directly 'in situ' (or in-plate). Its performance is demonstrated on distinct and relevant therapeutic targets currently being studied for ligand screening by X-ray crystallography using either a bending-magnet beamline or a rotating-anode generator. The possibility of using DMSO stock solutions of the ligands to be coated opens up a route to screening most chemical libraries

Refinement statistics for native SPAP.

<p>*The numbers between parentheses indicate the value in the outer resolution shell. R.m.s =  root mean square.</p

Lys171 in SPAP, a substitute for third metal: Superposition of SPAP (magenta) and ECAP (cyan).

<p>(A) amino acid substitutions that abolish Mg²⁺ binding site in SPAP. (B) Comparision of interactions of Mg²⁺ (ECAP) and Lys171 (SPAP) with substrate phosphate and active site aspartate.</p

Metal coordination distances in SPAP and ECAP.

<p>Metal coordination distances in SPAP and ECAP.</p

Active site geometry: co-ordination of zinc metal ions (slate) to protein residues (magenta) and to the substrate in the active site.

<p>The oxygen atom (O3) from the substrate-phosphate is shown in red.</p