The impact of molecular variants, crystallization conditions and the space group on ligand – protein complexes: a case study on bacterial phosphotriesterase

A bacterial phosphotriesterase was employed as an experimental paradigm to examine the effects of multiple factors, such as the molecular constructs, the ligands used during protein expression and purification, the crystallization conditions and the space group, on the visualization of molecular complexes of ligands with a target enzyme. In this case, the ligands used were organophosphates that are fragments of the nerve agents and insecticides on which the enzyme acts as a bioscavenger. 12 crystal structures of various phosphotriesterase constructs obtained by directed evolution were analyzed, with resolutions of up to 1.38   Å . Both apo forms and holo forms, complexed with the organophosphate ligands, were studied. Crystals obtained from three different crystallization conditions, crystallized in four space groups, with and without N-terminal tags, were utilized to investigate the impact of these factors on visualizing the organophosphate complexes of the enzyme. The study revealed that the tags used for protein expression can lodge in the active site and hinder ligand binding. Furthermore, the space group in which the protein crystallizes can significantly impact the visualization of bound ligands. It was also observed that the crystallization precipitants can compete with, and even preclude, ligand binding, leading to false positives or to the incorrect identification of lead drug candidates. One of the co-crystallization conditions enabled the definition of the space...

Source: Acta Crystallographica Section D - October 20, 2023 Category: Biochemistry Authors: Dym, O. Aggarwal, N. Ashani, Y. Leader, H. Albeck, S. Unger, T. Hamer-Rogotner, S. Silman, I. Tawfik, D.S. Sussman, J.L. Tags: crystallization conditions zinc ions phosphotriesterases organophosphates space groups bioscavengers expression systems research papers Source Type: research