Effect of crystal freezing and small-molecule binding on internal cavity size in a large protein: X-ray and docking studies of lipoxygenase at ambient and low temperature at 2.0 Å resolution

Abstract

Flash-freezing is a technique that is commonly used nowadays to collect diffraction data for X-ray structural analysis. It can affect both the crystal and molecular structure and the molecule's surface, as well as the internal cavities. X-ray structural data often serve as a template for the protein receptor in docking calculations. Thus, the size and shape of the binding site determines which small molecules could be found as potential ligands in silico, especially during high-throughput rigid docking. Data were analyzed for wild soybean lipoxygenase-3 (MW 97 kDa) at 293 and 93 K and compared with the results from studies of its molecular complexes with known inhibitors, structures published by others for a derivative of the same enzyme (98 K) or a topologically close isozyme lipoxygenase-1 (at ambient temperature and 100 K). Analysis of these data allows the following conclusions. (i) Very small changes in the relative orientation of the molecules in the crystal can cause major changes in the crystal reciprocal lattice. (ii) The volume of the internal cavities can ;shrink' by several percent upon freezing even when the unit-cell and the protein molecular volume show changes of only 1-2%. (iii) Using a receptor structure determined based on cryogenic data as a target for computational screening requires flexible docking to enable the expansion of the binding-site cavity and sampling of the alternative conformations of the crucial residues.

Human Platelet 12-Lipoxygenase, New Findings about Its Activity, Membrane Binding and Low-resolution Structure

“…(2) Do the differences between molecular conformers exist in solution or are they the artifacts of flashfreezing? Meticulous studies of soybean LOX-3 at ambient and cryogenic conditions 35,36 showed the two molecules related in crystal by the 2-fold axis to lose that symmetry upon cooling when molecules were pushed several angstroms closer by the shrinking volume of the crystal. It has also been proven that rapid freezing of crystals not only can impact the conformation of the more flexible peripheral loops, but also can change the relative orientation of the molecules and, as a result, change the geometry of the crystal lattice and impact the size and shape of the internal cavity as well.…”

Probing Dimerization and Structural Flexibility of Mammalian Lipoxygenases by Small-Angle X-ray Scattering

“…Also, rapid crystal freezing can affect not only crystal lattice but also molecules, especially external loops, hydrogen bonding network, and so on, even distribution of water in the cavities. 22 In this study, we first explored, by in silico modeling, possible associations of conformers (A The discrepancy factor χ indicates the difference between the experimental data and the fit of a linear combination of scattering profiles estimated from the monomer-dimer equilibrium as in the crystal structure and in the refined structure. The theoretical R g value calculated based on the crystal coordinates is 28.4 Å.…”

3D Modelling of Three Pro-Inflammatory Molecules in Selected Fish Species

“…It can be speculated that this molecular feature has been selected as an adaptation to lower water temperatures, in agreement with other studies on the thermo-adaptation of proteins to extreme environmental conditions. These molecules are usually characterised by a lower number of stabilizing factors compared to their counterparts from species living in more temperate environments and, therefore, by an higher flexibility [60][61][62]. Hovewer, our preliminary data [44] suggest that the structural differences between icefish IL-1 and sea bass and trout homologues are not linked to a different biological activity of this molecule as the regulation of icefish IL-1 after stimulation with lipopolysaccharide from E. coli and lectin from Phaseolus vulgaris Leucoagglutinin is comparable to other Teleosts.…”