Crystal packing modifies ligand binding affinity: The case of aldose reductase

Abstract

The relationship between the structures of protein–ligand complexes existing in the crystal and in solution, essential in the case of fragment-based screening by X-ray crystallography (FBS-X), has been often an object of controversy. To address this question, simultaneous co-crystallization and soaking of two inhibitors with different ratios, Fidarestat (FID; Kd = 6.5 nM) and IDD594 (594; Kd = 61 nM), which bind to h-aldose reductase (AR), have been performed. The subatomic resolution of the crystal structures allows the differentiation of both inhibitors, even when the structures are almost superposed. We have determined the occupation ratio in solution by mass spectrometry (MS) Occ(FID)/Occ(594) = 2.7 and by X-ray crystallography Occ(FID)/Occ(594) = 0.6. The occupancies in the crystal and in solution differ 4.6 times, implying that ligand binding potency is influenced by crystal contacts. A structural analysis shows that the Loop A (residues 122–130), which is exposed to the solvent, is flexible in solution, and is involved in packing contacts within the crystal. Furthermore, inhibitor 594 contacts the base of Loop A, stabilizing it, while inhibitor FID does not. This is shown by the difference in B-factors of the Loop A between the AR–594 and AR–FID complexes. A stable loop diminishes the entropic energy barrier to binding, favoring 594 versus FID. Therefore, the effect of the crystal environment should be taken into consideration in the X-ray diffraction analysis of ligand binding to proteins. This conclusion highlights the need for additional methodologies in the case of FBS-X to validate this powerful screening technique, which is widely used.

Fragments as Novel Starting Points for tRNA‐Guanine Transglycosylase Inhibitors Found by Alternative Screening Strategies

“…Studies on larger ligands have at least shown differences in the affinity in solution and solid state. [29] Additionally in crystallography, ligands have to pass through crystal packing channels to reach the binding site on the protein. These channels can block access, in the simplest case due to steric reason, but electrostatics or hydrophobic barriers can also hamper successful diffusion to the binding sites.…”

Protein–ligand interaction databases: advanced tools to mine activity data and interactions on a structural level

“…Especially surface‐exposed parts of the complex may undergo conformational changes due to interactions with adjacent molecules in the crystal . Also the binding mode of a ligand and the affinity of a ligand for a protein can be influenced by the crystal environment . Thus, when geometrical aspects of the binding between a ligand and a protein are analyzed, one should keep in mind that the binding can be an artifact of the crystal packing.…”

The emerging role of native mass spectrometry in characterizing the structure and dynamics of macromolecular complexes

“…By contrast, native MS was used to demonstrate that crystal packing environment alters the relative binding affinity of aldose reductase for two different inhibitors. 54 Native MS has also been successfully used to investigate the effect of inhibitors on a protein:RNA complex. 55 Another study that combined native MS with X-ray crystallography concerned two nickel import proteins of Staphylococcus aureus.…”

“…The authors used native MS to confirm the 2:1 binding stoichiometry for one of these compounds and to rule out a crystal packing artefact. By contrast, native MS was used to demonstrate that crystal packing environment alters the relative binding affinity of aldose reductase for two different inhibitors . Native MS has also been successfully used to investigate the effect of inhibitors on a protein:RNA complex .…”