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; K(d) = 6.5 nM) and IDD594 (594; K(d) = 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.

Figure 1

(a) Chemical formula of 594; (b) chemical formula of FID.

Figure 2

Competition experiments in solution monitored by native mass spectrometry. Holo AR was diluted to 10 µM in 10 mM ammonium acetate pH 6.8 either (a) alone or in the presence of (b) 10 µM FID + 10 µM 594, (c) 20 µM FID + 10 µM 594, (d) 10 µM FID + 20 µM 594, and (e) 10 µM FID + 30 µM 594. Mass spectra represent the +12 charge states of holo AR and holo AR/ligand complexes. Relative intensity of each species is given in brackets.

Figure 3

Difference map for the FID = 594 structure data shows the superposition of the inhibitors 594 and FID in the active site. The F_obs − F_calc density map in green is contoured at 3σ. The models of the inhibitors 594 and FID are colored in blue and magenta, respectively.

Figure 4

Difference in the binding of the inhibitors 594 and FID in the structure FID>594. Inhibitor FID (magenta) binds only to the active site while 594 (violet) binds both to the active site and to the specificity pocket (marked S). Residues Phe 122, Leu 130, and Leu 300 in the conformation that binds 594 are shown in cyan, and residue Leu 300 in the conformation that binds FID is shown in magenta. The cartoon representation of the fold in the conformation that binds 594 is shown in green.

Figure 5

Detail of the interaction of 594 with Phe 122 and Leu 300 shows their close contact.

Figure 6

Crystal packing contacts of Loop A (residues 125–127) in the structure FID>594. Two conformations are observed for these residues, one corresponding to the single-inhibitor complex AR–594 (1US0), shown in cyan, and the other to the single-inhibitor AR–FID (1PWM), shown in magenta. The symmetry-related residues Asp 36 and Glu 29 are shown in yellow, and labeled with “FID” and “594” according to their appearance in the structures of the single-inhibitor complexes AR–594 and AR–FID. Note that conformation B of Glu 129, making an H-bond with Ser 127, appears only in the single-inhibitor AR–594 complex and not in the single-inhibitor AR–FID complex.

Figure 7

Comparison of atomic B values in the loop 122–130 for the structures of single-inhibitor AR–FID, single-inhibitor AR–594, FID = 594 and FID < 594. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 8

Water molecules in the active site of FID > 594. The active site contains water molecules that were observed both in the single-inhibitor AR–594 complex (2I16) and in single-inhibitor AR–FID complex (1PWM). Water molecule 2227 is present in model 2I16 and not present in model 1PWM. Water molecules 4296, 6128, and 4238 are present in model 1PWM and not present in model 2I16. 2F_o − F_calc maps are shown for the models 2I16 (colored magenta), 1PWM (colored green), and FID>594 (colored blue). All three maps are contoured at 1.4 σ.