One Crystal, Two Temperatures: Cryocooling Penalties Alter Ligand Binding to Transient Protein Sites

Abstract

Interrogating fragment libraries by X-ray crystallography is a powerful strategy for discovering allosteric ligands for protein targets. Cryocooling of crystals should theoretically increase the fraction of occupied binding sites and decrease radiation damage. However, it might also perturb protein conformations that can be accessed at room temperature. Using data from crystals measured consecutively at room temperature and at cryogenic temperature, we found that transient binding sites could be abolished at the cryogenic temperatures employed by standard approaches. Changing the temperature at which the crystallographic data was collected could provide a deliberate perturbation to the equilibrium of protein conformations and help to visualize hidden sites with great potential to allosterically modulate protein function.

Keywords: X-ray diffraction; allosterism; biophysics; ligand discovery; structural biology; thermodynamics.

Figure 1

Protein structure is more perturbed by temperature and than by ligand binding. A) The distance of each protein residue from the protein center-of-mass is compared between two structures either at different temperatures (green line) or in different ligand states (apo versus with benzimidazole) at the same temperature (red=RT and blue=cryogenic temperature). All temperature pairs were collected consecutively on the same crystal. The amount of offset from zero reflects the expected thermal contraction of the protein upon freezing (green line). The broader distributions indicate structural heterogeneity upon ligand binding at cryogenic temperature (blue line) and structural heterogeneity of the same structure collected at different temperatures (green line). The narrow distribution of the different ligand states at RT (red line) suggests that, at cryogenic temperature, the protein structure is non-specifically perturbed by cryocooling rather than showing a response unique to ligand binding. B) Multiple protein sites display ligand electron density at different temperatures. Electron density was observed at both RT (red mesh) and cryogenic temperature (blue mesh) for the primary cavity site and the M119 surface site, whereas the heme proximal δ-site and the H96 cryptic binding site are temperature sensitive.

Figure 2

Benzimidazole binding to the transient cryptic site is only observed at RT. A) Electron density sampling around His96 Chi-2 using Ringer reveals an electron density peak (dotted circle) for the alternative conformation at room temperature (red line), but not cryogenic temperature (blue line). This minor “open” state would have been missed if the conventional 1σ cutoff to distinguish electron density signal from noise was used or only cryogenic data were available. b) Difference electron density contoured at 3σ (green and red mesh) confirms the presence of the alternative His rotamer and ligand presence at RT (red box). Both the ligand and the alternative His96 conformer were excluded from the crystallographic refinement but are shown in magenta as refined automatically to their final occupancies. At cryogenic temperature (blue box) we observed no ligand electron density but instead only the pocket-filling His96 rotamer and water molecules (blue spheres) that occluded the cryptic site. Note that observing ligand binding to the cryptic site only at RT is unexpected, because thermodynamics should favor binding at cryogenic temperatures.

Figure 3

Cryptic site binders are prevalent in the PDB. A) 2-amino-5-methylthiazole binds to the cryptic site at both temperatures, RT (red box) and cryogenic (blue box). Both the ligand and the alternative His96 conformation (included in refinement) refined to higher ligand occupancy at cryogenic temperatures (0.75 versus 0.54 at RT). B) Electron density maps in other CcP structures, as deposited in the PDB, show evidence of ligand binding with an “open” His96 conformer, either unmodeled (phenol in PDB structure 2AS3, and N-methyl-1H-benzimidazol-2-amine in 4JMZ) or modeled (3-fluorocatechol in 4JMA, and guaiacol in 4A6Z). 2 mF_o−DF_c maps shown as blue mesh (rendered at 1σ), mF_o−DF_c maps in green and red (3σ).