Effects of cryoprotectants on the structure and thermostability of the human carbonic anhydrase II-acetazolamide complex

Abstract

Protein X-ray crystallography has seen a progressive shift from data collection at cool/room temperature (277-298 K) to data collection at cryotemperature (100 K) because of its ease of crystal preparation and the lessening of the detrimental effects of radiation-induced crystal damage, with 20-25%(v/v) glycerol (GOL) being the preferred choice of cryoprotectant. Here, a case study of the effects of cryoprotectants on the kinetics of carbonic anhydrase II (CA II) and its inhibition by the clinically used inhibitor acetazolamide (AZM) is presented. Comparative studies of crystal structure, kinetics, inhibition and thermostability were performed on CA II and its complex with AZM in the presence of either GOL or sucrose. These results suggest that even though the cryoprotectant GOL was previously shown to be directly bound in the active site and to interact with AZM, it affects neither the thermostability of CA II nor the binding of AZM in the crystal structure or in solution. However, addition of GOL does affect the kinetics of CA II, presumably as it displaces the water proton-transfer network in the active site.

Keywords: acetazolamide; carbonic anhydrase; cryoprotectants; glycerol; sucrose.

Figure 1

Active sites of (a) CA II, showing ordered waters involved in proton transfer (PDB entry 3ks3; Avvaru et al., 2010 ▶), (b) CA II in complex with AZM (PDB entry 3v2j; this study) and (c) CA II in complex with AZM and GOL (PDB entry 3hs4; Sippel et al., 2009 ▶). Hydrogen bonds are shown as black dotted lines. Waters are colored in gradients of blue from dark (not displaced) to light (AZM displaced) to lighter (GOL displaced). The active-site Zn²⁺ ion is shown as a magenta sphere. This figure was produced using PyMOL (http://www.pymol.org).

Figure 2

Active site of CA II in complex with AZM at (a) RT (298 K), (b) cryo-temperature (100 K) with sucrose and (c) cryo-temperature (100 K) with GOL (PDB entry 3hs4; Sippel et al., 2009 ▶). (d) Surface representation of the three structures superposed. Residues are as labeled. The |2F _o − F _c| electron density is contoured at 1.0σ. Water molecules are shown as red spheres. Hydrogen bonds are shown as black dotted lines. The active-site Zn²⁺ ion is shown as a magenta sphere. This figure was produced using PyMOL (http://www.pymol.org).