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.

Structural, catalytic and stabilizing consequences of aromatic cluster variants in human carbonic anhydrase II

“…1B; Table 4). The promotion of more favorable C-H···π interactions among the F226 aromatic cluster upon CO 2 binding may contribute significantly to thermostability as has been seen in previous ligand-HCA II complexes (1, 34) and among other proteins (41-43). The extra stability inferred from reorientation of F226 upon ligation of CO 2 may provide additional protection against oxidative (44, 45) and acidic (46, 47) microenvironments encountered within the cell.…”

“…The proton-shuttle residue H64 (11) was seen to be in a dual conformation, ‘in’ and ‘out’, for the F226L and F226W variants, but was found only in the ‘in’ conformation (i.e., towards the active site) in the F226I variant. These variable conformations among crystallographic structures of HCA II are not an uncommon occurrence and are dependent on the crystallization conditions (1, 9, 10, 33, 34). …”

Structural insight into activity enhancement and inhibition of H64A carbonic anhydrase II by imidazoles

“…Although the binding sites relevant for drug design (sites 1-4) reported here are very similar among the human catalytic CAs, the fragment-addition approach could be combined with a tail approach to design more isoform-specific inhibitors, as discussed previously (Aggarwal, Kondeti et al, 2013b). This approach offers a great opportunity, as is shown by carbohydrate (glycerol; Aggarwal, Boone et al, 2013) binding near the active site of CA isoforms.…”

Carbonic Anhydrases and Their Biotechnological Applications

“…High-resolution X-ray [ 75 , 76 ] and neutron structures [ 29 ] of HCA II in complex with acetazolamide (Diamox), a tight binding inhibitor used in the treatment of glaucoma [ 12 , 77 ], has accelerated research into structural-based rational design of an isozyme specific inhibitor. Current research interest includes specific inhibition of HCA IX, which has been shown to be overexpressed in a wide array of cancer cell lines [ 11 , 39 , 78 , 79 , 80 ].…”