Production and X-ray crystallographic analysis of fully deuterated human carbonic anhydrase II

Abstract

Human carbonic anhydrase II (HCA II) is a zinc metalloenzyme that catalyzes the reversible hydration and dehydration of carbon dioxide and bicarbonate, respectively. The rate-limiting step in catalysis is the intramolecular transfer of a proton between the zinc-bound solvent (H2O/OH-) and the proton-shuttling residue His64. This distance (approximately 7.5 A) is spanned by a well defined active-site solvent network stabilized by amino-acid side chains (Tyr7, Asn62, Asn67, Thr199 and Thr200). Despite the availability of high-resolution (approximately 1.0 A) X-ray crystal structures of HCA II, there is currently no definitive information available on the positions and orientations of the H atoms of the solvent network or active-site amino acids and their ionization states. In preparation for neutron diffraction studies to elucidate this hydrogen-bonding network, perdeuterated HCA II has been expressed, purified, crystallized and its X-ray structure determined to 1.5 A resolution. The refined structure is highly isomorphous with hydrogenated HCA II, especially with regard to the active-site architecture and solvent network. This work demonstrates the suitability of these crystals for neutron macromolecular crystallography.

Active-site solvent replenishment observed during human carbonic anhydrase II catalysis

“…Neutron studies have been utilized to observe the protonation states and orientation of water molecules in proteins (Langan et al, 2008). Such experiments have determined that the water network in hCA II is pH-dependent, with an unbranched wire between W Zn and His64 at physiological pH that is broken at high pH owing to a rearrangement of the hydrogen bonds of W1 (Budayova-Spano et al, 2006;Fisher et al, 2011). The side chain of His64 is oriented in two conformations, termed the 'in' (pointing towards the active site) and 'out' (pointing away from the active site) positions, that are suggested to facilitate the proton-shuttling process (Tu et al, 1989;Fisher et al, 2005;Nair & Christianson, 1991;Lindskog, 1997;Avvaru et al, 2010).…”

Carbonic Anhydrase as a Model for Biophysical and Physical-Organic Studies of Proteins and Protein−Ligand Binding

“…X-ray structures of both native and mutant isozymes of HCA provide useful information on how inhibitors bind in the active site and at nearby hydrophobic sites (see section 4.6). Table 4 lists all of the structures in the PDB of native HCA with 45,128,162, and without 54,183,184,[239][240][241][242] bound ligands. Table 5 lists the structures of mutant HCAs and the motivation for constructing each mutation.…”

Interfacial assembly of proteins and peptides: recent examples studied by neutron reflection

“…With the benefits of deuteration becoming increasingly realizable, more research activities will be dedicated into this direction in the next few years S660 Review. Interfacial assembly of proteins X. Zhao et al (Meilleur et al 2005;Budayova-Spano et al 2006;Di-Costanzo et al 2007;Liu et al 2007;Laux et al 2008).…”