David Duda

The refined atomic structure of carbonic anhydrase II at 1.05 ˚ A resolution: implications of chemical rescue of proton transfer

Using synchrotron radiation and a CCD detector, X-ray data have been collected at 100 K for the His64Ala mutant of human carbonic anhydrase II complexed with 4-methylimidazole (4-MI) to a maximal 1.05 A resolution, allowing full anisotropic least-squares refinement. The refined model has a conventional R factor of 15.7% for all reflections. The C(alpha) coordinates of the model presented here have an r.m.s. deviation of 0.10 A relative to the previously determined structure at 1.6 A resolution. Several amino-acid residues (six of the 255 observed) have been identified with multiple rotamer side-chain conformations. C, N and O atoms can be differentiated with selective electron-density map contouring. The estimated standard deviations for all main-chain non-H atom bond lengths and angles are 0.013 and 0.030 A, respectively, based on unrestrained full-matrix least-squares refinement. This structure gives detailed information about the tetrahedrally arranged zinc ion coordinated by three histidine N atoms (His94 N(epsilon 2), His96 N(epsilon2) and His119 N(delta1)) and a water/hydroxide, the multiple binding sites of the proton chemical rescue molecule 4-MI and the solvent networks linking the zinc-bound water/hydroxide and 4-MI molecules. This structure presents the highest resolution structure of a carbonic anhydrase isozyme so far determined and adds to the understanding of proton-transfer processes.

Crystallization and preliminary X-ray analysis of human carbonic anhydrase III

Carbonic anhydrases catalyze the interconversion of carbon dioxide to bicarbonate. Human carbonic anhydrase isozyme III with a C-terminal hexahistidine tag was overexpressed in Eschericha coli, purified and crystallized. Diffraction data (93.4% completeness) were collected to 2.2 A resolution on an in-house R-AXIS IV++ image-plate system with Osmic mirrors and a Rigaku HU-H3R CU rotating-anode generator operating at 50 kV and 100 mA. A 60 degrees sweep of data were collected from a single crystal with a crystal-to-detector distance of 150 mm and a 0.5 degrees oscillation angle per frame using an exposure of 60 s per frame at 293 K. The crystals were shown to conform to the Laue hexagonal crystal system P6, with unit-cell parameters a = 44.7, c = 222.5 A and a scaling R(sym) of 0.087 for 11 962 unique reflections. Using the known crystal structure of the rat form of carbonic anhydrase isozyme III, a molecular-replacement model was built. This model was used for rotation and translation searches and uniquely defined the space group as P6(5). Rigid-body refinement of the model was used to generate an initial phased electron-density map with an R(work) of 31.17%.

Ultra-High Resolution X-Ray Diffraction From Crystals Of The Kinetic Mutant Of Human Carbonic Anhydrase Ii, His 64 Ala, And Its Complexes With Proton Acceptor Donors.

Crystals of human carbonic anhydrase II with a specific point mutation, His 64 to Ala, have been grown in a solution of ammonium sulfate in the presence of mercury chloride. The crystals appear in approximately two weeks and belong to the monoclinic space group P21, with unit cell parameters of a = 42.2 A, b = 41.4 A, c = 71.9 A , beta= 104.2 o and one carbonic anhydrase molecule in the asymmetric unit. The crystals diffract X-rays beyond 1.0 A resolution. These crystals, soaked with exogenous proton acceptor donors, will be used in X-ray and neutron diffraction studies to map the fine water structure “proton wire” in the active site of carbonic anhydrase and to assign the intra- and intermolecular proton transfer pathway(s) from the zinc-bound water out to the bulk solvent.