Cryoannealing has been demonstrated to improve the diffraction quality and resolution of crystals of the β-carbonic anhydrase psCA3 concomitant with a change in space group. After initial flash-cooling in a liquid-nitrogen cryostream an X-ray diffraction data set from a psCA3 crystal was indexed in space group P2₁2₁2 and was scaled to 2.6 Å resolution, but subsequent cryoannealing studies revealed induced protein rearrangements in the crystal contacts, which transformed the space group to I222, with a corresponding improvement of 0.7 Å in resolution. Although the change in diffraction resolution was significant, only minor changes in the psCA3 structure, which retained its catalytic `open' conformation, were observed. These findings demonstrate that cryoannealing can be successfully utilized to induce higher diffraction-quality crystals while maintaining enzymatically relevant conformations and may be useful as an experimental tool for structural studies of other enzymes where the initial diffraction quality is poor.

Human carbonic anhydrase (CA; EC 4.2.1.1) isoform IX (CA IX) is an extracellular zinc metalloenzyme that catalyzes the reversible hydration of CO₂ to HCO₃⁻, thereby playing a role in pH regulation. The majority of normal functioning cells exhibit low-level expression of CA IX. However, in cancer cells CA IX is upregulated as a consequence of a metabolic transition known as the Warburg effect. The upregulation of CA IX for cancer progression has drawn interest in it being a potential therapeutic target. CA IX is a transmembrane protein, and its purification, yield and crystallization have proven challenging to structure-based drug design, whereas the closely related cytosolic soluble isoform CA II can be expressed and crystallized with ease. Therefore, we have utilized structural alignments and site-directed mutagenesis to engineer a CA II that mimics the active site of CA IX. In this paper, the X-ray crystal structure of this CA IX mimic in complex with sucrose is presented and has been refined to a resolution of 1.5 Å, an R_cryst of 18.0% and an R_free of 21.2%. The binding of sucrose at the entrance to the active site of the CA IX mimic, and not CA II, in a non-inhibitory mechanism provides a novel carbohydrate moiety binding site that could be further exploited to design isoform-specific inhibitors of CA IX.

Glutathione transferase zeta 1 (GSTZ1-1) is a homodimeric enzyme found in the cytosol and mitochondrial matrix of the liver and other tissues. It catalyzes the glutathione-dependent isomerization of maleylacetoacetate to fumarylacetoacetate in the tyrosine catabolic pathway and can metabolize small halogenated carboxylic acids. GSTZ1a-1a crystals diffracted to a resolution of 3.1 Å and belonged to space group P1, with unit-cell parameters a = 42.0, b = 49.6, c = 54.6 Å, α = 82.9, β = 69.9, γ = 73.4°, with a calculated Matthews coefficient of 2.1 ų Da⁻¹ assuming a dimer in the crystallographic asymmetric unit. Refinement of the structure is currently in progress.

The binding of anions to carbonic anhydrase II (CA II) has been attributed to high affinity for the active-site zinc. An anion of interest is cyanate, for which contrasting binding modes have been reported in the literature. Previous spectroscopic data have shown cyanate behaving as an inhibitor, directly binding to the zinc, in contrast to previous crystallographic data that implied that cyanate acts as a substrate mimic that is not directly bound to the zinc but overlaps with the binding site of the substrate CO₂. Wild-type and the V207I variant of CA II have been expressed and X-ray crystal structures of their cyanate complexes have been determined to 1.7 and 1.5 Å resolution, respectively. The rationale for the V207I CA II variant was its close proximity to the CO₂-binding site. Both structures clearly show that the cyanate binds directly to the zinc. In addition, inhibition constants (∼40 µM) were measured using ¹⁸O-exchange mass spectrometry for wild-type and V207I CA II and were similar to those determined previously (Supuran et al., 1997). Hence, it is concluded that under the conditions of these experiments the binding of cyanate to CA II is directly to the zinc, displacing the zinc-bound solvent molecule, and not in a site that overlaps with the CO₂ substrate-binding site.