Table 1.
Crystallization, data collection and refinement statistics.
Figure 1.
Ribbon drawings showing the fold and oligomerization of Co-CA.
A: The monomer unit. The zinc ion is shown in cyan. For better orientation, the positions of some residues are indicated. B: The dimer unit. Contacts are mediated by strand β5, and helices α1 and α2 that wrap around the second monomer. C: The tetrameric structure. The tetramer is formed as a dimer of dimers. The chloride ions at the monomer-monomer interface are hidden in panel B, but visible in panel C as orange spheres.
Figure 2.
Interactions at the dimer-dimer interface involve residues at the C-terminal end of Co-CA.
A: Superposition of Co-CA monomer A in blue on Hi-CA monomer E in green (pdb code 2A8D [14]). Whereas the C-terminal helix in Hi-CA extends over 4 turns, the helix in Co-CA breaks after two turns at residue Arg-213. B: The final 24 residues at the C-terminal in Co-CA are directed over the tetramer interface and make extensive hydrophobic- and hydrogen-bond contacts with a symmetry-related monomer (pink). Residues involved in dimer-dimer contacts are shown as ball-and-sticks. Bonds from carbon atoms in monomer A are colored in yellow, and bonds from carbon atoms in the symmetry-related monomer C are colored in white.
Figure 3.
The buried hydrogen-bond network in Co-CA dimers.
A: Ribbon representation. B: Ball-and-stick representation of the channel. Zinc and chloride ions are shown as cyan and orange spheres, respectively; water molecules are shown as red spheres. The buried four water molecules in each monomer are present in all five Co-CA structures.
Figure 4.
The active site of acetazolamide-inhibited Co-CA (Co-CA-AZM).
A: 2|Fobs|-|Fcalc| map of the catalytic site contoured at 1σ level. For clarity the map is calculated only over the acetazolamide, the glycerol molecule, and the zinc ion. Residues are shown in ball-and stick, zinc ion in cyan, and water molecules as red spheres. The glycerol molecule is labeled GOL. Primed residue numbers indicate symmetry-related residues in the dimer. B: Detailed view over the active site and the hydrogen-bonding network in Co-CA-AZM.
Figure 5.
The active site of anion inhibited Co-CA.
2|Fobs|-|Fcalc| maps of the catalytic site in Co-CA contoured at 1σ level. For clarity are all maps calculated only over the inhibitors and the zinc ions. A: Co-CA-PO4. B: Co-CA-IOD. The green mesh corresponds to the difference |Fobs|-|Fcalc| map, calculated in the absence of the zinc-bound water molecule and contoured at 3σ level. C: Co-CA-AZI. D: Co-CA-SCN.
Figure 6.
Superposition of anion- and sulfonamide-inhibited Co-CA.
The color code is as follows: Co-CA-AZM, dark blue; Co-CA-PO4, green; Co-CA-IOD, cyan; Co-CA-AZI, dark purple; Co-CA-SCN, magenta. Zinc and iodide ions are shown as spheres, water molecules as crosses. Superpositions are based on the zinc-binding monomers only.
Figure 7.
Accessibility and electrostatic potentials at the active sites of Co-CA and human CAII.
Different CA enzymes have a differently shaped active site. The middle section i.e. the "channel gate" of the active site cavity in Co-CA is narrow. When the protein is in complex with the smallest inhibitors, i.e. azide ions, the "channel gate" seems closed due to the short distance between Tyr-88′, Gly-107 and Ala-108. A: Co-CA-AZI. B: Co-CA-AZI including the "stepping stone" water molecule. The water is shown as a red sphere with a radius of 1.2 Å. C: Co-CA-AZM with the acetazolamide shown as a ball-and-stick. D: The active site in the 1.1 Å structure of human CAII in complex with acetazolamide (pdb code 3HS4, [41]). The zinc ion is shown in cyan. Red and blue colors represent negative and positive potential, respectively. The elevated positive electrostatic potential at the active sites is due to the zinc ion included in the calculation.
Figure 8.
Superposition of the non-catalytic bicarbonate binding site in Hi-CA with the corresponding area in Co-CA.
The close proximity of the Tyr-44 and Val-52 side chains prevents bicarbonate binding in Co-CA. The structure of Co-CA has carbon bonds colored in yellow, whereas the Hi-CA structure is shown with carbon bonds colored in orange (pdb code 2A8D, [14]). Superposition is based on residues 42-56 and 37-51 in Co-CA and Hi-CA, respectively.
Figure 9.
Schematic view illustrating the similarities of acetazolamide and anion binding in the structures of human CAII and Co-CA.
For all structures the "door keeper" rule is fulfilled. The Co-CA representations show themirror-images of the active site to allow direct comparisons with the α-CA representations (adapted from [33]). hCAI and hCAII stand for human CA I and II, respectively.
Table 2.
The "door keeper" rule of inhibitor binding in Co-CA.
Table 3.
The width of the "channel gate" of the active site cavity of β-CA.
Figure 10.
The catalytic mechanism of Co-CA.
A: The CO2-HCO3- interconversion step. B: The proposed mechanism for the proton transfer step in Co-CA. C: The proton transfer step in human CAII. The coordinates are from the 0.9 Å structure showing a penta-coordinated zinc atom (pdb code 3KS3 [56]).