Fig 1.
Structures of sulfonamides discussed in this study.
(AAZ) acetazolamide, N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)acetamide; (MZA) methazolamide, (E)-N-(3-methyl-5-sulfamoyl-1,3,4-thiadiazol-2(3H)-ylidene)acetamide. The values for the inhibitory constants are as reported previously [18].
Table 1.
X-ray data collection statistics.
Table 2.
Properties of the final models.
Fig 2.
The crystal structure of HpαCA and comparison with other bacterial CAs.
A: Stereo diagram of the structure of the HpαCA monomer. Each element of the secondary structure is labeled. The zinc ion and AAZ molecule are shown in ball and in stick representation, respectively, to indicate the location of the active site. B, C, D, E: Dimers observed in the crystal structures of HpαCA, NgCA, SspCA and TaCA, respectively.
Fig 3.
Structure-based sequence alignment of HpαCA, NgCA, SspCA, TaCA and HCAII.
The elements of the secondary structure and the sequence numbering for HpαCA are shown above the alignment. Conserved residues are highlighted in red. The conserved histidine residues coordinating the zinc ion are marked by an asterisk. The proton shuttle residue of HCAII (His64) is marked with an open circle. The corresponding residue in HpαCA is His85. The conserved residues that form a hydrophobic pocket in the active site are labeled with filled squares. The location of the catalytically important HCAII residue Thr199 which forms a hydrogen bond to the zinc-bound water (Wat263, Fig 5), thereby orienting its two lone electron pairs toward the two neighbouring water molecules (Wat318 and Wat338) in the active site is shown by an open square. The corresponding residue in HpαCA is T191.
Fig 4.
Stereo view of the AAZ-binding site in HpαCA and comparison to other αCAs.
A: The (mFo-DFc) sigmaA-weighted [32] electron density for AAZ bound to HpαCA is shown in green. The map was calculated at 2.0-Å resolution and contoured at 3.0-σ level. The AAZ molecule is shown in all-atom ball-and-stick representation with carbon atoms coloured orange. Amino acid residues that interact with AAZ are shown in stick representation. The catalytic zinc ion that is coordinated tetrahedrally (black dash lines) is shown as a black sphere. Hydrogen bonds are shown as blue dashed lines. B: Stereo view of the inhibitor-binding sites of the superimposed HpαCA/AAZ (black), SspCA/AAZ (red), TaCA/AAZ (blue) and HCAII/AAZ (green) complexes. The diagram illustrates the remarkably similar mode of AAZ binding, particularly around the sulfonamide moiety which coordinates the zinc ion. Residues are labeled in HpαCA. C: Stereo view of the superposition of the active site regions of HpαCA (red) and HCAII (blue for the free enzyme, green for the complex with CO2). Residues and water molecules are labeled in HCAII. The “deep” water (Wat338), catalytic water (Wat263) and Wat318 observed in the active site of free HCAII are shown as blue spheres. The CO2 (substrate) observed in the crystal structure of the HCAII/CO2 complex is shown in green. The zinc ion (black sphere) is only shown in the free HCAII structure for clarity.
Fig 5.
Structural comparison between the MZA and AAZ complexes of HpαCA.
The MZA molecule is shown in all-atom representation with carbon atoms coloured in orange. The (mFo-DFc) sigmaA-weighted electron density for MZA is shown in green. The map was calculated at 2.2-Å resolution and contoured at 3.0-σ level. The AAZ molecule is shown in cyan. Protein surface is shown and coloured according to the electrostatic potential. The subtle rotation and shift of MZA with respect to AAZ does not break the hydrogen bond between the O3 atom of the carbonamide moiety and the side chain of Asn108. The weaker binding of MZA in comparison to AAZ is likely due to energetically unfavourable interaction between the additional aliphatic methyl group of MZA and partially negatively charged carbonyl oxygens of the main-chain peptides of Ala192 (C-O distance 4.3 Å) and Pro193 (C-O distance 3.5 Å).