Table 1.
Data collection and refinement statistics.
Fig 1.
Overall structure of PprCA and structural comparison with prokaryotic and human α–CA.
(A) Cartoon representation of the PprCA monomer with central ten-stranded β-sheets colored gold. An intra-molecular disulfide bond that stabilizes the monomer is shown as stick model. Catalytic zinc ions are shown as gray spheres while the chloride ion is shown in green (B) Cartoon representation of the PprCA dimer with α-helices and β-sheets colored according to structural conservation. The dimer interface chloride ion is shown in green. The active site zinc ions are shown as grey spheres. (C) Comparison on PprCA monomer with hCAII. hCAII is shown in green while PprCA is shown in blue. Surface loops regions of hCAII are colored red. (D) Chloride ion coordination by four water molecules and Asn 178/ND2 from Chain A (green) and Chain B (blue). 2Fo-Fc electron density contoured at 8.0σ (green) and 1.0σ (blue).
Fig 2.
Cartoon representation of the PprCA active site and oligomerization status of PprCA.
(A) The highly conserved active site residues of PprCA showing Zinc ion in the center of the active site (gray sphere). The hydrophobic (Val117, Val127, Leu181, Val190 and Trp192), the hydrophilic (Tyr17, Asn69, Gln74, Thr182 and Thr183), and zinc coordinating residues (His96, His98 and His115) are similar to other well characterized α–CA’s. (B) Active site water network involved in proton transfer is shown as red spheres. 2Fo-Fc electron density contoured at 8.0σ (green) and 1.0σ in blue. (C) Purified PprCA was analyzed on a Hiprep 16/60 Sephacryl S-200 size-exclusion column that was equilibrated with 20 mM Tris pH 7.5 and 150 mM NaCl. Peaks A and B correspond to dimer and monomer, respectively. The elution fractions were analyzed by SDS-PAGE and stained with Coomassie Blue. A single ~35 kDa band was observed for both dimeric and monomeric PprCA. MW–Molecular-weight marker, lanes 43 to 51 (elution fractions representing PprCA dimer) and lanes 52 to 64 (elution fractions representing PprCA monomer). (D) Purified PprCA was separated under both reducing and non-reducing conditions on a SDS-PAGE. Lanes were loaded with PprCA from monomer fraction, dimer fraction and from a sample containing a mixture of monomers and dimers. The gels were stained either by Coomassie blue or subjected to protonography. Protonography was performed according to De Luca et al, 2015 [9]. The gel was incubated in CO2 enriched water for 5 to 15 seconds at room temperature. Appearance of distinctive yellow bands in gels subjected to protonography indicates both monomeric (~27 kDa) and dimeric (~58 kDa) PprCA is catalytically active.