Crystal structure of the secretory form of membrane-associated human carbonic anhydrase IV at 2.8-Å resolution

Abstract

It has recently been demonstrated that the C-terminal deletion mutant of recombinant human carbonic anhydrase IV (G267X CA IV) converts the normally glycosylphosphatidylinositol-anchored enzyme into a soluble secretory form which has the same catalytic properties as the membrane-associated enzyme purified from human tissues. We have determined the three-dimensional structure of the secretory form of human CA IV by x-ray crystallographic methods to a resolution of 2.8 Å. Although the zinc binding site and the hydrophobic substrate binding pocket of CA IV are generally similar to those of other mammalian isozymes, unique structural differences are found elsewhere in the active site. Two disufide linkages, Cys-6-Cys-11G and Cys-23-Cys-203, stabilize the conformation of the N-terminal domain. The latter disulfide additionally stabilizes an active site loop containing a cis-peptide linkage between Pro-201 and Thr-202 (this loop contains catalytic residue Thr-199). On the opposite side of the active site, the Val-131-Asp-136 segment adopts an extended loop conformation instead of an ␣-helix conformation as found in other isozymes. Finally, the C terminus is surrounded by a substantial electropositive surface potential, which is likely to stabilize the interaction of CA IV with the negatively charged phospholipid headgroups of the membrane. These structural features are unique to CA IV and provide a framework for the design of sulfonamide inhibitors selective for this particular isozyme.

Carbonic anhydrase inhibitors

“…2,7,[15][16][17] In all these adducts, the deprotonated sulfonamide is coordinated to the Zn(II) ion of the enzyme, and its NH moiety participates in a hydrogen bond with the Og of Thr 199, which in turn is engaged in another hydrogen bond to the carboxylate group of Glu 106. [15][16][17] One of the oxygen atoms of the SO 2 NH moiety also participates in a hydrogen bond with the backbone NH moiety of Thr 199. [15][16][17] In Figure 5, the crystal structures of the hCA II adducts with the simplest compounds incorporating a sulfamoyl moiety (sulfamide and sulfamic acid, respectively) are shown.…”

Three-dimensional structure of a halotolerant algal carbonic anhydrase predicts halotolerance of a mammalian homolog

“…This similarity is exemplified by the structure-based sequence alignment of dCA II (274 aa) with the best-characterized human isozyme, hCA II (259 aa), which shows 24% sequence identity and a rms deviation of 1.4 Å for 176 C ␣ atoms. Like other membrane-attached CAs (34,35), dCA II contains a single disulfide linkage between Cys-31 and Cys-221 (equivalent to Cys-23 and Cys-201 in hCA XII).…”

Pathogenesis of retinitis pigmentosa associated with apoptosis-inducing mutations in carbonic anhydrase IV

“…The loss in activity for the R69H mutation was thus proportional to the reduced level of the protein expressed. However, the R219S substitution nearly abolished the catalytic activity of the CA IV protein expressed, most likely because of its proximity to the active-site cleft (10). The fact that reduction in activity is barely detectable for the R69H mutant argues that loss of CA IV activity itself does not explain the retinal pathology in RP17.…”