title: Expression, assay, and structure of the extracellular domain of murine carbonic anhydrase XIV: implications for selective inhibition of membrane-associated isozymes.
abstract: Carbonic anhydrase (CA) XIV is the most recently identified mammalian carbonic anhydrase isozyme, and its presence has been demonstrated in a number of tissues. Full-length CA XIV is a transmembrane protein composed of an extracellular catalytic domain, a single transmembrane helix, and a short intracellular polypeptide segment. The amino acid sequence identity of human CA XIV relative to the other membrane-associated isozymes (CA IV, CA IX, and CA XII) is 34-46%. We report here the expression and purification of both the full-length enzyme and a truncated, secretory form of murine CA XIV. Both forms of this isozyme are highly active, and both show an abrogation of activity in the presence of 0.2% SDS, in contrast to the behavior of murine CA IV. We also report the crystal structure of the extracellular domain of murine CA XIV at 2.8 A resolution and of an enzyme-acetazolamide complex at 2.9 A resolution. The structure shows a monomeric glycoprotein with a topology similar to that of other mammalian CA isozymes. Based on the x-ray crystallographic results, we compare and contrast known structures of membrane-associated CA isozymes to rationalize the structural elements responsible for the SDS resistance of CA IV and to discuss prospects for the design of selective inhibitors of membrane-associated CA isozymes.
authors.affiliation: Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Douglas A
authors.initials: DA
authors.lastname: Whittington
authors.forename: Jeffrey H
authors.initials: JH
authors.lastname: Grubb
authors.forename: Abdul
authors.initials: A
authors.lastname: Waheed
authors.forename: Gul N
authors.initials: GN
authors.lastname: Shah
authors.forename: William S
authors.initials: WS
authors.lastname: Sly
authors.forename: David W
authors.initials: DW
authors.lastname: Christianson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002138:Calibration; D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000235:genetics*; D002384:Catalysis; D020134:Catalytic Domain; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D018360:Crystallography, X-Ray; D018076:DNA, Complementary / Q000378:metabolism; D006801:Humans; D007477:Ions; D051379:Mice; D008810:Mice, Inbred C57BL; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D015032:Zinc / Q000737:chemistry
chemical_list: D018076:DNA, Complementary; D007477:Ions; D020033:Protein Isoforms; D002256:Carbonic Anhydrases; C466621:carbonic anhydrase XIV; D015032:Zinc