Evolution reversed: the ability to bind iron restored to the N-lobe of the murine inhibitor of carbonic anhydrase by strategic mutagenesis.
The murine inhibitor of carbonic anhydrase (mICA) is a member of the superfamily related to the bilobal iron transport protein transferrin (TF), which binds a ferric ion within a cleft in each lobe. Although the gene encoding ICA in humans is classified as a pseudogene, an apparently functional ICA gene has been annotated in mice, rats, cows, pigs, and dogs. All ICAs lack one (or more) of the amino acid ligands in each lobe essential for high-affinity coordination of iron and the requisite synergistic anion, carbonate. The reason why ICA family members have lost the ability to bind iron is potentially related to acquiring a new function(s), one of which is inhibition of certain carbonic anhydrase (CA) isoforms. A recombinant mutant of the mICA (W124R/S188Y) was created with the goal of restoring the ligands required for both anion (Arg124) and iron (Tyr188) binding in the N-lobe. Absorption and fluorescence spectra definitively show that the mutant binds ferric iron in the N-lobe. Electrospray ionization mass spectrometry confirms the presence of both ferric iron and carbonate. At the putative endosomal pH of 5.6, iron is released by two slow processes indicative of high-affinity coordination. Induction of specific iron binding implies that (1) the structure of mICA resembles those of other TF family members and (2) the N-lobe can adopt a conformation in which the cleft closes when iron binds. Because the conformational change in the N-lobe indicated by metal binding does not impact the inhibitory activity of mICA, inhibition of CA was tentatively assigned to the C-lobe. Proof of this assignment is provided by limited trypsin proteolysis of porcine ICA.
Mason
Anne B
AB
Department of Biochemistry, College of Medicine, University of Vermont, Burlington, Vermont 05405, USA. anne.mason@uvm.edu
Judson
Gregory L
GL
Bravo
Maria Cristina
MC
Edelstein
Andrew
A
Byrne
Shaina L
SL
James
Nicholas G
NG
Roush
Eric D
ED
Fierke
Carol A
CA
Bobst
Cedric E
CE
Kaltashov
Igor A
IA
Daughtery
Margaret A
MA
D000818:Animals; D001665:Binding Sites; D005075:Biological Evolution; D024402:Carbonic Anhydrase II / Q000037:antagonists & inhibitors / Q000378:metabolism; D002256:Carbonic Anhydrases / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000378:metabolism*; D008024:Ligands; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization
D008024:Ligands; D011994:Recombinant Proteins; D007501:Iron; D024402:Carbonic Anhydrase II; D002256:Carbonic Anhydrases