The structure and evolution of the murine inhibitor of carbonic anhydrase: a member of the transferrin superfamily

Abstract

The original signature of the transferrin (TF) family of proteins was the ability to bind ferric iron with high affinity in the cleft of each of two homologous lobes. However, in recent years, new family members that do not bind iron have been discovered. One new member is the inhibitor of carbonic anhydrase (ICA), which as its name indicates, binds to and strongly inhibits certain isoforms of carbonic anhydrase. Recently, mouse ICA has been expressed as a recombinant protein in a mammalian cell system. Here, we describe the 2.4 Å structure of mouse ICA from a pseudomerohedral twinned crystal. As predicted, the structure is bilobal, comprised of two α-β domains per lobe typical of the other family members. As with all but insect TFs, the structure includes the unusual reverse γ-turn in each lobe. The structure is consistent with the fact that introduction of two mutations in the N-lobe of murine ICA (mICA) (W124R and S188Y) allowed it to bind iron with high affinity. Unexpectedly, both lobes of the mICA were found in the closed conformation usually associated with presence of iron in the cleft, and making the structure most similar to diferric pig TF. Two new ICA family members (guinea pig and horse) were identified from genomic sequences and used in evolutionary comparisons. Additionally, a comparison of selection pressure (dN/dS) on functional residues reveals some interesting insights into the evolution of the TF family including that the N-lobe of lactoferrin may be in the process of eliminating its iron binding function.

Figure 1

(A) Ribbon diagram of the crystal structure of mICA. Subdomains N1 (red), N2 (yellow), C1 (blue), and C2 (green) are highlighted. The linker between lobes for which density was not observed is indicated by the dashed line. (B) The N-lobe cleft of mICA. Shown is the N1 subdomain (pink), N2 subdomain (yellow), mICA residues (black), residues for porcine TF (PDB: 1H76, orange), and residues for human TF (PDB: 1A8E) including the Fe atom (green) after least squares superposition. Electron density shown is 2F_o − F_c. All structural images produced using PyMOL. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 2

(A) Least squares superposition of the structure of mICA with subdomain N1 (red), N2 (yellow), C1 (blue), and C2 (green) and the iron-bound form of porcine TF (PDB: 1H76) in white. (B) Detailed examination of the region indicated by the box in (A), which is implicated in binding to the TF receptor. Porcine TF in pink, human TF (PDB: 1A8E, yellow), mICA chain A in green, and mICA chain C in cyan. The largest positional shift relative to hTF were for the N-terminal residues of the sequence with 8.8 Å and 4.7 Å changes for mICA chain A and C, respectively. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 3

Stereo images of the superposition of the iron binding sites for (A) N-lobes and (B) C-lobes of porcine TF (PDB: 1H76) (white) including the iron shown as a sphere, human TF (PDB: 1A8E) (grey), and mICA (black). Numbering in both panels is for human TF. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]