<i>Grxcr2</i> is paralogous to the previously identified deafness-associated gene <i>Grxcr1</i>.

(A) Regions of mouse chromosomes 5 and 18 that contain the Grxcr1 and Grxcr2, respectively, also contain additional conserved gene pairs (Kctd8 and Kctd16; Yipf7 and Yipf5) that are indicative of an ancestral duplication. Cen, centromere; tel, telomere. (B) Amino acid sequence comparison of GRXCR2 orthologs in a range of vertebrate species and of mouse GRXCR1 indicates strong conservation of the N- and C- termini, while the central domains are more divergent. Gray bar, thioredoxin-like domain of GRXCR1; bracket, putative GRXCR1 active site. Asterisks, conserved bipartite arrangement of cysteine residues. Black or gray shading indicates positions at which residues from at least four species are identical or biochemically similar, respectively. (C) Predicted secondary structure of the C-terminal Cys-rich domain of GRXCR2 exhibits significant similarity to Cys-rich domains present in E. coli dnaJ/Hsp40 (P08622) and human DNAJ3 (NP_005138) proteins. PSI-BLAST alignments are shown along with secondary structural information (H, alpha helix; E, beta strand; and C, coil) and associated confidence scores for the GRXCR2 predictions derived from analysis with Phyre2 (9, highest; 0, lowest). E values supporting significance of the PSI-BLAST alignments (three iterations) were 4x10-10 (dnaJ/Hsp40) and 2x10-8 (DNAJ3). (D) Structure of the dnaJ/Hsp40 Cys-rich domain bound to two zinc ions is similar to the top scoring model of the GRXCR2 C-terminal domain predicted by I-TASSER using the MUSTER threading algorithm [36]. The confidence score supporting this GRXCR2 model was -0.41, consistent with an RMSD of 3.7 ± 2.5 angstroms with respect to dnaJ/Hsp40. Secondary structure is indicated by color: red, alpha helix; yellow, beta strand; and green, coil. Zinc ions are indicated in blue and Cys residues comprising zinc binding domain I (ZBDI) and ZBDII are indicated in orange. (E) RT-PCR products corresponding to full-length Grxcr2 transcripts were amplified from cochlear RNA, but not from RNA derived from a variety of other adult tissues. Molecular weight markers are indicated at right, in kilobases. RT, reverse transcriptase.