HOPX: The Unusual Homeodomain-Containing Protein Anita Mariotto, Olesya Pavlova, Hyun-Sook Park, Marcel Huber, Daniel Hohl  Journal of Investigative Dermatology  Volume 136, Issue 5, Pages 905-911 (May 2016) DOI: 10.1016/j.jid.2016.01.032 Copyright © 2016 The Authors Terms and Conditions

Figure 1 Three-dimensional reconstruction of ordinary homeodomain (HD)-binding DNA and atypical homeodomain-only protein homeobox (HOPX) HD. (a) Co-crystal structure of ordinary HD in complex with DNA (PDB ID 3HDD). The C-terminal part of helix α3 is responsible for direct interaction with the major groove of DNA. The N-terminal tail (depicted in violet) and recognition helix α3 (shown in red) of HD interact with the TAAT-binding consensus motif of DNA. Amino acids R/K(3) and R(5) (light blue pearls) of the N-terminal arm of helix α1 is involved in shape readout of the minor groove and bind nucleotides T1 and A2, whereas amino acids I/V(47) and N(51) of helix α3 (light blue pearls) contact with the major groove by binding the nucleotides T4 and A3, respectively (all interactions indicated by dotted arrows). T1, A2, A3, and T4 indicate base pairs making up the TAAT motif. I, isoleucine; K, lysine; N, asparagine; R, arginine; V, valine. (b) The crystal structure of the human HOPX protein containing an atypical HD. Top: Three α-helices. Bottom: HOPX protein amino acid sequence with the mutations in key residues (27, 49, 53, and 55, depicted in red) responsible for DNA binding. HOPX contains histidine (H) and leucine (L) in positions 27 and 55, respectively. HDs contain at these positions tyrosine (Y) and arginine (R), which are important for contacts with the sugar-phosphate backbone of DNA. HOPX also contains lysine (K) and glutamine (Q) in positions 49 and 53, respectively, instead of the highly conserved isoleucine (I) and asparagine (N) normally present in HDs for contacts with the major groove of DNA. Journal of Investigative Dermatology 2016 136, 905-911DOI: (10.1016/j.jid.2016.01.032) Copyright © 2016 The Authors Terms and Conditions

Figure 2 General features of human and mouse homeodomain-only protein homeobox (HOPX/Hopx) proteins. (a) Top: Low-resolution structure of human chromosome 4. Localization of the human HOPX gene is shown with the red rectangle. Middle: Seven exons (E1–E7) of the human HOPX gene are presented with different colors. Introns are depicted as thin dark-gray rectangles between coloured exons. Bottom: Four exons (E1, E5, E6, and E7) participate in alternative splicing and generate five messenger RNA transcript variants that give rise to three different proteins (isoforms a, b, and c). Transcript variants 2, 3, and 4 produce the same protein (isoform b). National Center for Biotechnology Information reference sequences (NP_number) for each isoform with protein length and molecular weight are shown at the right. (b) Sequence alignment of three human HOPX isoforms. Green color indicates highly similar amino acid sequences common for all three isoforms. Red color indicates amino acids presented just in isoform c, which indicates a highly distinct C-terminal sequence compared to the other protein isoforms. (c) Top: Low-resolution structure of mouse chromosome 5. Localization of the mouse Hopx gene is depicted with the red rectangle. Middle: Structure of the mouse Hopx gene is composed of three exons (E1–E3), shown with different colors. Introns are illustrated as thin dark-gray rectangles between coloured exons. Bottom: Alternative splicing generates three transcript variants that give rise to the same 73-amino-acid protein (right). Protein length and molecular size are indicated. (d) Similar sequences of human and mouse HOPX/Hopx proteins, which share 92% identity. Both human and mouse HOPX/Hopx proteins consist of 73 amino acids (molecular weight is ≈8.3 kDa). Helix 1 (H1, green), helix 2 (H2, blue), and helix 3 (H3, violet) are shown. Amino acids that are different between two species are indicated in red. aa, amino acid. Journal of Investigative Dermatology 2016 136, 905-911DOI: (10.1016/j.jid.2016.01.032) Copyright © 2016 The Authors Terms and Conditions