Structure of the substrate-binding b' domain of the Protein Disulfide Isomerase-Like protein of the Testis

Abstract

Protein Disulfide Isomerase-Like protein of the Testis (PDILT) is a testis-specific member of the PDI family. PDILT displays similar domain architecture to PDIA1, the founding member of this protein family, but lacks catalytic cysteines needed for oxidoreduction reactions. This suggests special importance of chaperone activity of PDILT, but how it recognizes misfolded protein substrates is unknown. Here, we report the high-resolution crystal structure of the b' domain of human PDILT. The structure reveals a conserved hydrophobic pocket, which is likely a principal substrate-binding site in PDILT. In the crystal, this pocket is occupied by side chains of tyrosine and tryptophan residues from another PDILT molecule, suggesting a preference for binding exposed aromatic residues in protein substrates. The lack of interaction of the b' domain with the P-domains of calreticulin-3 and calmegin hints at a novel way of interaction between testis-specific lectin chaperones and PDILT. Further studies of this recently discovered PDI member would help to understand the important role that PDILT plays in the differentiation and maturation of spermatozoids.

Figure 1. Crystal structure of the b′ domain of PDILT.

(a) Domain organization of human PDILT and PDIA1. Catalytic motifs are shown in a and a′ thioredoxin-like domains. (b) Structure of PDILT b′ domain displays a typical thioredoxin-fold with a central five-stranded β-sheet (β1–β5) and four flanking α-helices (α1–α4). An overlay of chains A (green) and C (magenta) shows divergence in the C-terminal region (C-term) of the construct. (c) Structure-based sequence alignment of the b′ domains of several members of PDI family. Amino acids forming the hydrophobic pocket in the PDILT structure and conserved between aligned proteins are highlighted in gray. Residues of ERp57 involved in the interaction with calreticulin (CRT) and calnexin (CNX) are highlighted in cyan. Secondary structure corresponds to the PDILT structure.

Figure 2. A large hydrophobic pocket on the b′ domain of PDILT is able to interact with aromatic side chains.

(a) The residues Y383 and W384 from the C-terminal tail of the crystallized fragment bind to a hydrophobic pocket located between helices α1 and α3 of an adjacent PDILT molecule. (b) The base of the large pocket is lined with hydrophobic residues with the exception of H277, which forms a hydrogen bond with the hydroxyl group of Y383. (c) Overlay of the crystal structures of b′ domain from PDILT (green) and PDIA1 (yellow; PDB accession number 3BJ5) reveals differing orientations of helix α1, resulting in a larger substrate-binding pocket in PDILT. (d) Overlay of the crystal structures of b′ domain from PDILT (green) and the C-terminal domain of ERp27 (orange; PDB accession number 4F9Z).

Figure 3. Structural comparison between PDILT and ERp57 b′ domains.

Cartoon representations and electrostatic surfaces for ERp57 (a) and PDILT (b) are displayed in the same orientation. The residues K274 and R282 of ERp57 b′ domain essential for binding to CNX and CRT are shown as stick representations. The positively charged CNX-binding site (circled) is not conserved in PDILT.