Crystal Structure and Functional Analysis of the Protein Disulfide Isomerase-Related Protein ERp29

Abstract

The protein disulfide isomerase-related protein ERp29 is a putative chaperone involved in processing and secretion of secretory proteins. Until now, however, both the structure and the exact nature of interacting substrates remained unclear. We provide for the first time a crystal structure of human ERp29, refined to 2.9 Å, and show that the protein has considerable structural homology to its Drosophila homolog Wind. We show that ERp29 binds directly not only to thyroglobulin and thyroglobulin-derived peptides in vitro but also to the Wind client protein Pipe and Pipe-derived peptides, although it fails to process Pipe in vivo. A monomeric mutant of ERp29 and a D domain mutant in which the second peptide binding site is inactivated also bind protein substrates, indicating that the monomeric thioredoxin domain is sufficient for client protein binding. Indeed, the b domains of ERp29 or Wind, expressed alone, are sufficient for binding proteins and peptides. Interacting peptides have in common two or more aromatic residues, with stronger binding for sequences with overall basic character. Thus, the data allow a view of the two putative peptide binding sites of ERp29 and indicate that the apparent, different processing activity of the human and Drosophila proteins in vivo does not stem from differences in peptide binding properties.

Introduction

Chaperones within the endoplasmic reticulum (ER) assist in the maturation and transport¹ of many secretory proteins.2, 3 One of the major protein families in the ER is the protein disulfide isomerase (PDI)-related family. PDI-related proteins have various functions,⁴ including disulfide bond formation and reduction as well as a noncovalent chaperone activity. Chaperone activity in PDI-related proteins generally involves interaction of a substrate to one or more thioredoxin domains, with the strongest binding in PDI occurring with a redox-inactive b-type domain.⁵

A subgroup of PDI proteins found in most species to date is the PDI-D (PDI-related protein containing a D domain) proteins. Characterized by the presence of a D domain, a C-terminal, α-helical fold unique to this class of proteins, the family members fall into two classes. PDI-Dα proteins contain at least one N-terminal redox-active a-type thioredoxin domain, whereas PDI-Dβ proteins contain only redox-inactive b-type domains. Human ERp29, a PDI-Dβ protein,6, 7 was reported to be essential for posttranslational processing and/or secretion of natural substrates such as thyroglobulin.⁸ Furthermore, the protein may be required for murine polyoma virus entry into animal cells.⁹ Interactions with other secretory and ER-lumenal resident proteins have also been described.6, 10

The Drosophila melanogaster PDI-Dβ protein Wind is an ERp29 ortholog with similar importance in secretory protein processing.7, 11, 12, 13 Based partially on the solved crystal structure, two putative peptide binding sites, one within the b domain involving a tyrosine cluster and the second within the D domain, were identified in this protein.14, 15 In vivo, both binding sites are required for substrate processing. In vitro, client protein interacting sequences were found to contain specific sequences of aromatic residues, suggested to occur as either -(F,Y)-(F,Y)- or -(F,Y)-X-(F,Y)- motifs. The nature of the residue X is unclear, with at least an uncharged polar residue (glutamine) allowed.¹⁴

Interestingly, although ERp29 cannot replace Wind in Pipe substrate processing, the D domains of both proteins can be exchanged, indicating functional conservation between at least these domains of the proteins.¹⁵

With an established role in protein processing and secretion, the need for accurate structural data becomes apparent. This is especially important as variant dimerization models for the b domain and dramatically different tertiary structures of the D domains of mammalian orthologs compared to Wind have been suggested and have been interpreted as an indication of variant functional roles of the two proteins.16, 17, 18 NMR structures of the individual b and D domains of rat ERp29, reported by Liepinsh et al.,¹⁶ indicated involvement of residues Asp71, Phe118, Arg122, Asp123, and Trp144 in dimerization. Furthermore, a conserved proline residue, Pro116, found in the a domains of most PDI proteins in cis conformation, where it plays a structurally and possibly functionally important role, was suggested to be in trans conformation in ERp29, indicating gross structural and functional differences in this domain beyond the variant dimerization properties.

However, the dimerization model does not agree with biophysical and functional analyses from our group on ERp29 and Wind14, 15 and has recently been called into question by us and others.18, 19 Thus, we could show, using mutagenesis and biophysical studies, that the dimerization interface of ERp29 is likely highly similar to that of Wind, with a critical role in dimer formation in the human protein played by the conserved residue Asp42²⁰ (Wind Asp31). Furthermore, as in Wind, dimerization of ERp29 appears to be essential for functional activity in processing of thyroglobulin and polyoma virus proteins.14, 18, 19

Given the remaining differences in reported structures and function properties, one might expect differences in the protein and peptide binding properties of ERp29 compared to Wind. Thus, it would be of value to compare binding of ERp29 and Wind to natural substrates such as Pipe and thyroglobulin (to which no direct interaction by ERp29 has yet been shown) and to binding of smaller proteins and peptides with fewer potential binding motifs.

Here, we provide X-ray crystallographic data and evidence from in vitro functional assays proving that the overall structures of the b domains of ERp29 and Wind are indeed highly similar, and we show that the D domain of ERp29 strongly resembles that of Wind. Furthermore, we investigate the peptide binding properties of human ERp29 and show that the protein recognizes similar sequences as Wind, supporting our view of conserved functional properties of both the b and D domains in PDI-Dβ proteins.