The Eps1p Protein Disulfide Isomerase Conserves Classic Thioredoxin Superfamily Amino Acid Motifs but Not Their Functional Geometries

Abstract

The widespread thioredoxin superfamily enzymes typically share the following features: a characteristic α-β fold, the presence of a Cys-X-X-Cys (or Cys-X-X-Ser) redox-active motif, and a proline in the cis configuration abutting the redox-active site in the tertiary structure. The Cys-X-X-Cys motif is at the solvent-exposed amino terminus of an α-helix, allowing the first cysteine to engage in nucleophilic attack on substrates, or substrates to attack the Cys-X-X-Cys disulfide, depending on whether the enzyme functions to reduce, isomerize, or oxidize its targets. We report here the X-ray crystal structure of an enzyme that breaks many of our assumptions regarding the sequence-structure relationship of thioredoxin superfamily proteins. The yeast Protein Disulfide Isomerase family member Eps1p has Cys-X-X-Cys motifs and proline residues at the appropriate primary structural positions in its first two predicted thioredoxin-fold domains. However, crystal structures show that the Cys-X-X-Cys of the second domain is buried and that the adjacent proline is in the trans, rather than the cis isomer. In these configurations, neither the “active-site” disulfide nor the backbone carbonyl preceding the proline is available to interact with substrate. The Eps1p structures thus expand the documented diversity of the PDI oxidoreductase family and demonstrate that conserved sequence motifs in common folds do not guarantee structural or functional conservation.

Intestinal Gel-Forming Mucins Polymerize by Disulfide-Mediated Dimerization of D3 Domains

“…10). Although amino acid sequence homology is a powerful predictor of structure conservation [33], residues that are apparently aligned in primary structures occasionally appear in functionally different tertiary structural contexts [34]. Another example of this phenomenon is the change in connectivity of a conserved cysteine, as described above for MUC2 C1137.…”

Salmonella enterica BcfH Is a Trimeric Thioredoxin-Like Bifunctional Enzyme with Both Thiol Oxidase and Disulfide Isomerase Activities

“…(A) BcfH ensures Bcf fimbriae mediated biofilm formation in S. Typhimurium. Biofilm formation by wild-type SL1344 ([1] w.t.-circles), an isogenic mutant lacking dsbA, srgA, and dsbLI (SL1344D3KO) [2] complemented with empty vectors (pWSK29/pSU2718-triangles), [3] Bcf with BcfH (pBcf/pBcfH-inverted triangles) or [4] Bcf without BcfH (pBcf/pSU2718-squares). Biofilm-associated bacteria were recovered and enumerated as described in Materials and Methods.…”

Chemistry and Enzymology of Disulfide Cross-Linking in Proteins

“…S. cerevisiae Eps1p contains a domain with a CXXC motif in the appropriate primary structural context, but the structure of the enzyme shows the disulfide to be buried and unreactive, and the adjacent proline residue, while maintained, is found in the trans rather than the cis configuration. 141…”

“…Furthermore, a proline is present in the Eps1p amino acid sequence at the appropriate place to correspond to a conserved cis-proline near the activesite cysteines in the structures of redox-active trx domains, but this proline in the Eps1p structure is in the trans rather than the cis configuration observed in functional trx folds. 141 Most fundamentally, PDI and other enzymes that undergo thiol−disulfide exchange with substrate proteins should conform to the steric requirements of the reaction. 142 In-line attack in the context of structured globular proteins would predict alignment of the catalytic disulfide axis, as depicted schematically in Figure 7A.…”

“…Saccharomyces cerevisiae Eps1p contains a domain with a CXXC motif in the appropriate primary structural context, but the structure of the enzyme shows the disulfide to be buried and unreactive. Furthermore, a proline is present in the Eps1p amino acid sequence at the appropriate place to correspond to a conserved cis -proline near the active-site cysteines in the structures of redox-active trx domains, but this proline in the Eps1p structure is in the trans rather than the cis configuration observed in functional trx folds …”