Volume 24, Issue 10, Pages 1755-1765 (October 2016) Crystal Structure of the ERp44-Peroxiredoxin 4 Complex Reveals the Molecular Mechanisms of Thiol-Mediated Protein Retention  Kai Yang, De-Feng Li, Xi’e Wang, Jinzhao Liang, Roberto Sitia, Chih-chen Wang, Xi Wang  Structure  Volume 24, Issue 10, Pages 1755-1765 (October 2016) DOI: 10.1016/j.str.2016.08.002 Copyright © 2016 Elsevier Ltd Terms and Conditions

Structure 2016 24, 1755-1765DOI: (10.1016/j.str.2016.08.002) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 Reaction between Prx4 and ERp44 (A) Secretion profiles of Prx4 and its mutants. Wild-type Prx4-HA (WT) or the indicated cysteine mutants were expressed in HeLa cells without (left) and with (right) HA-ERp44. Two days after transfection, aliquots from cell lysates and culture media were blotted and decorated with αHA or αGAPDH. (B) ERp44 reacts with Prx4 in a pH-dependent manner. Oxidized (ox) or reduced (red) Prx4-C14S or Prx4-C87S/C208S (10 μM) were incubated with ERp44 (5 μM) at the indicated pH values for 30 min at 25°C, blocked with 20 mM NEM, resolved by non-reducing SDS-PAGE, and visualized by Coomassie blue staining. (C) Redox dependence of the ERp44-Prx4 reaction. Prx4 mutants (10 μM) were incubated with ERp44 (5 μM) at pH 6.5 in the absence (left) and presence (right) of 25 μM H2O2 at 25°C for 30 min, and analyzed by non-reducing SDS-PAGE with Coomassie blue staining. Structure 2016 24, 1755-1765DOI: (10.1016/j.str.2016.08.002) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 Preparation of the ERp44-Prx4 Complex (A) Schematic view of ERp44-Prx4 complex preparation by DTNB modification. (B and C) Reaction of DTNB-modified Prx4-T118E/C14S/C87S or Prx4-T118E/C14S/C208S with ERp44. DTNB-modified Prx4 proteins (10 μM) were incubated with ERp44 (12 μM) in buffer A at room temperature for 30 min and analyzed by non-reducing SDS-PAGE (B) and gel filtration (C). Structure 2016 24, 1755-1765DOI: (10.1016/j.str.2016.08.002) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 Structure of the ERp44-Prx4 Complex A Prx4 homodimer (chains A in pink and B in purple) and two ERp44 molecules (chains C in cyan and D in blue) are shown in ribbon representation. The regions around the two intermolecular disulfides (in yellow) between Cys29 of ERp44 and Cys208 of Prx4 are shown as sticks, and their 2Fo-Fc electron density map are meshed in gray and contoured to 1σ in the extended boxes. Structure 2016 24, 1755-1765DOI: (10.1016/j.str.2016.08.002) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 Structural Comparisons of ERp44 in the Complex and the Free State ERp44 in the complex (chain C) is shown in cyan and free ERp44 in yellow (PDB: 2R2J). The a and b domains of ERp44 are shown in cylindrical cartoon representation, while domain b′ is shown in ribbon representation. In the complex, the b′ domain in chain C rotates ∼40° and the visible part of the C-tail (residues 351–372) in free ERp44 colored in green becomes untraceable. This part of the C-tail and the b′ domain of ERp44 would make a steric hindrance to the access of the 98–105 β sheet and the 203–211 helix of Prx4, colored in red (inset). Structure 2016 24, 1755-1765DOI: (10.1016/j.str.2016.08.002) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 5 Structural Comparisons of Prx4 in the Complex with Free Prx4 in the Different Redox States (A and B) Prx4 in the complex structure (colored as in Figure 3) was superposed with that in the free oxidized (A, forest, PDB: 3TJG) or reduced state (B, purple blue, PDB: 3TKP). Structure 2016 24, 1755-1765DOI: (10.1016/j.str.2016.08.002) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 6 Interfaces between ERp44 and Prx4 (A) A close-up view of the interface between one ERp44 molecule and the bound Prx4 dimer, colored as in Figure 3. Red dashed lines indicate either hydrogen bonds or salt bridges. (B) In vitro reaction of ERp44 mutants with Prx4. ERp44 mutants (12 μM) were incubated with oxidized Prx4-C14S (10 μM) in buffer A at room temperature for 30 min, blocked with 20 mM NEM, and analyzed by non-reducing SDS-PAGE. The mutants ERp44-T369A and ERp44-C29S (Wang et al., 2008) were used as positive and negative controls, respectively. (C) The relative amounts of the complex of Prx4 with various ERp44 mutants were quantified by densitometry (area of the complex bands divided by area of the total bands in B), and expressed as average of six experiments ± SD. Mutants with a negative effect are labeled in red, and those with an enhanced effect or no significant effect in blue or black, respectively. (D) Intermolecular disulfide bonding patterns between ERp44 mutants and Ero1α. The reactions of ERp44 mutants (6 μM) and Ero1α (5 μM) were carried out as described in (B). (E) Covalent complexes with ERp44 in HeLa cells. Lysates from HeLa cells transiently transfected with indicated ERp44 plasmids were prepared 24 hr after transfection, blocked with 20 mM NEM, and resolved by SDS-PAGE under non-reducing or reducing conditions, and blots decorated with αHA or αGAPDH. Structure 2016 24, 1755-1765DOI: (10.1016/j.str.2016.08.002) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 7 Reduction of the ERp44-Prx4 Complex (A–C) ERp44-Prx4 complexes (1 μM) were incubated with 1 μM Ero1α, 1 μM denatured and reduced RNase A, 10 mM GSH or 10 mM GSH and 1 μM PDI (A), 10 mM GSH and 1 μM PDI family members (B), and 10 mM GSH and 2 μM PDIa or its mutants (C) at 25°C for 10 min. Aliquots from each reaction at indicated time points were subjected to non-reducing SDS-PAGE. Structure 2016 24, 1755-1765DOI: (10.1016/j.str.2016.08.002) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 8 Schematic Model of the Prx4 Retention and Release Cycle Mediated by ERp44 in the Early Secretory Pathway Reduced Prx4 is oxidized by H2O2. Movements of the C-tail and domain b′ in ERp44 expose the active site, which can recognize oxidized but not reduced Prx4 and form a disulfide with Cys208 via its Cys29. The intermolecular disulfides can be reduced by GSH and PDI family members, allowing the two molecules to recycle. Structure 2016 24, 1755-1765DOI: (10.1016/j.str.2016.08.002) Copyright © 2016 Elsevier Ltd Terms and Conditions