Fig. 4

Zn²⁺-dependent conformational changes enhance the ERp44–Ero1α interaction. a Comparison of the His-cluster (site 1) conformation in the unbound (left), Zn²⁺-bound states (center), and their superposition (right). Yellow dashed lines in the right panel indicate van der Waals contact among the three histidine and Pro353. b Molecular surface properties of the Zn²⁺-bound ERp44. (Upper left) Ribbon representation as a reference; (upper right) Electrostatic surface potential, in which positively and negatively charged regions are shown in blue and red, respectively. (Lower) Hydrophobic regions viewed from two different angles, in which hydrophobic residues (except main-chain oxygen and nitrogen atoms) are shown in green. c SEC analysis for the mixture of ERp44 (40 µM) and ZnCl₂ (80 µM) (dashed blue line), Ero1α (40 µM) alone (dashed green line), and the pre-mixture of ERp44 (40 µM) and ZnCl₂ (80 µM) supplemented with Ero1α (40 µM) (red line) at pH 6.2. Peak fractions were analyzed by SDS-PAGE under non-reducing or reducing conditions. d SEC analysis of ERp44 (40 µM), Ero1α (40 µM), and equimolar mixtures of ERp44 and Ero1α (40 µM each) with or without Zn²⁺ ions (80 µM) at pH 6.2. e ITC raw data (upper) and binding isotherm data (lower) for titration of Ero1α into ERp44 at pH 6.2 (left) or titration of ERp44 into a mixture of Ero1α and Zn²⁺ at pH 6.2 (right). The calculated K_d is shown with 68.3% confidence interval in brackets