Fig. 4: Application of GSNO affect the disulfide bonds distribution in AtERO1.

a Structural modeling of AtERO1, with disulfide bonds shown by purple sticks. b Superimposed structure of modeled AtERO1 (cyan) with the inactive form of hsEro1α (brown, PDB code: 3AHR). Inset highlights the close-up view of Cys71-Cys337 disulfide bond in AtERO1 (purple sticks), and the disulfide bridges formed in the same region of hsEro1α (blue sticks). Green sticks represent the FAD molecule. c Comparison of the disulfide bonds in N-terminal of AtERO1 and hsEro1α. The disulfides Cys35-Cys48 and C37-Cys46 in hsEro1α, and Cys71-Cys337 in AtERO1 are highlighted. d The MS/MS spectrum of Cys71-Cys337 disulfide bond identified in AtERO1. The α and β indicates two peptides contain Cys71 and Cys337, respectively. e The relative intensity of Cys71-Cys337, Cys222-Cys231, Cys375-Cys378 disulfide in ERO1 protein without or with incubation of GSNO. The trypsin/Glu-C digested AtERO1 peptides were subjected into LC-MS/MS analysis, the relative intensity of disulfide peptides and ¹³⁸KPFVPGLPSDDL were obtained from the result of pLink and Proteome Discovery analyses, respectively. Error bars, SD (n = 3 biological replicates). *p < 0.05, **p < 0.01, two-tail unpaired t test. f Schematic representation of cysteine positions in AtERO1 and hsEro1α. Lines indicate disulfide bonds. The Cys108-Cys372 disulfide that not detected by LC-MS/MS is indicated by dot line. g Alignment of ERO1 protein sequences in different model species. The alignment was performed with MUSCLE (https://www.ebi.ac.uk/Tools/msa/muscle). The cysteine positions in AtERO1 are indicated by black arrows, and the residues corresponding to outer and inner active sites are highlighted by red and blue arrows, respectively.