Structure of Yeast Sulfhydryl Oxidase Erv1 Reveals Electron Transfer of the Disulfide Relay System in the Mitochondrial Intermembrane Space

Peng-Chao Guo,Jin-Di Ma,Yong-Liang Jiang,Shu-Jie Wang,Zhang-Zhi Bao,Xiao-Jie Yu,Yuxing Chen,Cong-Zhao Zhou

doi:10.1074/jbc.m112.394759

Abstract

The disulfide relay system in the mitochondrial intermembrane space drives the import of proteins with twin CX(9)C or twin CX(3)C motifs by an oxidative folding mechanism. This process requires disulfide bond transfer from oxidized Mia40 to a substrate protein. Reduced Mia40 is reoxidized/regenerated by the FAD-linked sulfhydryl oxidase Erv1 (EC 1.8.3.2). Full-length Erv1 consists of a flexible N-terminal shuttle domain (NTD) and a conserved C-terminal core domain (CTD). Here, we present crystal structures at 2.0 Å resolution of the CTD and at 3.0 Å resolution of a C30S/C133S double mutant of full-length Erv1 (Erv1FL). Similar to previous homologous structures, the CTD exists as a homodimer, with each subunit consisting of a conserved four-helix bundle that accommodates the isoalloxazine ring of FAD and an additional single-turn helix. The structure of Erv1FL enabled us to identify, for the first time, the three-dimensional structure of the Erv1NTD, which is an amphipathic helix flanked by two flexible loops. This structure also represents an intermediate state of electron transfer from the NTD to the CTD of another subunit. Comparative structural analysis revealed that the four-helix bundle of the CTD forms a wide platform for the electron donor NTD. Moreover, computational simulation combined with multiple-sequence alignment suggested that the amphipathic helix close to the shuttle redox enter is critical for the recognition of Mia40, the upstream electron donor. These findings provide structural insights into electron transfer from Mia40 via the shuttle domain of one subunit of Erv1 to the CTD of another Erv1 subunit.

Highlights

Mia40 is regenerated by the sulfhydryl oxidase Erv1 in the disulfide relay system
Computational simulation combined with multiple-sequence alignment suggested that the amphipathic helix close to the shuttle redox enter is critical for the recognition of Mia40, the upstream electron donor
Each asymmetric unit consists of one Erv1CTD molecule, which adopts an all-␣ overall structure (Fig. 1A), similar to previous core domain structures

Summary

Background

Mia is regenerated by the sulfhydryl oxidase Erv in the disulfide relay system. Results: Crystal structures of the Erv core domain and full length of Erv were determined. The disulfide relay system in the mitochondrial intermembrane space drives the import of proteins with twin CX9C or twin CX3C motifs by an oxidative folding mechanism This process requires disulfide bond transfer from oxidized Mia to a substrate protein. Computational simulation combined with multiple-sequence alignment suggested that the amphipathic helix close to the shuttle redox enter is critical for the recognition of Mia, the upstream electron donor These findings provide structural insights into electron transfer from Mia via the shuttle domain of one subunit of Erv to the CTD of another Erv subunit. In addition to the conserved core redox center, Erv1/ALR proteins, except for the viral homologs, possess another cysteine pair This is at the N-terminal domain (NTD) in fungi and mammals and at the C-terminal segment in plants [33].

EXPERIMENTAL PROCEDURES

RESULTS AND DISCUSSION

Protein Data Bank entry