Structure and Electron-Transfer Pathway of the Human Methionine Sulfoxide Reductase MsrB3

Abstract

Aims: The post-translational oxidation of methionine to methionine sulfoxide (MetSO) is a reversible process, enabling the repair of oxidative damage to proteins and the use of sulfoxidation as a regulatory switch. MetSO reductases catalyze the stereospecific reduction of MetSO. One of the mammalian MetSO reductases, MsrB3, has a signal sequence for entry into the endoplasmic reticulum (ER). In the ER, MsrB3 is expected to encounter a distinct redox environment compared with its paralogs in the cytosol, nucleus, and mitochondria. We sought to determine the location and arrangement of MsrB3 redox-active cysteines, which may couple MsrB3 activity to other redox events in the ER. Results: We determined the human MsrB3 structure by using X-ray crystallography. The structure revealed that a disulfide bond near the protein amino terminus is distant in space from the active site. Nevertheless, biochemical assays showed that these amino-terminal cysteines are oxidized by the MsrB3 active site after its reaction with MetSO. Innovation: This study reveals a mechanism to shuttle oxidizing equivalents from the primary MsrB3 active site toward the enzyme surface, where they would be available for further dithiol-disulfide exchange reactions. Conclusion: Conformational changes must occur during the MsrB3 catalytic cycle to transfer oxidizing equivalents from the active site to the amino-terminal redox-active disulfide. The accessibility of this exposed disulfide may help couple MsrB3 activity to other dithiol-disulfide redox events in the secretory pathway.

Conformational switches and redox properties of the colon cancer‐associated human lectin ZG16

“…Aliquots were taken and the reaction quenched by addition of mal‐PEG. Gel shifts corresponding to 10–15 kD are typically seen for each modification with mal‐PEG 5 kD [ 43 ], so the observed shift is consistent with modification of the two cysteines in reduced ZG16. No appreciable oxidation of ZG16 occurs over this time period in the absence of QSOX1 (not shown).…”

The Colon Mucosal Sialylglycome Is Redox-Regulated by the Golgi Enzyme QSOX1

“…St6gal1 with one pair of free cysteines is modified by two PEG-mal additions (2PEG), whereas protein with two pairs of free cysteines gets four PEG-mal additions (4PEG). The change in migration per two PEG-mal modifications appears greater than 4 kD as expected due to the differences in hydrodynamic properties and SDS binding of PEG vs. protein 44 . f, QSOX1 oxidation of St6gal1 mutants.…”

“…4a-c). It should be noted that PEG conjugation retards protein migration to a greater extent than addition of the equivalent mass of protein 44 . As a control, treatment with the small (125 Da) cysteine alkylating agent N-ethylmaleimide (NEM) did not cause appreciable differences in glycosyltransferase migration (Fig.…”

The disulfide catalyst QSOX1 maintains the colon mucosal barrier by regulating Golgi glycosyltransferases

“…In all three cases, species that migrated more slowly, indicating the presence of cysteines conjugated to PEG‐mal, were observed for QSOX1 KO samples (Fig 4A–C ). It should be noted that PEG conjugation retards protein migration to a greater extent than addition of the equivalent mass of protein (Javitt et al , 2020b ). As a control showing that the differences in glycosyltransferase migration between WT and KO are due to the addition of PEG‐mal, treatment with the small (125 Da) cysteine alkylating agent N‐ethylmaleimide (NEM) did not result in appreciable differences in migration (Fig 4A–C ).…”