Abstract
We studied two pathways that involve the transfer of persulfide sulfur in humans, molybdenum cofactor biosynthesis and tRNA thiolation. Investigations using human cells showed that the two-domain protein MOCS3 is shared between both pathways. MOCS3 has an N-terminal adenylation domain and a C-terminal rhodanese-like domain. We showed that MOCS3 activates both MOCS2A and URM1 by adenylation and a subsequent sulfur transfer step for the formation of the thiocarboxylate group at the C terminus of each protein. MOCS2A and URM1 are β-grasp fold proteins that contain a highly conserved C-terminal double glycine motif. The role of the terminal glycine of MOCS2A and URM1 was examined for the interaction and the cellular localization with MOCS3. Deletion of the C-terminal glycine of either MOCS2A or URM1 resulted in a loss of interaction with MOCS3. Enhanced cyan fluorescent protein and enhanced yellow fluorescent protein fusions of the proteins were constructed, and the fluorescence resonance energy transfer efficiency was determined by the decrease in the donor lifetime. The cellular localization results showed that extension of the C terminus with an additional glycine of MOCS2A and URM1 altered the localization of MOCS3 from the cytosol to the nucleus.
Highlights
E1-like proteins are required for activation and thiocarboxylation of -grasp fold proteins involved in sulfur transfer to cofactors and tRNA
Expression and Purification of Human MOCS3 from Sf9 Cells— Earlier attempts to purify the holo-MOCS3 protein in an active form failed after expression in heterologous systems like E. coli, Pichia pastoris, or S. cerevisiae
The results show that the effects of the variation of the C-terminal glycine of MOCS2A and URM1 variants observed above are based on an impaired ability of the proteins to form a stable complex with MOCS3
Summary
E1-like proteins are required for activation and thiocarboxylation of -grasp fold proteins involved in sulfur transfer to cofactors and tRNA. We showed that MOCS3 activates both MOCS2A and URM1 by adenylation and a subsequent sulfur transfer step for the formation of the thiocarboxylate group at the C terminus of each protein. For Moco biosynthesis in humans, the E1-like protein MOCS3 forms a thiocarboxylate group at the C-terminal glycine of the -grasp fold protein MOCS2A (4 – 6). Interaction of MOCS3 with MOCS2A and URM1 the second step of the Moco biosynthesis is found in the underlying sulfur chemistry, because Ub activation involves thioester formation, and the thiocarboxylate formation is based on persulfide group transfer [12]. We proposed that MOCS3 connects the two distinct processes in the cell, for Moco biosynthesis and tRNA thiolation, especially because it is the only E1-like activating enzyme that is present in humans for these pathways [5]. Gly motif in terms of its role for the localization of the proteins
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