Sumoylation has emerged as an indispensable post‐translational modification that modulates the functions of a broad spectrum of proteins. Recent studies have demonstrated that reactive oxygen species influence the equilibrium of sumoylation‐desumoylation. We show herein that H ₂ O ₂ induces formation of an intermolecular disulfide linkage of human SUMO protease SENP1 via the active‐site Cys 603 and a unique residue Cys 613. Such reversible modification confers a higher recovery of enzyme activity, which is also observed in yeast Ulp1, but not in human SENP2, suggesting its protective role against irreversible sulfhydryl oxidation. In vivo formation of a disulfide‐linked dimer of SENP1 is also detected in cultured cells in response to oxidative stress. The modifications are further elucidated by the crystal structures of Ulp1 with the catalytic cysteine oxidized to sulfenic, sulfinic, and sulfonic acids. Our findings suggest that, in addition to SUMO conjugating enzymes, SUMO proteases may act as redox sensors and effectors modulating the des‐umoylation pathway and specific cellular responses to oxidative stress.— Xu, Z., Lam, L. S. M., Lam, L. H., Chau, S. F., Ng, T. B., Au, S. W. N. Molecular basis of the redox regulation of SUMO proteases: a protective mechanism of intermolecular disulfide linkage against irreversible sulfhydryl oxidation. FASEB J. 22, 127–137 (2008)

SUMO (small ubiquitin-related modifier) is a member of the ubiquitin-like protein family that regulates cellular function of a variety of target proteins. SUMO proteins are expressed as their precursor forms. Cleavage of the residues after the ‘GG’ region of these precursors by SUMO-specific proteases in maturation is a prerequisite for subsequent sumoylation. To understand further this proteolytic processing, we expressed and purified SENP1 (sentrin-specific protease 1), one of the SUMO-specific proteases, using an Escherichia coli expression system. We show that SENP1 is capable of processing all SUMO-1, -2 and -3 in vitro; however, the proteolytic efficiency of SUMO-1 is the highest followed by SUMO-2 and -3. We demonstrate further that the catalytic domain of SENP1 (SENP1C) alone can determine the substrate specificity towards SUMO-1, -2 and -3. Replacement of the C-terminal fragments after the ‘GG’ region of SUMO-1 and -2 precursors with that of the SUMO-3, indicates that the C-terminal fragment is essential for efficient maturation. In mutagenesis analysis, we further map two residues immediately after the ‘GG’ region, which determine the differential maturation. Distinct patterns of tissue distribution of SENP1, SUMO-1, -2 and -3 are characterized. Taken together, we suggest that the observed differential maturation process has its physiological significance in the regulation of the sumoylation pathway.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, has developed multiple strategies to adapt to the human host. The five type VII secretion systems, ESX-1–5, direct the export of many virulence-promoting protein effectors across the complex mycobacterial cell wall. One class of ESX substrates is the PE–PPE family of proteins, which is unique to mycobacteria and essential for infection, antigenic variation, and host–pathogen interactions. The genome of Mtb encodes 168 PE–PPE proteins. Many of them are thought to be secreted through ESX-5 secretion system and to function in pairs. However, understanding of the specific pairing of PE–PPE proteins and their structure–function relationship is limited by the challenging purification of many PE–PPE proteins, and our knowledge of the PE–PPE interactions therefore has been restricted to the PE25–PPE41 pair and its complex with the ESX-5 secretion system chaperone EspG5. Here, we report the crystal structure of a new PE–PPE pair, PE8–PPE15, in complex with EspG5. Our structure revealed that the EspG5-binding sites on PPE15 are relatively conserved among Mtb PPE proteins, suggesting that EspG5–PPE15 represents a more typical model for EspG5–PPE interactions than EspG5–PPE41. A structural comparison with the PE25–PPE41 complex disclosed conformational changes in the four-helix bundle structure and a unique binding mode in the PE8–PPE15 pair. Moreover, homology-modeling and mutagenesis studies further delineated the molecular determinants of the specific PE–PPE interactions. These findings help develop an atomic algorithm of ESX-5 substrate recognition and PE–PPE pairing.

SUMO (small ubiquitin-related modifier)-specific proteases catalyse the maturation and de-conjugation processes of the sumoylation pathway and modulate various cellular responses including nuclear metabolism and cell cycle progression. The active-site cysteine residue is conserved among all known SUMO-specific proteases and is not substitutable by serine in the hydrolysis reactions demonstrated previously in yeast. We report here that the catalytic domain of human protease SENP1 (SUMO-specific protease 1) mutant SENP1CC603S carrying a mutation of cysteine to serine at the active site is inactive in maturation and de-conjugation reactions. To further understand the hydrolytic mechanism catalysed by SENP1, we have determined, at 2.8 Å resolution (1 Å=0.1 nm), the X-ray structure of SENP1CC603S–SUMO-1 complex. A comparison of the structure of SENP2–SUMO-1 suggests strongly that SUMO-specific proteases require a self-conformational change prior to cleavage of peptide or isopeptide bond in the maturation and de-conjugation processes respectively. Moreover, analysis of the interface of SENP1 and SUMO-1 has led to the identification of four unique amino acids in SENP1 that facilitate the binding of SUMO-1. By means of an in vitro assay, we further demonstrate a novel function of SENP1 in hydrolysing the thioester linkage in E1-SUMO and E2-SUMO complexes. The results disclose a new mechanism of regulation of the sumoylation pathway by the SUMO-specific proteases.

Summary: FliG and FliM are switch proteins that regulate the rotation and switching of the flagellar motor. Several assembly models for FliG and FliM have recently been proposed; however, it remains unclear whether the assembly of the switch proteins is conserved among different bacterial species. We applied a combination of pull‐down, thermodynamic and structural analyses to characterize the FliM–FliG association from the mesophilic bacterium Helicobacter pylori. FliM binds to FliG with micromolar binding affinity, and their interaction is mediated through the middle domain of FliG (FliG_M), which contains the EHPQR motif. Crystal structures of the middle domain of H. pylori FliM (FliM_M) and FliG_M–FliM_M complex revealed that FliG binding triggered a conformational change of the FliM α3‐α1′ loop, especially Asp130 and Arg144. We furthermore showed that various highly conserved residues in this region are required for FliM–FliG complex formation. Although the FliM–FliG complex structure displayed a conserved binding mode when compared with Thermotoga maritima, variable residues were identified that may contribute to differential binding affinities across bacterial species. Comparison of the thermodynamic parameters of FliG–FliM interactions between H. pylori and Escherichia coli suggests that molecular basis and binding properties of FliM to FliG is likely different between these two species.

The bacterial flagellar motor is a self-assembling supramolecular nanodevice. Its spontaneous biosynthesis is initiated by the insertion of the MS ring protein FliF into the inner membrane, followed by attachment of the switch protein FliG. Assembly of this multiprotein complex is tightly regulated to avoid nonspecific aggregation, but the molecular mechanisms governing flagellar assembly are unclear. Here, we present the crystal structure of the cytoplasmic domain of FliF complexed with the N-terminal domain of FliG (FliF_C-FliG_N) from the bacterium Helicobacter pylori. Within this complex, FliF_C interacted with FliG_N through extensive hydrophobic contacts similar to those observed in the FliF_C-FliG_N structure from the thermophile Thermotoga maritima, indicating conservation of the FliF_C-FliG_N interaction across bacterial species. Analysis of the crystal lattice revealed that the heterodimeric complex packs as a linear superhelix via stacking of the armadillo-repeat-like motifs (ARM) of FliG_N. Notably, this linear helix was similar to that observed for the assembly of the FliG middle domain. We validated the in vivo relevance of the FliG_N stacking by complementation studies in Escherichia coli. Furthermore, structural comparison with apo FliG from the thermophile Aquifex aeolicus indicated that FliF regulates the conformational transition of FliG and exposes the complementary ARM-like motifs of FliG_N, containing conserved hydrophobic residues. FliF apparently both provides a template for FliG polymerization and spatiotemporally controls subunit interactions within FliG. Our findings reveal that a small protein fold can serve as a versatile building block to assemble into a multiprotein machinery of distinct shapes for specific functions.