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Structure determination of the small ubiquitin-related modifier SUMO-1

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Abstract

The recently discovered small ubiquitin-related modifier SUMO-1 belongs to the growing family of ubiquitin-related proteins involved in postranslational protein modification. Unlike ubiquitin, SUMO-1 does not appear to target proteins for degradation but seems to be involved in the modulation of protein-protein interactions. Independent studies demonstrate an essential function of SUMO-1 in the regulation of nucleo-cytoplasmic transport, and suggest a role in cell-cycle regulation and apoptosis. Here, we present the first three-dimensional structure of SUMO-1 solved by NMR. Although having only 18% amino acid sequence identity with ubiquitin, the overall structure closely resembles that of ubiquitin, featuring the ββαββαβ fold of the ubiquitin protein family. In addition, the position of the two C-terminal Gly residues required for isopeptide bond formation is conserved between ubiquitin and SUMO-1. The most prominent feature of SUMO-1 is a long and highly flexible N terminus, which protrudes from the core of the protein and which is absent in ubiquitin. Furthermore, ubiquitin Lys48, required to generate ubiquitin polymers, is substituted in SUMO-1 by Gln69 at the same position, which provides an explanation of why SUMO-1 has not been observed to form polymers. Moreover, the hydrophobic core of SUMO-1 and ubiquitin is maintained by conserved hydrophobic residues, whereas the overall charge topology of SUMO-1 and ubiquitin differs significantly, suggesting specific modifying enzymes and target proteins for both proteins.

Introduction

Postranslational modifications are important means to regulate protein activity, function, stability and intracellular localization. The best characterized modification that involves covalent attachment of one protein to another is the ubiquitin system. The conjugation of ubiquitin to other proteins is a complex process that requires multiple steps, each facilitated by a specific set of enzymes (reviewed by Hochstrasser 1995, Hochstrasser 1996, Jentsch and Schlenker 1995, Hershko 1996, Varshavsky 1997). First, ubiquitin is activated by a ubiquitin-activating enzyme, E1. E1 adenylates the C terminus of ubiquitin, which becomes attached to a Cys residue of E1 by a high-energy thiolester bond. Ubiquitin is then transferred to a Cys residue of E2, the ubiquitin-conjugating enzyme. The enzyme E2 catalyses the isopeptide bond formation between the C terminus of ubiquitin and the ϵ-amino group of a Lys residue of a receiving protein.

The best characterized functions of ubiquitylation is the targeting of substrate proteins to the 26 S proteasome, which subsequently leads to the degradation of these ubiquitin-tagged proteins (reviewed by Ciechanover 1994, Hochstrasser 1995, Hochstrasser 1996, Jentsch and Schlenker 1995). Apart from the proteasome-dependent degradation of proteins, ubiquitin is also attached to proteins that are not rapidly degraded, like histone H2A (Goldknopf & Busch, 1977) and actin of Drosophila flight muscles (Ball et al., 1987). Furthermore, ubiquitin is required for the internalization and targeting of cell surface receptors to the lysosome or vacuole (Hicke & Riezman 1996). In vitro, ubiquitin is able to activate a protein kinase that phosphorylates IκBα, an inhibitor of the NFκB transcription factor (Chen et al., 1996). Therefore, ubiquitylation appears to serve a great variety of different cellular functions.

Recently, several proteins have been identified that are related to ubiquitin (reviewed by Johnson and Hochstrasser 1997, Saitoh et al 1997. These proteins can be divided into two groups, those that are closely related to ubiquitin with identities >35% and those that are only remotely related to ubiquitin with identities <20%. To the first group belong proteins like the nucleotide excision repair protein Rad23p (Watkins et al., 1993), Dsk2p, which is involved in spindle pole duplication (Biggins et al., 1996) and the interferon-inducible ubiquitin cross-reactive protein UCRP (Loeb & Haas 1992). To the second group belong proteins like Smt3p (Meluh & Koshland 1995), the recently discovered SUMO-1 Matunis et al 1996, Mahajan et al 1997 and its close relatives Mannen et al 1996, Lapenta et al 1997. However, among these ubiquitin-related proteins, only SUMO-1, Smt3p and UCRP are covalently conjugated to other proteins in a ubiquitin-like fashion Mahajan et al 1997, Johnson et al 1997, Loeb and Haas 1994. The ability to conjugate with other proteins is dependent on two C-terminal Gly residues, which are conserved in SUMO-1, Smt3p and UCRP but not in Rad23p or Dsk2p.

A number of studies indicate that SUMO-1 modifies multiple, predominantly nuclear, proteins Matunis et al 1996, Mahajan et al 1997, Kamitani et al 1997. However, the only known substrate is RanGAP1 Mahajan et al 1997, Matunis et al 1996, the GTPase-activating protein of Ran (Bischoff et al., 1995). Both Ran and RanGAP1 are essential for nuclear import in mammalian cells Melchior et al 1993, Moore and Blobel 1993, Corbett et al 1995, Mahajan et al 1997. The postranslational modification by SUMO-1 translocates RanGAP1 to the nuclear envelope, where it binds to RanBP2/NUP358 (Matunis et al., 1996; Mahajan et al., 1977), a component of the cytoplasmic fibrils of the nuclear pore complex Wu et al 1995, Yokoyama et al 1995. The translocation of RanGAP1 to the nuclear pore is crucial for nuclear import (Mahajan et al., 1997). While in mammalian cells the modification of RanGAP1 by SUMO-1 is essential for nuclear import (Mahajan et al., 1997), the biological function of the only SUMO-1 homologue in yeast, Smt3p, is not defined. The target of SUMO-1 modification is Lys526 in the C-terminal domain of RanGAP1 Mahajan et al 1998, Matunis et al 1998. This C-terminal domain of RanGAP1, which is not needed for GTPase activation (J.B. unpublished results), and absent in the yeast counterpart, Rna1p (Becker et al., 1995), and there is no evidence for a modification of Rna1p at an alternative site Melchior et al 1993, Corbett et al 1995, Johnson et al 1997. Given the ability of SUMO-1 to modify proteins other than RanGAP1 Matunis et al 1996, Mahajan et al 1997, Kamitani et al 1997, Johnson et al 1997 it is likely that Smt3p and SUMO-1 will have additional functions unrelated to nucleo-cytoplasmic transport.

Some of the potential targets for modification by SUMO-1 were revealed in a number of two-hybrid interaction studies that led to the independent discovery of SUMO-1 under the names PIC1 (Boddy et al., 1996), UBL1 Shen et al 1996a, Shen et al 1996b or sentrin (Okura et al., 1996). In the first study, SUMO-1 was shown to interact with the proto-oncogene PML (Boddy et al., 1996). In patients with acute promyelocytic leukemia, the PML gene is fused with the gene encoding the retinoic acid receptor a. As a result, SUMO-1, which normally colocalizes with PML to the nuclear bodies, is no longer present in these structures. In the second study, SUMO-1 was found to interact with RAD51 and RAD52, proteins involved in DNA repair (Shen et al., 1996a). In the third study, SUMO-1 was shown to interact with the so-called “death domains” of FAS/APO-1 and tumor necrosis factor, which are involved in apoptosis (Okura et al., 1996). Overexpression of SUMO-1 prevents antiFAS or tumor necrosis factor induced cell death. In addition, yeast Smt3p was originally isolated as high-copy suppressor of the centromere protein MIF2 (Meluh & Koshland, 1995). These findings suggest additional roles for SUMO-1 modification in cell-cycle regulation, DNA repair and apoptosis.

Here, we present the first three-dimensional structure of SUMO-1 solved by NMR spectroscopy. Despite the limited sequence identity of 18% between SUMO-1 and ubiquitin, we find that the two proteins share the same protein fold. SUMO-1 presents the two conserved C-terminal Gly residues essential for the modification of other proteins in the same spatial arrangement as that in ubiquitin. In contrast to ubiquitin, SUMO-1 contains an N-terminal extension, which forms a flexible appendix that protrudes from the core protein. In place of the conserved Lys48 of ubiquitin, which is responsible for the formation of ubiquitin polymers, SUMO-1 contains a Gln69 residue. A comparison of the surface charge distribution of ubiquitin and SUMO-1 suggests that the two proteins interact, at least partially, with different modifying enzymes or substrate proteins. The structure of SUMO-1 provides the basis for future studies to determine the specific interactions of SUMO-1 with modifying enzymes and target proteins.

Section snippets

Results and discussion

At concentrations higher than 350 μM, as used for NMR studies, we observed line-broadening of 1D spectra within 48 hours at 300 K. Most likely, the line-broadening effect could be due to oligomerization or aggregation of the protein. However, the protein is extremely soluble and stayed in solution for the duration of the experiments, even in the presence of 70% saturated ammonium sulphate. Gel-filtration (data not shown) and sedimentation analysis proved SUMO-1 to be monomeric at concentrations

Cloning procedures and protein purification

SUMO-1 was cloned into pGEX-2 T as described (Mahajan et al., 1997). pGEX-SUMO-1 was transformed into BL21 cells. Expression was induced at A600 0.7 by adding 100 μM IPTG for 16 hours. Cells were lysed with a sonifier and a 100,000 g supernatant was used for protein purification. The fusion protein was purified on a GSH-Sepharose (Pharmacia) column according to the manufacture’s specification. The GST-SUMO-1 fusion protein was digested with thrombin and the final protein product purified on a

Supplementary Files

Acknowledgements

We thank Markus Rudolph for the help with UV spectra, Bernhard Griewel for technical assistance with the NMR, Heino Prinz for help with the ESI-MS measurements, Ingrid Vetter, Frank Schmitz and Louis Renault for advice on computer programs, and Roger Goody, Fred Wittinghofer, Hans Georg Kräußlich and Larry Gerace for continuous support. This work was supported in part by DFG grant Be1423/2-1 (J.B.) and Ja18/34 (R.J.), by BMBF (P.B.), and by Fonds der Chemischen Industrie (S.M.).

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