Structural basis for SUMO-E2 interaction revealed by a complex model using docking approach in combination with NMR data

The interaction between small ubiquitin-related modifier SUMO and its conjugating-enzyme Ubc9 (E2) is an essential step in SUMO conjugation cascade. However, an experimental structure of such a transient complex is still unavailable. Here, a structural model of SUMO-3-Ubc9 complex was obtained with HADDOCK, combining NMR chemical shift mapping information. Docking calculations were performed using SUMO-3 and Ubc9 structures as input. The resulting complex reveals that the complementary surface electrostatic potentials contribute dominantly to the specific interaction. At the interface, similar numbers of oppositely-charged conserved residues are identified on the respective binding partners. Hydrogen bonds are formed in the vicinity of the interface to stabilize the complex. Comparison of the structure of SUMO-3-Ubc9 complex generated by HADDOCK and the experimental structures in free form indicates that SUMO-3 and Ubc9 maintain their respective fold as a whole after docking. However, the N-terminal helix alpha1 and its subsequent L1 loop of Ubc9 experience sizeable changes upon complex formation. They cooperatively move towards the hydrophilic side of the beta-sheet of SUMO-3. Our observations are consistent with the data from previous Ubc9 mutational analysis and conformational flexibility studies. Together, we have proposed that the SUMO-3-Ubc9 interaction is strongly electrostatically driven and the N terminus of Ubc9 shifts to SUMO-3 to facilitate the interaction. The NMR-based structural model, which provides considerable insights into the molecular basis of the specific SUMO-E2 recognition and interaction, implicates the general interaction mode between SUMO-3 and Ubc9 homologues from yeast to humans.     

Role of two residues proximal to the active site of Ubc9 in substrate recognition by the Ubc9.SUMO-1 thiolester complex

The small ubiquitin-like modifier SUMO-1 is covalently attached to lysine residues on target proteins by a specific conjugation pathway involving the E1 enzyme SAE1/SAE2 and the E2 enzyme Ubc9. In an ATP-dependent manner, the C-terminus of SUMO-1 forms consecutive thiolester bonds with cysteine residues in the SAE2 subunit and Ubc9, before the Ubc9.SUMO-1 thiolester complex catalyzes the formation of an isopeptide bond between SUMO-1 and the epsilon-amino group of the target lysine residue on the protein substrate. The SUMO-1 conjugation pathway bears many similarities with that of ubiquitin and other ubiquitin-like protein modifiers (Ubls), and because of its production of a singly conjugated substrate and the lack of absolute requirement in vitro for E3 enzymes, the SUMO-1/Ubc9 system is a good model for the analysis of protein conjugation pathways that share this basic chemistry. Here we describe methods of both steady-state and half-reaction kinetic analysis of Ubc9, and use these techniques to determine the role of two residues, Asp(100) and Lys(101) of Ubc9 which are not found in E2 enzymes from other protein conjugation pathways. These residues are found close to the active site Cys in the tertiary structure of Ubc9, and although they are shown to inhibit the transesterification reaction from SAE1/SAE2, they are important for substrate recognition in the context of the thiolester complex with SUMO-1.     

A non-covalent interaction between small ubiquitin-like modifier-1 and Zac1 regulates Zac1 cellular functions

Zac1, a zinc-finger protein that regulates apoptosis and cell cycle arrest 1, such as p53, can induce cell-cycle arrest and apoptosis. The transactivation and coactivation functions of Zac1 may occur at non-promyelocytic leukemia nuclear body (PML-NB) sites in the presence of other PML-NB components, including ubiquitin-conjugating 9 (Ubc9). It is unclear whether post-translational modification of Zac1 by the small ubiquitin-like modifier SUMO plays a role in the coactivation functions of Zac1 for the regulation of the p21 gene. Mutagenesis experiments revealed that the two SUMO-binding lysine residues of Zac1, K237 and K424, repress the transactivation activity of Zac1. Studies using a SUMO-1 C-terminal di-glycine motif mutant that is deficient in the ability to form covalent bonds with lysines, SUMO-1 (GA), and a dominant-negative Ubc9 construct (C93S) indicated that SUMO-1 might regulate Zac1 transactivation and coactivation via a non-covalent interaction. Unlike the wild-type Zac1, which induced apoptosis, the Zac1 (K237/424R) double mutant had the ability to induce autophagy. The functional role of p21 remains to be investigated. SUMO-1 selectively suppressed the induction of the p21 gene and protein by wild-type Zac1 but not by the Zac1 (K237/424R) double mutant. Moreover, wild-type Ubc9 but not Ubc9 (C93S) further potentiated the suppression of SUMO-1 in all Zac1-induced p21 promoter activities. Our data reveal that p21 may be an important factor for the prevention of Zac1-induced apoptosis without affecting autophagosome formation. This work indicates that Zac1 functions are regulated, at least in part, via non-covalent interactions with SUMO-1 for the induction of p21, which is important for the modulation of apoptosis.     

Phosphorylation of the human Fhit tumor suppressor on tyrosine 114 in Escherichia coli and unexpected steady state kinetics of the phosphorylated forms

The human tumor suppressor Fhit is a homodimeric histidine triad (HIT) protein of 147 amino acids which has Ap(3)A hydrolase activity. We have recently discovered that Fhit is phosphorylated in vivo and is phosphorylated in vitro by Src kinase [Pekarsky, Y., Garrison, P. N., Palamarchuk, A., Zanesi, N., Aqeilan, R. I., Huebner, K., Barnes, L. D., and Croce, C. M. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 3775-3779]. Now we have coexpressed Fhit with the elk tyrosine kinase in Escherichia coli to generate phosphorylated forms of Fhit. Unphosphorylated Fhit, Fhit phosphorylated on one subunit, and Fhit phosphorylated on both subunits were purified to apparent homogeneity by column chromatography on anion-exchange and gel filtration resins. MALDI-TOF and HPLC-ESI tandem mass spectrometry of intact Fhit and proteolytic peptides of Fhit demonstrated that Fhit is phosphorylated on Y(114) on either one or both subunits. Monophosphorylated Fhit exhibited monophasic kinetics with K(m) and k(cat) values approximately 2- and approximately 7-fold lower, respectively, than the corresponding values for unphosphorylated Fhit. Diphosphorylated Fhit exhibited biphasic kinetics. One site had K(m) and k(cat) values approximately 2- and approximately 140-fold lower, respectively, than the corresponding values for unphosphorylated Fhit. The second site had a K(m) approximately 60-fold higher and a k(cat) approximately 6-fold lower than the corresponding values for unphosphorylated Fhit. The unexpected kinetic patterns for the phosphorylated forms suggest the system may be enzymologically novel. The decreases in the values of K(m) and k(cat) for the phosphorylated forms in comparison to those of unphosphorylated Fhit favor the formation and lifetime of the Fhit-Ap(3)A complex, which may enhance the tumor suppressor activity of Fhit.     

Unique binding interactions among Ubc9, SUMO and RanBP2 reveal a mechanism for SUMO paralog selection

The conjugation of small ubiquitin-like modifiers SUMO-1, SUMO-2 and SUMO-3 onto target proteins requires the concerted action of the specific E1-activating enzyme SAE1/SAE2, the E2-conjugating enzyme Ubc9, and an E3-like SUMO ligase. NMR chemical shift perturbation was used to identify the surface of Ubc9 that interacts with the SUMO ligase RanBP2. Unlike known ubiquitin E2-E3 interactions, RanBP2 binds to the beta-sheet of Ubc9. Mutational disruption of Ubc9-RanBP2 binding affected SUMO-2 but not SUMO-1 conjugation to Sp100 and to a newly identified RanBP2 substrate, PML. RanBP2 contains a binding site specific for SUMO-1 but not SUMO-2, indicating that a Ubc9-SUMO-1 thioester could be recruited to RanBP2 via SUMO-1 in the absence of strong binding between Ubc9 and RanBP2. Thus we show that E2-E3 interactions are not conserved across the ubiquitin-like protein superfamily and identify a RanBP2-dependent mechanism for SUMO paralog-specific conjugation.     

Activity-dependent SUMOylation of the brain-specific scaffolding protein GISP

G-protein coupled receptor interacting scaffold protein (GISP) is a multi-domain, brain-specific protein derived from the A-kinase anchoring protein (AKAP)-9 gene. Using yeast two-hybrid screens to identify GISP interacting proteins we isolated the SUMO conjugating enzyme Ubc9. GISP interacts with Ubc9 in vitro, in heterologous cells and in neurons. SUMOylation is a post-translational modification in which the small protein SUMO is covalently conjugated to target proteins, modulating their function. Consistent with its interaction with Ubc9, we show that GISP is SUMOylated by both SUMO-1 and SUMO-2 in both in vitro SUMOylation assays and in mammalian cells. Intriguingly, SUMOylation of GISP in neurons occurs in an activity-dependent manner in response to chemical LTP. These data suggest that GISP is a novel neuronal SUMO substrate whose SUMOylation status is modulated by neuronal activity.     

The small ubiquitin-like modifier-1 (SUMO-1) consensus sequence mediates Ubc9 binding and is essential for SUMO-1 modification

SUMO-1 is an ubiquitin-related protein that is covalently conjugated to a diverse assortment of proteins. The consequences of SUMO-1 modification include the regulation of protein-protein interactions, protein-DNA interactions, and protein subcellular localization. At present, very little is understood about the specific mechanisms that govern the recognition of proteins as substrates for SUMO-1 modification. However, many of the proteins that are modified by SUMO-1 interact directly with the SUMO-1 conjugating enzyme, Ubc9. These interactions suggest that Ubc9 binding may play an important role in substrate recognition as well as in substrate modification. The SUMO-1 consensus sequence (SUMO-1-CS) is a motif of conserved residues surrounding the modified lysine residue of most SUMO-1 substrates. This motif conforms to the sequence "PsiKXE," where Psi is a large hydrophobic residue, K is the lysine to which SUMO-1 is conjugated, X is any amino acid, and E is glutamic acid. In this study, we demonstrate that the SUMO-1-CS is a major determinant of Ubc9 binding and SUMO-1 modification. Mutating residues in the SUMO-1-CS abolishes both Ubc9 binding and substrate modification. These findings have important implications for how SUMO-1 substrates are recognized and for how SUMO-1 is ultimately transferred to specific lysine residues on these substrates.     

Polymeric chains of SUMO-2 and SUMO-3 are conjugated to protein substrates by SAE1/SAE2 and Ubc9.

Conjugation of the small ubiquitin-like modifier SUMO-1/SMT3C/Sentrin-1 to proteins in vitro is dependent on a heterodimeric E1 (SAE1/SAE2) and an E2 (Ubc9). Although SUMO-2/SMT3A/Sentrin-3 and SUMO-3/SMT3B/Sentrin-2 share 50% sequence identity with SUMO-1, they are functionally distinct. Inspection of the SUMO-2 and SUMO-3 sequences indicates that they both contain the sequence psiKXE, which represents the consensus SUMO modification site. As a consequence SAE1/SAE2 and Ubc9 catalyze the formation of polymeric chains of SUMO-2 and SUMO-3 on protein substrates in vitro, and SUMO-2 chains are detected in vivo. The ability to form polymeric chains is not shared by SUMO-1, and although all SUMO species use the same conjugation machinery, modification by SUMO-1 and SUMO-2/-3 may have distinct functional consequences.     

Solution structure of human SUMO-3 C47S and its binding surface for Ubc9

Small ubiquitin-related modifier SUMO-3 is a member of a growing family of ubiquitin-like proteins (Ubls). So far, four isoforms of SUMO have been identified in humans. It is generally known that SUMO modification regulates protein localization and activity. Previous structure and function studies have been mainly focused on SUMO-1. The sequence of SUMO-3 is 46% identical with that of SUMO-1; nevertheless, functional heterogeneity has been found between the two homologues. Here we report the solution structure of SUMO-3 C47S (residues 14-92) featuring the beta-beta-alpha-beta-beta-alpha-beta ubiquitin fold. Structural comparison shows that SUMO-3 C47S resembles ubiquitin more than SUMO-1. On the helix-sheet interface, a strong hydrophobic interaction contributes to formation of the globular and compact fold. A Gly-Gly motif at the C-terminal tail, extending away from the core structure, is accessible to enzymes and substrates. In vivo, SUMO modification proceeds via a multistep pathway, and Ubc9 plays an indispensable role as the SUMO conjugating enzyme (E2) in this process. To develop a better understanding of SUMO-3 conjugation, the Ubc9 binding surface on SUMO-3 C47S has been detected by chemical shift perturbation using NMR spectroscopy. The binding site mainly resides on the hydrophilic side of the beta-sheet. Negatively charged and hydrophobic residues of this region are highly or moderately conserved among SUMO family members. Notably, the negatively charged surface of SUMO-3 C47S is highly complementary in its electrostatic potentials and hydrophobicity to the positively charged surface of Ubc9. This work indicates dissimilarities between SUMO-3 and SUMO-1 in tertiary structure and provides insight into the specific interactions of SUMO-3 with its modifying enzyme.     

Covalent modification of the Werner's syndrome gene product with the ubiquitin-related protein, SUMO-1

Werner's syndrome is a potential model of accelerated human aging. The gene responsible for Werner's syndrome encodes a protein that has a helicase domain homologous to Escherichia coli RecQ. To identify binding partners that regulate the function in concert with Wrn, we screened for proteins using the yeast two-hybrid system with mouse Wrn as bait and found three. One was a novel protein, and the other two were mouse Ubc9 and SUMO-1. Ubc9 also interacted with the mouse homologue of the Bloom's syndrome gene product, another eukaryotic RecQ-type helicase, but not mouse DNA helicase Q1/RecQL (RecQL1). Deletion experiments indicated that both proteins interacted with the N-terminal segment of Wrn (amino acid 272-514). The interaction between Wrn and SUMO-1 was weaker than that between Wrn and Ubc9. Positive interaction was observed in the heterogeneous combination of Wrn and yeast Ubc9 (yUbc9), as well as yUbc9 and SUMO-1, in the two-hybrid system. The interaction between yUbc9 and SUMO-1 was abolished by deleting the C-terminal Gly residue of SUMO-1, which is reportedly required for the formation of Ubc9-SUMO-1 thioester linkage. The interaction of Wrn and SUMO-1 was also abolished by deleting the Gly residue, indicating that the interaction of Wrn and SUMO-1 is mediated by yUbc9 in the two-hybrid system. Finally, we confirmed by immunoblotting with an anti-SUMO-1 antibody that Wrn was covalently attached with SUMO-1.     

Enzymes of the SUMO modification pathway localize to filaments of the nuclear pore complex

SUMOs are small ubiquitin-related polypeptides that are reversibly conjugated to many nuclear proteins. Although the number of identified substrates has grown rapidly, relatively little is still understood about when, where, and why most proteins are modified by SUMO. Here, we demonstrate that enzymes involved in the SUMO modification and demodification of proteins are components of the nuclear pore complex (NPC). We show that SENP2, a SUMO protease that is able to demodify both SUMO-1 and SUMO-2 or SUMO-3 protein conjugates, localizes to the nucleoplasmic face of the NPC. The unique amino-terminal domain of SENP2 interacts with the FG repeat domain of Nup153, indicating that SENP2 associates with the nucleoplasmic basket of the NPC. We also investigated the localization of the SUMO conjugating enzyme, Ubc9. Using immunogold labeling of isolated nuclear envelopes, we found that Ubc9 localizes to both the cytoplasmic and the nucleoplasmic filaments of the NPC. In vitro binding studies revealed that Ubc9 and SUMO-1-modified RanGAP1 bind synergistically to form a trimeric complex with a component of the cytoplasmic filaments of the NPC, Nup358. Our results indicate that both SUMO modification and demodification of proteins may occur at the NPC and suggest a connection between the SUMO modification pathway and nucleocytoplasmic transport.     

SUMOylation Is Required for Glycine-Induced Increases in AMPA Receptor Surface Expression (ChemLTP) in Hippocampal Neurons

Multiple pathways participate in the AMPA receptor trafficking that underlies long-term potentiation (LTP) of synaptic transmission. Here we demonstrate that protein SUMOylation is required for insertion of the GluA1 AMPAR subunit following transient glycine-evoked increase in AMPA receptor surface expression (ChemLTP) in dispersed neuronal cultures. ChemLTP increases co-localisation of SUMO-1 and the SUMO conjugating enzyme Ubc9 and with PSD95 consistent with the recruitment of SUMOylated proteins to dendritic spines. In addition, we show that ChemLTP increases dendritic levels of SUMO-1 and Ubc9 mRNA. Consistent with activity dependent translocation of these mRNAs to sites near synapses, levels of the mRNA binding and dendritic transport protein CPEB are also increased by ChemLTP. Importantly, reducing the extent of substrate protein SUMOylation by overexpressing the deSUMOylating enzyme SENP-1 or inhibiting SUMOylation by expressing dominant negative Ubc9 prevent the ChemLTP-induced increase in both AMPAR surface expression and dendritic SUMO-1 mRNA. Taken together these data demonstrate that SUMOylation of synaptic protein(s) involved in AMPA receptor trafficking is necessary for activity-dependent increases in AMPAR surface expression.     

Contractile function of rat myocardium is less susceptible to hypoxia/reoxygenation after acute infarction

The FHIT (fragile histidine triad) gene located at chromosome 3p14.2 has been proposed as a candidate tumor suppressor gene in human cancers. Fhit protein with the diadenosine 5',5'-P¹,P³-triphosphate (Ap₃A) hydrolase activity is the protein product of FHIT gene. The way in which Fhit exerts its tumor suppressor activity and the relationship of the Ap₃A hydrolase activity to tumor suppression are not known. As a step toward understanding of the Fhit function in the cell we have explored its intracellular localization and distribution in the rat tissues. Data obtained from immunoblot analysis showed that Fhit protein was most abundant in spleen and brain. Moderate amount of Fhit was detected in kidney and liver, whereas the level of Fhit protein in heart, skeletal muscle and kidney glomeruli was undetectable. RT-PCR performed on RNA isolated from these tissues showed no product, whereas the level of Fhit mRNA in spleen, brain, kidney, liver and lung correlated with the Fhit protein level. The immunoblot analysis performed on subcellular fractions of various rat tissues obtained by differential and density-gradient centrifugation showed that Fhit protein was localized exclusively in nucleus and at the plasma membrane. Presented data showing nuclear and plasma membrane localization of Fhit may support the hypothesis concerning Fhit as a signaling molecule.     

Role of an N-terminal site of Ubc9 in SUMO-1, -2, and -3 binding and conjugation

Covalent posttranslational modification of target proteins with ubiquitin and ubiquitin-like proteins regulates many important cellular processes. However, the molecular mechanisms by which these proteins are activated and conjugated to substrates has yet to be fully understood. NMR studies have shown that the ubiquitin-like proteins SUMO-1, -2, and -3 interact with the same N-terminal region of the E2 conjugating enzyme Ubc9 with similar affinities. This is correlated to their almost identical utilization by Ubc9 in the SUMO conjugation pathway. To investigate the functional significance of this interaction, site-directed mutagenesis was used to alter residues in the SUMO binding surface of Ubc9, and the effect of the amino acid substitutions on binding and conjugation to SUMO-1 and target protein RanGAP1 was investigated by isothermal titration calorimetry and biochemical analysis. R13A/K14A and R17A/K18A mutations in Ubc9 disrupted the interaction with SUMO-1 but did not completely abolish the interaction with E1. While these Ubc9 mutants displayed a significantly reduced efficiency in the transfer of SUMO-1 from E1 to E2, their ability to recognize substrate and transfer SUMO-1 from E2 to the target protein was unaffected. These results suggest that the noncovalent binding site of SUMO-1 on Ubc9, although distant from the active site, is important for the transfer of SUMO-1 from the E1 to the E2. The conservation of E2 enzymes across the ubiquitin and ubiquitin-like protein pathways indicates that analogous N-terminal sites of E2 enzymes are likely to have similar roles in general.