A Central Cysteine Residue Is Essential for the ... - ACS Publications

Article pubs.acs.org/bc

A Central Cysteine Residue Is Essential for the Thermal Stability and Function of SUMO‑1 Protein and SUMO‑1 Peptide−Protein Conjugates Hervé Drobecq,† Emmanuelle Boll,† Magalie Sénéchal,† Rémi Desmet,† Jean-Michel Saliou,‡ Jean-Jacques Lacapère,§ Alexandra Mougel,† Jérôme Vicogne,*,† and Oleg Melnyk*,† †

M3TMechanisms of Tumorigenesis and Target Therapies, Université de Lille, CNRS, Institut Pasteur de Lille, UMR 8161, F-59000 Lille, France ‡ Université de Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019-UMR 8204, F-59000 Lille, France § Sorbonne Universités, UPMC Université Paris 06, École Normale Supérieure, PSL Research University, CNRS UMR 7203 LBM, F-75005, Paris, France S Supporting Information *

ABSTRACT: SUMOylation constitutes a major post-translational modification (PTM) used by the eukaryote cellular machinery to modulate protein interactions of the targeted proteins. The small ubiquitin-like modifier-1 (SUMO-1) features a central and conserved cysteine residue (Cys52) that is located in the hydrophobic core of the protein and in tight contact with Phe65, suggesting the occurrence of an S/π interaction. To investigate the importance of Cys52 on SUMO-1 thermal stability and biochemical properties, we produced by total chemical synthesis SUMO-1 or SUMO-1 Cys52Ala peptide−protein conjugates featuring a native isopeptidic bond between SUMO-1 and a peptide derived from p53 tumor suppressor protein. The Cys52Ala modification perturbed SUMO-1 secondary structure and resulted in a dramatic loss of protein thermal stability. Moreover, the cleavage of the isopeptidic bond by the deconjugating enzyme Upl1 was significantly less efficient than for the wild-type conjugate. Similarly, the in vitro SUMOylation of RanGap1 by E1/E2 conjugating enzymes was significantly less efficient with the SUMO-1 C52A analog compared to wild-type SUMO-1. These data demonstrate the critical role of Cys52 in maintaining SUMO-1 conformation and function and the importance of keeping this cysteine intact for the study of SUMO-1 protein conjugates.

■

INTRODUCTION

Ubiquitin1 (Ub, 76 AA) and small ubiquitin-like modifiers (SUMO,2 92−96 AA) are highly dynamic and reversible post-translational modifications (PTMs) that are used by the cell machinery to regulate a vast array of cellular processes. These modifiers are attached to the target proteins through an isopeptidic bond involving the C-terminus of the modifier and the ε-amino group of a lysine residue (Lys). Ubiquitination is principally a signal for protein degradation by the proteasome. In contrast, the main role of SUMOylation is to change the localization, the stability, or the activity of the target protein by altering protein−protein interactions or by competing with other PTMs such as ubiquitin.3,4 SUMO-1 is the founding member of the SUMO family which contains three isoforms. SUMO-1 is involved in the regulation of some kinases such as cyclin-dependent kinase 65 and several transcription factors.6−8 One of these transcription factors is the tumor suppressor p53 protein,9 which plays a major role in cell cycle regulation and is considered as the “guardian of the genome”. p53 triggers cell apoptosis in response to various stresses, while its inactivation or mutation promotes tumorigenesis.10 p53 is the target of numerous PTMs including SUMO-1, which is linked to Lys386 within the C-terminal p53 © XXXX American Chemical Society

Figure 1. NMR structure of SUMO-1 (PDB entry 1A5R14) shows that Cys52 is buried in the interior of the protein and in contact with Phe65.

regulatory domain.6 In this context, the access to homogeneous SUMO-1 peptide−protein conjugates derived from p53 should be of great value for understanding the impact of the SUMO-1 modifier on the structure and biological function of p53. However, which sequence should we use for the SUMO-1 domain? The obvious answer is the wild-type sequence. However, previous work mentioned that the central Cys at position 52 Received: April 27, 2016 Revised: May 17, 2016

A

DOI: 10.1021/acs.bioconjchem.6b00211 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Article

Bioconjugate Chemistry resulted in difficulties in manipulating this protein due to the formation of intermolecular S−S bridges during purification or analysis steps.11 Intriguingly, the recombinant SUMO-1 protein used in this study was delivered as a mixed disulfide with β-mercaptoethanol, confirming the propensity of SUMO-1 to form disulfides in certain conditions (see Supporting Information).

As a consequence, several biochemical and structural studies used the SUMO-1 C52A mutant to overcome this problem.11−13 However, the impact of the C52A mutation on the stability and structure of SUMO-1 has not been examined so far. The core of SUMO-1 shows a typical ubiquitin fold with an α-helix (α1, L44-Q55) packed against a four-stranded β-sheet (Figure 1).14 Importantly, the side chain thiol of Cys52, which is located in the α1 helix, is in close contact with the phenyl ring of Phe65 and buried in the interior of the protein (Figure 1). The proximity of Cys52 and Phe65 suggests the occurrence of an S/π interaction within SUMO-1, i.e., a type of interaction frequently found in proteins and whose contribution to protein folding and stabilization is not fully understood.15,16 The location of Cys52 in the hydrophobic core of SUMO-1 and the Cys52/Phe65 contact prompted us to examine the effect of the C52A modification on SUMO-1 structure, thermal stability, and enzymatic processing at the level of the SUMO-1 protein or a SUMO-1 p53 peptide−protein conjugate.

Scheme 1. General Strategy for Accessing Native Ub or SUMO-1 Conjugates Using the NCL Reaction and δ-Mercaptolysine

■

RESULTS AND DISCUSSION Total Synthesis of p53 SUMO-1 Peptide Protein Conjugates. We used the power of chemical synthesis to access the homogeneous p53 SUMO-1 peptide−protein conjugates needed for our study in milligram quantities. Following the pioneering work of Ramage and co-workers,17 the total or semisynthesis of Ub or Ub-like modified polypeptides has been the subject of intense research during the past decade.18−29 29 One frequently used strategy for preparing Ub conjugates or polyubiquitin chains consists of reacting a Ub thioester molecule with a polypeptide featuring a γ- or δ-mercaptolysine19−21 using the native chemical ligation (NCL30) or expressed protein ligation (EPL25) (Scheme 1, illustrated with δ-mercaptolysine21). The Ub thioester itself can be prepared by recombinant techniques, by solid phase peptide synthesis (SPPS)20 or assembled using the Scheme 2. Synthesis of p53 SUMO-1 Peptide−Protein Conjugates

B


Article

Bioconjugate Chemistry NCL reaction.31 The later strategy requires replacing one internal alanine residue (Ala) by Cys, typically Ala46, since native Ub is devoid of Cys residues. The final step of the synthesis is a desulfurization of the mercaptolysine into lysine.32 When Ub(A46C) thioester is used in the assembly of the conjugate, the desulfurization step also converts the internal Cys46 residue into Ala. This strategy is immediately applicable to the synthesis of SUMO-1 peptide−protein conjugates featuring the C52A modification. In contrast, the access to native SUMO-1 conjugates implies that the desulfurization must be selective for the δ-mercaptolysine residue (Scheme 1). Ovaa and co-workers reported the semisynthesis of a SUMO-1 analog by performing the ligation of a recombinant and wild-type SUMO-1 thioester molecule with a δ-thiolysine dipeptide derivative and a desulfurization step in one pot.33 Surprisingly, MS analysis of the final product showed the loss of one sulfur atom only. Although these experiments suggested that the desulfurization reaction might be selective for the δ-mercaptolysine residue, no thorough characterization of the selectivity has been reported. Moreover, the total synthesis of native SUMO-1 peptide−protein conjugates on a milligram scale by selective desulfurization has not been described so far. We reasoned that the selectivity of the desulfurization step might arise from the masking of the side chain thiol of Cys52 by the hydrophobic core of SUMO-1, while the lysines modified by SUMO modifiers are usually solvent-exposed. Two different p53 SUMO-1 conjugates were considered in this study as shown in Scheme 2. In conjugate 5a, the SUMO-1 modifier is linked to Lys386 through a Cys residue, while in 5b it is directly attached to the δ- mercaptolysine residue. The p53 SUMO-1 conjugate 5a, which is more easily accessible than 5b, was envisioned as a good starting point for addressing the selectivity of the desulfurization reaction. Then, we verified the viability of this approach for accessing the native p53 SUMO-1 conjugate 6b in homogeneous form and milligram quantities. p53 SUMO-1 peptide−protein conjugates 5a,b, whose molecular weight is ∼14 kDa, were synthesized using the onepot three-peptide segment process depicted in Scheme 2.27,28,34 In this procedure, the SUMO-1−SEAoff thioester surrogate35 3 was assembled using an NCL reaction in the presence of 4-mercaptophenylacetic acid (MPAA36). Subsequent ligation with the target p53 peptide 4a or 4b in the presence of MPAA and tris(2-carboxyethyl)phosphine (TCEP) yielded p53 SUMO-1 conjugates 5a,b with equal efficiency and in highly homogeneous form (27−38% yield). Their identity and branched structure were demonstrated by high resolution mass spectrometry (HRMS), MALDI-TOF in source fragmentation, and proteomic analysis (see Supporting Information). The desulfurization reactions were performed by treating the conjugates with TCEP in the presence of the radical initiator VA-044 and glutathione (GSH) at pH 7.2 and 25 °C.32 We first examined the desulfurization of p53 SUMO-1 conjugate 5a in phosphate buffer in the presence or absence of guanidine hydrochloride (Gdn·HCl) used as a denaturant. The reactions were monitored by matrix-assisted laser desorption ionization (MALDI) mass spectrometry (see Supporting Information Figure S10). As expected, the reaction in denaturing conditions (6 M Gdn·HCl) yielded cleanly conjugate 7a due to the concomitant desulfurization of the SUMO Cys52 and linker Cys residues. In contrast, the desulfurization of p53 SUMO-1 conjugate 5a in the absence of denaturant resulted in the loss of only one sulfur atom even after prolonged reaction times

Figure 2. Desulfurization of synthetic p53 SUMO-1 conjugate 5b (VA-044 1.33 mM, TCEP 200 mM, GSH 100 mM, 25 °C, pH 7.2). MALDI-TOF analysis of the desulfurization reaction using sinapinic acid as matrix (a, starting conjugate 5b; b, 6 M Gdn·HCl; c−e, native conditions) and LC and HRMS analysis of purified 6b (f−h).

(>48 h). p53 SUMO-1 conjugate 6a was isolated and submitted to extensive proteomic analyses. These analyses included the digestion of 6a by trypsin and the separation and identification of the peptides corresponding to the central region of SUMO-1 and to the linker region between SUMO-1 and p53 peptide. These analyses showed the presence of an Ala residue between the SUMO-1 domain and the target p53 peptide and that the internal Cys52 of SUMO-1 domain was unaffected (see Supporting Information Figures S11−S16). Thus, our data demonstrate that the desulfurization reaction is highly selective for the Cys residue in the linker region. C


Article

Bioconjugate Chemistry

Figure 3. Preparation and characterization of hSUMO-1 proteins 8 and 9.

We next examined the reactivity of the δ-thiolysine analog 5b in similar experimental conditions. Here again, the desulfurization reaction in native conditions resulted in the loss of only one sulfur atom as for 5a (Figure 2a−e). Likewise, thorough proteomic analyses demonstrated the high selectivity of the desulfurization reaction for the δ-mercaptolysine residue. Native p53 SUMO-1 conjugate 7b was isolated in 80% yield and displayed the expected mass by high resolution mass spectrometry (Figure 2f,h). Preparation of Wild-Type and Cys52Ala SUMO-1 Domains. As mentioned before, the recombinant SUMO-1 protein used in this study was delivered as a mixed disulfide with β-mercaptoethanol. Therefore, the recombinant SUMO-1 β-mercaptoethanol mixed disulfide was solubilized in 6 M guanidinium hydrochloride, reduced with TCEP, and purified by HPLC to produce the wild-type SUMO-1 protein 8 as a lyophilized powder (Figure 3). The use of the radical initiator VA-044 and glutathione (GSH) in addition to TCEP furnished SUMO-1 C52A analog 9 which was also purified by HPLC and lyophilized. Both proteins were analyzed by LC−MS and high resolution mass spectrometry to demonstrate their purity and identity (Figure 3). The proteins were subsequently solubilized in the appropriate buffer and tested as discussed hereinafter. It is important to mention at this point that both proteins were exposed to the same solvent conditions. Moreover, hSUMO-1 is known to be stable upon lyophilization and to refold spontaneously upon dissolution in water at neutral pH. Analysis of p53 SUMO-1 Conjugates by Thermal Shift Assay and Circular Dichroism spectroscopy. We first examined the thermal stability of SUMO-1 proteins 8,9 and p53 SUMO-1 conjugates 6b,7b at pH 7.2 by thermal shift assay (TSA,37 see Supporting Information Figures S26 and S27). The native SUMO-1 protein 8 and p53 SUMO-1 conjugate 6b displayed cooperative thermal unfolding curves with a Tm of 65.8 ± 1.2 °C and 64.4 ± 1.9 °C, respectively. These Tm values are consistent with the reported Tm value for SUMO-1 (∼62 °C, pH 5.6) as determined by circular dichroism (CD).38 In contrast,

no Tm could be measured for the C52A analogs 9 and 7b using this technique. We next analyzed SUMO-1 proteins 8,9 and p53 SUMO-1 conjugates 6b,7b by CD spectroscopy at pH 7.2 and 20 °C (Figure 4). While the four proteins showed an α-helical

Figure 4. CD analysis of SUMO-1 proteins 8 and 9 and of p53 SUMO-1 conjugates 6b and 7b (10 μM, pH 7.2, 20 °C).

content of ∼8% at 20 °C, a value that is consistent with the α-helical content calculated from the NMR solution structure of SUMO-1,14 the far-UV CD spectra for C52A modified proteins 9 and 7b were significantly different from the wild-type analogs 8 and 6b. No significant change in the ellipticity at 222 nm was observed upon thermal denaturation, while the ellipticity at 202 nm was found to decrease almost linearly over the temperature range studied (20−80 °C; see Supporting Information Figure S28). Thus, to the contrary of the wild-type SUMO-1 protein which unfolds in a cooperative manner upon D


Article

Bioconjugate Chemistry heating,38 the thermal denaturation of C52A analogs 9 and 7b is clearly noncooperative. The only structures available for SUMO-1 C52A mutants were determined by X-ray crystallography.12,13 Koide and co-workers determined the structure of a full length SUMO-1 C52A analog (SUMO-1 1−97 C52A with a GS N-terminal extension) in complex with a monobody.12 Very recently, Cappadocia and co-workers determined the structure of a core SUMO-1 C52A analog (SUMO-1 18−97 C52A with a GSK N-terminal extension) in complex with different peptides derived from the promyelocytic leukemia protein (PML).13 In each case, the structure of the SUMO-1 domain was similar to the published X-ray crystallographic structures for wild-type SUMO-1. In contrast, the CD and TSA experiments presented above suggest that in solution the structure of C52A analogs 9 and 7b is significantly perturbed at 20 °C and above. One reason for this difference might be due to the stabilization of SUMO1 C52A mutated proteins by their interaction with PML or monobody polypeptides. Another explanation resides in a limitation of the cryocooling method used in the above X-ray crystallographic studies, which can sometimes distort the repertoire of accessible conformations.39 Functionality of Wild-Type and Cys52Ala SUMO-1 Domains. We next reasoned that the structural differences between wild-type and C52A modified SUMO-1 domains might impact the capacity of p53 SUMO-1 conjugates 6b and 7b to be cleaved by Ulp1 deconjugating enzyme. Indeed, Ulp1 is an excellent sensor of SUMO tertiary structure, since the deconjugation reaction catalyzed by Ulp1 is critically dependent on an extensive contact surface between the enzyme and SUMO.40 The cleavage at two different temperatures (20 and 37 °C) was monitored by gel electrophoresis using Coomassie staining (Figure 5A) or MALDI-TOF mass spectrometry (see

conformation in comparison with wild-type conjugate 6b due to the denaturing conditions used for the desulfurization step, rather than to the C52A modification. However, the denaturation of human SUMO-1 by heat or urea has been shown to be reversible.38 This is confirmed by the study of SUMO-1 protein 8 by CD and TSA. A comparison of SUMO-1 proteins 8 and 9 by these techniques gave us already the opportunity to clearly establish the impact of the C52A modification on the conformation and thermal stability of the SUMO-1 domain. Nevertheless, we further tested the biochemical conjugation of SUMO-1 proteins 8 and 9 to RanGap1 protein using E1/E2 conjugating enzymes (Figure 5B). The Western blot analysis of the SUMOylation reaction with an anti-SUMO antibody showed that the conjugation of SUMO-1 9 was markedly less efficient compared to wild-type SUMO-1 8. Taken together, the TSA, CD, and biochemical data presented above show that the conformation of SUMO-1 C52A analogs is significantly perturbed in comparison with wild-type proteins and highlight the critical role played by Cys52 in stabilizing SUMO-1 structure.

■

CONCLUSIONS The replacement of Cys52 in SUMO-1 by Ala results in a significant perturbation of protein thermal stability, secondary structure, and biochemical properties. This effect was observed with recombinant SUMO-1 proteins or synthetic p53 SUMO-1 conjugates. Although previous studies using SUMO-1 C52A mutants yielded useful structural informations, this work shows that the C52A modification has a major impact on SUMO-1 that cannot be neglected. Cys52 presents a free thiol group in tight contact with Phe65 within the hydrophobic core of SUMO-1. The fact that the loss of one sulfur atom can have such a large impact on protein thermal stability and folding cooperativity has been rarely observed. Up to now, several in silico studies using protein structures from the Protein Data Bank suggested the occurrence of Cys/aromatic contacts in proteins. However, the contribution of this interaction to protein stability is in debate. As a perspective, the ease of access to SUMO-1 analogs by chemical synthesis makes SUMO-1 a good model for studying the importance of Cys/aromatic contacts on protein stability. Importantly, this work highlights the relevance of keeping the central cysteine residue of SUMO1 intact for the studies aimed at exploring the properties and functions of SUMO-1 protein conjugates.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.6b00211. Procedures and characterization for all new compounds (PDF)

Figure 5. (A) Cleavage of conjugates 6b and 7b by Ulp 1; SDS− PAGE analysis using Coomassie staining. (B) In vitro transfer of SUMO proteins on RanGap1; SUMO-1 8 or 9 were transferred on RanGap1 using purified recombinant E1−E2 SUMO transferases for 10, 20, and 30 min at 37 °C. NR: no E1, E2 and ATP reagents. ATP−: ATP was omitted from the reaction mixture. Positive and negative controls from original SUMOylation kit containing (+) or not containing (−) recombinant SUMO-1 6×His tag. Mixtures were analyzed by SDS−PAGE and Western blot using a specific antiSUMO-1 antibody.

■

AUTHOR INFORMATION

Corresponding Authors

*J.V.: e-mail, [email protected]; phone, +33 (0)3 20 87 12 49. *O.M.: e-mail, [email protected]; Web site, http:// olegmelnyk.cnrs.fr; phone, +33 (0)3 20 87 12 14.

Supporting Information Figure S29). These analyses revealed that the cleavage of the wild-type conjugate 6b proceeded significantly faster than the cleavage of C52A analog 7b at both temperatures, confirming that the latter has a perturbed conformation. The C52A analog 7b might have a perturbed

Notes

The authors declare no competing financial interest. E


Article

Bioconjugate Chemistry

■

(18) Chatterjee, C., McGinty, R. K., Pellois, J. P., and Muir, T. W. (2007) Auxiliary-mediated site-specific peptide ubiquitylation. Angew. Chem., Int. Ed. 46, 2814−8. (19) Yang, R., Pasunooti, K. K., Li, F., Liu, X. W., and Liu, C. F. (2009) Dual native chemical ligation at lysine. J. Am. Chem. Soc. 131, 13592−3. (20) El Oualid, F., Merkx, R., Ekkebus, R., Hameed, D. S., Smit, J. J., de Jong, A., Hilkmann, H., Sixma, T. K., and Ovaa, H. (2010) Chemical synthesis of ubiquitin, ubiquitin-based probes, and diubiquitin. Angew. Chem., Int. Ed. 49, 10149−53. (21) Ajish Kumar, K. S., Haj-Yahya, M., Olschewski, D., Lashuel, H. A., and Brik, A. (2009) Highly efficient and chemoselective peptide ubiquitylation. Angew. Chem., Int. Ed. 48, 8090−8094. (22) Fierz, B., Chatterjee, C., McGinty, R. K., Bar-Dagan, M., Raleigh, D. P., and Muir, T. W. (2011) Histone H2B ubiquitylation disrupts local and higher-order chromatin compaction. Nat. Chem. Biol. 7, 113−9. (23) McGinty, R. K., Köhn, M., Chatterjee, C., Chiang, K. P., Pratt, M. R., and Muir, T. W. (2009) Structure-activity analysis of semisynthetic nucleosomes: Mechanistic insights into the stimulation of Dot1L by ubiquitylated histone H2B. ACS Chem. Biol. 4, 958−968. (24) McGinty, R. K., Chatterjee, C., and Muir, T. W. (2009) Semisynthesis of ubiquitylated proteins. Methods Enzymol. 462, 225− 43. (25) Flavell, R. R., and Muir, T. W. (2009) Expressed protein ligation (EPL) in the study of signal transduction, ion conduction, and chromatin biology. Acc. Chem. Res. 42, 107−16. (26) McGinty, R. K., Kim, J., Chatterjee, C., Roeder, R. G., and Muir, T. W. (2008) Chemically ubiquitylated histone H2B stimulates hDot1L-mediated intranucleosomal methylation. Nature 453, 812−6. (27) Boll, E., Drobecq, H., Ollivier, N., Blanpain, A., Raibaut, L., Desmet, R., Vicogne, J., and Melnyk, O. (2015) One-pot chemical synthesis of small ubiquitin-like modifier (SUMO) protein-peptide conjugates using bis(2-sulfanylethyl)amido peptide latent thioester surrogates. Nat. Protoc. 10, 269−292. (28) Boll, E., Drobecq, H., Ollivier, N., Raibaut, L., Desmet, R., Vicogne, J., and Melnyk, O. (2014) A novel PEG-based solid support enables the synthesis of >50 amino-acid peptide thioesters and the total synthesis of a functional SUMO-1 peptide conjugate. Chem. Sci. 5, 2017−2022. (29) Wucherpfennig, T. G., Pattabiraman, V. R., Limberg, F. R. P., Ruiz-Rodríguez, J., and Bode, J. W. (2014) Traceless Preparation of CTerminal α-Ketoacids for Chemical Protein Synthesis by α-Ketoacid− Hydroxylamine Ligation: Synthesis of SUMO2/3. Angew. Chem., Int. Ed. 53, 12248−12252. (30) Dawson, P. E., Muir, T. W., Clark-Lewis, I., and Kent, S. B. (1994) Synthesis of proteins by native chemical ligation. Science 266, 776−779. (31) Erlich, L. A., Kumar, K. S., Haj-Yahya, M., Dawson, P. E., and Brik, A. (2010) N-methylcysteine-mediated total chemical synthesis of ubiquitin thioester. Org. Biomol. Chem. 8, 2392−6. (32) Wan, Q., and Danishefsky, S. J. (2007) Free-radical-based, specific desulfurization of cysteine: a powerful advance in the synthesis of polypeptides and glycopolypeptides. Angew. Chem., Int. Ed. 46, 9248−52. (33) de Jong, A., Merkx, R., Berlin, I., Rodenko, B., Wijdeven, R. H., El Atmioui, D., Yalcin, Z., Robson, C. N., Neefjes, J. J., and Ovaa, H. (2012) Ubiquitin-based probes prepared by total synthesis to profile the activity of deubiquitinating enzymes. ChemBioChem 13, 2251−8. (34) Ollivier, N., Vicogne, J., Vallin, A., Drobecq, H., Desmet, R., ElMahdi, O., Leclercq, B., Goormachtigh, G., Fafeur, V., and Melnyk, O. (2012) A one-pot three-segment ligation strategy for protein chemical synthesis. Angew. Chem., Int. Ed. 51, 209−213. (35) Ollivier, N., Dheur, J., Mhidia, R., Blanpain, A., and Melnyk, O. (2010) Bis(2-sulfanylethyl)amino native peptide ligation. Org. Lett. 12, 5238−41. (36) Johnson, E. C., and Kent, S. B. (2006) Insights into the mechanism and catalysis of the native chemical ligation reaction. J. Am. Chem. Soc. 128, 6640−6.

ACKNOWLEDGMENTS We thank the CSB facility for technical help and the Laboratoire d’Excellence (LabEx) ParaFrap (ANR-11-LABX0024), the Métropole Européene de Lille (MEL), the Fonds Européen de Développement Economique Régionale (FEDERERDF), and the Région Nord Pas de Calais for financial support. We thank A. Brik (Technion-Israel Institute of Technology) for the generous gift of δ-mercaptolysine.

■

REFERENCES

(1) Goldstein, G., Scheid, M., Hammerling, U., Schlesinger, D. H., Niall, H. D., and Boyse, E. A. (1975) Isolation of a polypeptide that has lymphocyte-differentiating properties and is probably represented universally in living cells. Proc. Natl. Acad. Sci. U. S. A. 72, 11−5. (2) Matunis, M. J., Coutavas, E., and Blobel, G. (1996) A novel ubiquitin-like modification modulates the partitioning of the RanGTPase-activating protein RanGAP1 between the cytosol and the nuclear pore complex. J. Cell Biol. 135, 1457−70. (3) Muller, S., Hoege, C., Pyrowolakis, G., and Jentsch, S. (2001) SUMO, ubiquitin’s mysterious cousin. Nat. Rev. Mol. Cell Biol. 2, 202− 10. (4) Johnson, E. S. (2004) Protein modification by SUMO. Annu. Rev. Biochem. 73, 355−82. (5) Bellail, A. C., Olson, J. J., and Hao, C. (2014) SUMO1 modification stabilizes CDK6 protein and drives the cell cycle and glioblastoma progression. Nat. Commun. 5, 4234. (6) Gostissa, M., Hengstermann, A., Fogal, V., Sandy, P., Schwarz, S. E., Scheffner, M., and Del Sal, G. (1999) Activation of p53 by conjugation to the ubiquitin-like protein SUMO-1. EMBO J. 18, 6462−71. (7) Muller, S., Berger, M., Lehembre, F., Seeler, J. S., Haupt, Y., and Dejean, A. (2000) c-Jun and p53 activity is modulated by SUMO-1 modification. J. Biol. Chem. 275, 13321−9. (8) Lehembre, F., Muller, S., Pandolfi, P. P., and Dejean, A. (2001) Regulation of Pax3 transcriptional activity by SUMO-1-modified PML. Oncogene 20, 1−9. (9) Zakut-Houri, R., Bienz-Tadmor, B., Givol, D., and Oren, M. (1985) Human p53 cellular tumor antigen: cDNA sequence and expression in COS cells. EMBO J. 4, 1251−5. (10) Chene, P. (2003) Inhibiting the p53-MDM2 interaction: an important target for cancer therapy. Nat. Rev. Cancer 3, 102−9. (11) Tatham, M. H., Jaffray, E., Vaughan, O. A., Desterro, J. M., Botting, C. H., Naismith, J. H., and Hay, R. T. (2001) Polymeric chains of SUMO-2 and SUMO-3 are conjugated to protein substrates by SAE1/SAE2 and Ubc9. J. Biol. Chem. 276, 35368−74. (12) Koide, A., Wojcik, J., Gilbreth, R. N., Hoey, R. J., and Koide, S. (2012) Teaching an old scaffold new tricks: monobodies constructed using alternative surfaces of the FN3 scaffold. J. Mol. Biol. 415, 393− 405. (13) Cappadocia, L., Mascle, X. H., Bourdeau, V., Tremblay-Belzile, S., Chaker-Margot, M., Lussier-Price, M., Wada, J., Sakaguchi, K., Aubry, M., Ferbeyre, G., et al. (2015) Structural and functional characterization of the phosphorylation-dependent interaction between PML and SUMO1. Structure 23, 126−38. (14) Bayer, P., Arndt, A., Metzger, S., Mahajan, R., Melchior, F., Jaenicke, R., and Becker, J. (1998) Structure determination of the small ubiquitin-related modifier SUMO-1. J. Mol. Biol. 280, 275−86. (15) Beno, B. R., Yeung, K. S., Bartberger, M. D., Pennington, L. D., and Meanwell, N. A. (2015) A Survey of the Role of Noncovalent Sulfur Interactions in Drug Design. J. Med. Chem. 58, 4383−438. (16) Hussain, H. B., Wilson, K. A., and Wetmore, S. D. (2015) Serine and Cysteine pi-Interactions in Nature: A Comparison of the Frequency, Structure, and Stability of Contacts Involving Oxygen and Sulfur. Aust. J. Chem. 68, 385−395. (17) Ramage, R., Green, J., and Ogunjobi, O. M. (1989) Solid phase peptide synthesis of ubiquitin. Tetrahedron Lett. 30, 2149−2152. F


Article

Bioconjugate Chemistry (37) Niesen, F. H., Berglund, H., and Vedadi, M. (2007) The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nat. Protoc. 2, 2212−21. (38) Kumar, A., Srivastava, S., and Hosur, R. V. (2007) NMR characterization of the energy landscape of SUMO-1 in the native-state ensemble. J. Mol. Biol. 367, 1480−93. (39) Fraser, J. S., van den Bedem, H., Samelson, A. J., Lang, P. T., Holton, J. M., Echols, N., and Alber, T. (2011) Accessing protein conformational ensembles using room-temperature X-ray crystallography. Proc. Natl. Acad. Sci. U. S. A. 108, 16247−52. (40) Mossessova, E., and Lima, C. D. (2000) Ulp1-SUMO crystal structure and genetic analysis reveal conserved interactions and a regulatory element essential for cell growth in yeast. Mol. Cell 5, 865− 76.

G


A Central Cysteine Residue Is Essential for the ... - ACS Publications

Recommend Documents