Acetylation of SUMO1 Alters Interactions with the SIMs of PML and Daxx in a Protein-Specific Manner

Article

Acetylation of SUMO1 Alters Interactions with the SIMs of PML and Daxx in a Protein-Specific Manner Graphical Abstract

Authors Xavier H. Mascle, Christina Gagnon, Haytham M. Wahba, Mathieu Lussier-Price, Laurent Cappadocia, Kazuyasu Sakaguchi, James G. Omichinski

Correspondence [email protected]

In Brief Mascle et al. describe structural details of how acetylation of SUMO1 at lysine residues in its SIM-binding region alters interactions with the phosphorylated SIMs of PML and Daxx. In particular, acetylation at K37 alters interaction with the Daxx-SIM, but not with the PML-SIM, which highlights the plasticity of SUMOSIM interactions.

Highlights d

Structures of PML and Daxx phosphoSIMs bound to acetylated SUMO1 are presented

d

Acetylation of SUMO1 at key Lys residues alters interactions with the phosphoSIMs

d

SUMO1 acetylation at K39 or K46 inhibits binding to phosphoSIMs of PML and Daxx

d

SUMO1 acetylation at K37 inhibits binding only to the phosphoSIM of Daxx

Mascle et al., 2020, Structure 28, 1–12 February 4, 2020 ª 2019 Elsevier Ltd. https://doi.org/10.1016/j.str.2019.11.019

Please cite this article in press as: Mascle et al., Acetylation of SUMO1 Alters Interactions with the SIMs of PML and Daxx in a Protein-Specific Manner, Structure (2019), https://doi.org/10.1016/j.str.2019.11.019

Structure

Article Acetylation of SUMO1 Alters Interactions with the SIMs of PML and Daxx in a Protein-Specific Manner Xavier H. Mascle,1,5 Christina Gagnon,1,5 Haytham M. Wahba,1,2,5 Mathieu Lussier-Price,1 Laurent Cappadocia,1,4 Kazuyasu Sakaguchi,3 and James G. Omichinski1,6,* 1De ´ partement de Biochimie et Me´dicine Mole´culaire, Universite´ de Montre´al, C.P. 6128 Succursale Centre-Ville, Montre´al, QC H3C 3J7, Canada 2Department of Biochemistry, Beni-Suef University, Beni-Suef 62521, Egypt 3Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan 4Present address: De ´ partement de Chimie, Universite´ de Que´bec a` Montre´al, Montre´al, QC H2X 2J6, Canada 5These authors contributed equally 6Lead Contact *Correspondence: [email protected] https://doi.org/10.1016/j.str.2019.11.019

SUMMARY

The interactions between SUMO proteins and SUMO-interacting motif (SIM) in nuclear bodies formed by the promyelocytic leukemia (PML) protein (PML-NBs) have been shown to be modulated by either phosphorylation of the SIMs or acetylation of SUMO proteins. However, little is known about how this occurs at the atomic level. In this work, we examined the role that acetylation of SUMO1 plays on its binding to the phosphorylated SIMs (phosphoSIMs) of PML and Daxx. Our results demonstrate that SUMO1 binding to the phosphoSIM of either PML or Daxx is dramatically reduced by acetylation at either K39 or K46. However, acetylation at K37 only impacts binding to Daxx. Structures of acetylated SUMO1 variants bound to the phosphoSIMs of PML and Daxx demonstrate that there is structural plasticity in SUMO-SIM interactions. The plasticity observed in these structures provides a robust mechanism for regulating SUMO-SIM interactions in PML-NBs using signaling generated post-translational modifications.

INTRODUCTION Small ubiquitin-like modifiers (SUMOs) are a family of proteins that are found predominantly in the nucleus of eukaryotic organisms (Geiss-Friedlander and Melchior, 2007). Humans express five SUMO paralogs (SUMO1-5) that can be divided into two groups based on their primary sequence (Liang et al., 2016). The sequence of SUMO1 is highly homologous to that of both SUMO4 and SUMO5, whereas SUMO2 and SUMO3 share over 95% sequence identity, but only 45% with SUMO1 (Geiss-Friedlander and Melchior, 2007; Guo et al., 2004; Liang et al., 2016). Like ubiquitin, SUMO proteins are conjugated

to lysine (Lys) residues in substrates as a post-translational modification (PTM), a process referred to as SUMOylation. The SUMOylation reaction requires an E1-activating enzyme (SAE1-SAE2), an E2-conjugating enzyme (UBC9), and in most cases a SUMO E3 ligase, such as the protein inhibitor of activated STATs family members (Schmidt and Muller, 2003). Although it is still not clear why humans express multiple paralogs, it is established that the SUMO proteins play important roles in regulating many cellular processes, including transcription, DNA repair, and ribosome biogenesis (Haindl et al., 2008; Muller et al., 2004). SUMO proteins typically carry out their functions by participating in protein-protein interactions either in the unconjugated form or following SUMOylation of a target protein. In both cases, the SUMO proteins participate in non-covalent interactions with specialized SUMO-binding domains (SBDs) located in their binding partners (Geiss-Friedlander and Melchior, 2007). At present, the best characterized SBD is the SUMO-interacting motif (SIM), which has been defined to minimally contain a short hydrophobic core sequence of the form cccc, ccxc, or cxcc (where c is V, I, or L and x is typically D, E, S, or T) usually in an intrinsically disordered region (Beauclair et al., 2015; Gareau and Lima, 2010; Song et al., 2004). In addition, SIMs often contain negatively charged amino acids and/or potential phosphorylation sites (Ser or Thr) located immediately adjacent to the hydrophobic core sequence either at its N- or C-terminal side (Cappadocia and Lima, 2018; Chang et al., 2011; Cho et al., 2009; Lin et al., 2006; Negorev et al., 2001; Rasheed et al., 2002; Scaglioni et al., 2006; Stehmeier and Muller, 2009; Sung et al., 2011; Ullmann et al., 2012). Upon binding to a SUMO family protein, the SIM adopts a b strand conformation that is either in a parallel or an antiparallel orientation relative to the second b strand of the SUMO protein, and it appears that the orientation is dictated by whether the negatively charged amino acids are located on the N-terminal (antiparallel) or C-terminal (parallel) side of the hydrophobic core (Gareau and Lima, 2010; Song et al., 2005). Some of the best characterized SUMO-SIM interactions involve proteins located in promyelocytic leukemia nuclear bodies (PML-NBs). PML-NBs are subnuclear structures that Structure 28, 1–12, February 4, 2020 ª 2019 Elsevier Ltd. 1

Please cite this article in press as: Mascle et al., Acetylation of SUMO1 Alters Interactions with the SIMs of PML and Daxx in a Protein-Specific Manner, Structure (2019), https://doi.org/10.1016/j.str.2019.11.019

were first observed in tumor cells (Cho et al., 2009; Ishov et al., 1999; Lin et al., 2006; Rasheed et al., 2002; Sung et al., 2011). Subsequent studies revealed that the main component of PML-NBs was the promyelocytic leukemia (PML) protein (Negorev et al., 2001). PML-NBs are now known to function as recruitment and storage centers for a vast array of nuclear proteins in response to a variety of stress factors and most of the proteins that reside in PML-NBs are either SUMOylated and/or contain a SIM (Dellaire and Bazett-Jones, 2007; Dellaire et al., 2006; Ishov et al., 1999; Jensen et al., 2001; Lallemand-Breitenbach and de The, 2010; Maroui et al., 2012; Salomoni et al., 2005; Salomoni et al., 2012; Zhong et al., 2000). SUMO-SIM interactions have been shown to play a key role in defining the composition of PML-NBs, and PML itself is both SUMOylated at multiple Lys residues (Kamitani et al., 1998) and six of its seven isoforms contain a conserved SIM (Jensen et al., 2001; Kerscher, 2007). The SIM of PML is composed of a core of four hydrophobic residues (VVVI; residues 556–559 in human PML isoform I) followed by a cluster of Ser residues on the C-terminal side, and interactions involving PML have been shown to be regulated by phosphorylation of its SIM (Cappadocia et al., 2015; Stehmeier and Muller, 2009). This occurs in a casein kinase 2 (CK2)dependent manner, which phosphorylates four Ser residues (S560, S561, S562, and S565) within the phosphoSIM of PMLI (Kamitani et al., 1998; Scaglioni et al., 2006, 2008). In addition, several other proteins contain a phosphoSIM similar to the PML-SIM that regulates their function within PML-NBs, including the histone chaperone Daxx (Chang et al., 2011; Cho et al., 2009; Lin et al., 2006; Negorev et al., 2001; Rasheed et al., 2002; Sung et al., 2011). In Daxx, the phosphoSIM is located at the C terminus and it contains two CK2-phosphorylation sites (S737 and S739 in human Daxx) (Chang et al., 2011). The presence of Daxx in PML-NB is dependent on SUMOylation of PML and enhanced by phosphorylation of the two Ser residues in the phosphoSIM of Daxx (Chang et al., 2011; Lin et al., 2006; Stehmeier and Muller, 2009; Ullmann et al., 2012). Biochemical and structural studies with the phosphoSIMs of PML and Daxx have shown that the phosphorylated residues within their SIM make specific contacts with positively charged Lys residues that are highly conserved in SUMO proteins and that these interactions increase the binding affinity (Cappadocia et al., 2015; Chang et al., 2011; Lin et al., 2006; Stehmeier and Muller, 2009; Ullmann et al., 2012). In the SUMO1:PML-SIMPO4 complex (Cappadocia et al., 2015), it was shown that phosphoserines (pS) at positions 560, 561, and 562 of the SIM form contacts with K46, K39, and H43 of SUMO1, respectively. In the case of the SUMO1:Daxx-SIM-PO4 complex, pS737 and pS739 interact with K46 and H43 of SUMO1 as seen in the PML-SIM-PO4 complex, but D738 of Daxx forms an interaction with T42 of SUMO1(Cappadocia et al., 2015; Chang et al., 2011). These results suggested that different spacing patterns of the negatively charged and/or phosphorylated residues within the phosphoSIMs play an important role in regulating the interaction with SUMO family proteins. In addition to the effect of phosphorylation, it has been shown that SUMO-SIM interactions may be further regulated by acetylation of several key Lys residues within the SIM-binding region of SUMO proteins (Cheema et al., 2010; Choudhary et al., 2009; Ullmann et al., 2012). More specifically, it was shown that acetylation of either 2 Structure 28, 1–12, February 4, 2020

SUMO1 at K37 or SUMO2 at K33 decreases the non-covalent interaction with the phosphoSIMs of both PML and Daxx in vitro, and that substituting K37 of SUMO1 with the acetylmimetic Gln inhibits Daxx recruitment to PML-NBs in cellular localization studies (Ullmann et al., 2012). Based on these results, it was postulated that either acetylation of SUMO1 or phosphorylation of the SIMs can play a crucial role in regulating SUMO-SIM interactions (Ullmann et al., 2012). Given the potential role that post-translational modifications can play in regulating SUMO-SIM interactions and the impact these pathways have on biological functions, we have characterized the interactions between the phosphoSIMs of PML (PMLSIM-PO4) and Daxx (Daxx-SIM-PO4) with acetylated variants of SUMO1 (K37Ac, K39Ac, K45Ac, K46Ac) using a combination of isothermal titration calorimetery (ITC), bioluminescence resonance energy transfer (BRET), and X-ray crystallography studies. We establish that the interactions between SUMO1 and either the PML-SIM-PO4 or Daxx-SIM-PO4 can be significantly reduced by acetylation at several Lys residues in the SIM-binding region of SUMO1, but that acetylation at K37 has a varying impact on the binding to the two phosphoSIMs. Structural characterization of the acetylated variants of SUMO1 in complex with PML-SIM-PO4 or Daxx-SIM-PO4 highlights how acetylation of a specific Lys impacts the positioning of negatively charged Asp and/or phosphorylated Ser residues of the phosphoSIMs relative to the positively charged residues of SUMO1. Together, these results provide an atomic level description of the role that phosphorylation and acetylation play in regulating SUMO-SIM interactions, as well as important insights into how the acetylation of SUMO1 can regulate Daxx and PML interactions within PML-NBs. RESULTS Acetylated SUMO1 Variants Have Lower Affinities for the phosphoSIM of PML It has previously been demonstrated that phosphorylation of residues within SIMs typically enhances their affinity for SUMO proteins (Cappadocia et al., 2015), whereas acetylation of key Lys residues in SUMO proteins decreases their binding affinity to SIMs (Ullmann et al., 2012). To determine the roles that these two post-translational modifications have on the interaction between SUMO1 and the PML-SIM, we generated acetyl-Lys variants of SUMO1 and a phosphomimetic (S-to-D substitutions) PML-SIM peptide. In the case of SUMO1, four Lys residues within the SIM-binding region (Figure 1A) have been shown to be acetylated either in vitro or in vivo (Cheema et al., 2010; Choudhary et al., 2009; Ullmann et al., 2012) and we generated four acetyl-Lys variants (K37Ac, K39Ac, K45Ac, K46Ac) using a bacterial system that allows for site selective incorporation of acetyl-Lys (Neumann et al., 2008). The incorporation of the acetyl-Lys was done in a truncated SUMO1 protein missing the disordered N-terminal region (residues 17–97 of human SUMO1), as this form will be used in subsequent crystallography studies. For the phosphomimetic PML-SIM peptide (residues 547–573 of human PML isoform I), the four Ser residues that undergo phosphorylation were substituted as a group to generate the PML-SIM-4SD (S560D/S561D/S562D/S565D) peptide (Figure 1B). To characterize the binding of the four acetyl-Lys

Please cite this article in press as: Mascle et al., Acetylation of SUMO1 Alters Interactions with the SIMs of PML and Daxx in a Protein-Specific Manner, Structure (2019), https://doi.org/10.1016/j.str.2019.11.019

Table 1. ITC Results for DN-SUMO1 Acetylated Variant Binding to phosphoSIMs PML-SIM-4SD

Daxx-SIM-PO4

SUMO1

KD (mM)

Fold Da

KD (mM)

Fold Da

WT

0.52 ± 0.08

–

0.10 ± 0.02

–

K37Ac

1.6 ± 0.1

3.1

1.0 ± 0.2

10

K39Ac

11 ± 0.7

21.1

1.9 ± 0.3

19

K45Ac

2.0 ± 0.2

3.8

0.39 ± 0.04

3.9

K46Ac

11 ± 2.4

21.1

4.4 ± 0.2

44

Relative change in binding affinity compared with wild-type (WT) DNSUMO1. The standard deviations listed represent the values around the mean of two or more independent measurements.

a

and K46 in forming key interactions with phosphoserine residues at the interface of the SUMO1:PML-SIM-PO4 complex.

Figure 1. Post-translational Modifications Alter Interactions between SUMO1 and phosphoSIMs (A) Cartoon representation of the structure of DN-SUMO1 (light gray; PDB: 2UYZ), with the four Lys residues (K37, K39, K45, K46) at the SIM-binding interface that undergo acetylation highlighted. (B) Sequences of SIMs of PML and Daxx used in the studies. The hydrophobic core regions of the SIMs are in italics and Ser residues that are phosphorylated are in bold. (C) ITC thermogram for the interaction between PML-SIM-4SD and either the wild-type (left panel) or the K46Ac (right panel) SUMO1. See also Figure S1.

SUMO1 variants to the PML-SIM-4SD peptide, ITC experiments were performed to determine the dissociation constant (KD) for complex formation. In the ITC experiments (Table 1; Figures 1C and S1), the K39Ac (KD = 11 mM) and K46Ac (KD = 11 mM) variants of SUMO1 reduce the binding affinity to the PML-SIM-4SD peptide by 21- and 21-fold, respectively. In contrast, a more modest decrease in binding affinity is observed with the K37Ac (3.1-fold; KD = 1.6 mM) and the K45Ac (3.8-fold; KD = 2.0 mM) variants. Taken together, the results suggest that, contrary to acetylation at either K37 or K45, acetylation at either K39 or K46 in SUMO1 significantly decreases its affinity for the phosphoSIM of PML. In addition, they are consistent with the role of K39

Acetylation Does Not Alter the Overall Fold of SUMO1 To verify that acetylation of the Lys residues in SUMO1 does not significantly alter the structure of the protein, the four variants of SUMO1 were crystalized in complex with the un-phosphorylated PML-SIM peptide. The crystals of all four complexes diffract between 1.30 and 1.63 A˚ resolution and belong to the P212121 space group (Table 2). The structures of the four acetylated SUMO1 variants in complex with the PML-SIM peptide are virtually identical to the previously determined structure of the wildtype SUMO1 in complex with the identical PML-SIM peptide (Figure 2) (Cappadocia et al., 2015). These results are consistent with the fact that only the residues in the hydrophobic core of the PML-SIM peptide contributes to the binding interface with SUMO1 in the absence of phosphorylation and there are no interactions involving the positively charged side chains of K37, K39, K45, and K46 from SUMO1. This is further supported by ITC studies showing that insertion of a Gln residue as an acetyl-mimetic at any of the four positions (K37Q, K39Q, K45Q, and K46Q) does not significantly alter the affinity of the SUMO1 K-to-Q variants for the PML-SIM peptide (Table S1). Together, the structures of these variants with the PML-SIM clearly indicate that acetylation at any of the four Lys residues in the SIM-binding region induces minimal structural changes in SUMO1, which is consistent with the fact that the side chains of these Lys residues are all located on the surface of the protein. Structures of the K39Ac and K46Ac SUMO1 Variants in Complex with PML-SIM-PO4 In an attempt to understand the dramatic reduction in binding affinity observed with the K39Ac and K46Ac variants in the ITC studies at the atomic level, these SUMO1 variants were crystallized with a tetra-phosphorylated PML-SIM peptide (pS560/pS561/pS562/pS565; PML-SIM-PO4) and the resulting structures compared with our previous structure of wildtype SUMO1 in complex with the same peptide (Cappadocia et al., 2015). The crystals of the SUMO1K39Ac:PML-SIM-PO4 complex diffracted to a resolution of 1.66 A˚, whereas the crystals of the SUMO1K46Ac:PML-SIM-PO4 complex diffracted to a resolution of 1.40 A˚ (Table 3). The structure of the SUMO1K39Ac:PML-SIM-PO4 complex displays some Structure 28, 1–12, February 4, 2020 3

Please cite this article in press as: Mascle et al., Acetylation of SUMO1 Alters Interactions with the SIMs of PML and Daxx in a Protein-Specific Manner, Structure (2019), https://doi.org/10.1016/j.str.2019.11.019

Table 2. Data Collection and Refinement Statistics for Structures with PML-SIM Dataset

DN-SUMO1K37Ac: PML-SIM

DN-SUMO1K39Ac: PML-SIM

DN-SUMO1K45Ac: PML-SIM

DN-SUMO1K46Ac: PML-SIM

Data Collection Beamline

08ID-1, CLS

08ID-1, CLS

08ID-1, CLS

08ID-1, CLS

Wavelength (A˚)

0.97949

0.97949

0.97949

0.97949

Space group

P212121

P212121

P212121

P212121

Unit cell parameters a, b, c (A˚)

38.27, 38.57, 142.93

38.34, 47.43, 62.19

38.23, 47.07, 63.52

38.37, 47.07, 63.17

90, 90, 90

90, 90, 90

90, 90, 90

90, 90, 90

36.98–1.63 (1.69–1.63)

32.64–1.41 (1.46–1.41)

32.76–1.50 (1.55–1.50)

29.74–1.30 (1.34–1.30)

a, b, g ( ) Resolution range (A˚) No. of unique reflections

25,537 (1,618)

21,894 (2,017)

18,491 (1,601)

28,366 (2,449)

Multiplicity

4.4 (2.9)

5.7 (3.9)

7.0 (4.4)

6.0 (2.9)

Completeness (%)

94.78 (60.17)

98.85 (93.01)

97.56 (85.65)

98.36 (85.27)

Rmerge

0.062 (0.913)

0.053 (1.06)

0.077 (1.06)

0.057 (1.058)

CCmerge

0.999 (0.620)

0.999 (0.336)

0.999 (0.462)

0.999 (0.392)

I/s(I)

12.21 (0.70)

17.25 (1.18)

16.41 (1.15)

16.26 (0.89)

Refinement Statistics Resolution (A˚)

36.98–1.63

32.64–1.41

32.76–1.50

29.74–1.30

Reflections (total/test)a

25,476/1,992

21,872/2,000

18,489/1,850

28,359/2,000

Rwork/Rfree (%)

17.59/19.57

15.82/17.26

15.09/17.48

15.61/17.33

CCwork

0.967 (0.742)

0.965 (0.703)

0.964 (0.752)

0.962 (0.699)

CCfree

0.948 (0.785)

0.962 (0.551)

0.957 (0.679)

0.969 (0.600)

No. of atoms (excluding hydrogens) Protein

1,417

754

750

763

Water

163

106

132

126

Protein

36.11

23.40

16.80

17.76

Water

43.48

37.66

33.50

33.10

B factors

Root-mean-square deviation Bond length (A˚) Bond angle ( )

0.009

0.009

0.009

0.008

1.29

1.35

1.29

1.31

Ramachandran (%)b Favored

100

100

100

100

Outliers

0

0 P

0

0

P Values in parentheses are for highest-resolution shell. Rsym = hkl i|Ihkl,i , where Ihkl,i is the intensity of an individual measurement of the P P reflection with Miller indices hkl and Ihkl is the mean intensity of the reflection. Rwork = hkl||Fo| |Fc|| / hkl |Fo|, where |Fo| is the observed structurefactor amplitude and |Fc| is the calculated structure-factor amplitude. Rfree is the R factor based on at least 500 test reflections that were excluded from the refinements. CLS, Canadian Light Source. a Reflection for Fo > 0. b MolProbity analysis.

similarities to the wild-type SUMO1:PML-SIM-PO4 complex, but also some key differences (Figures 3A and S2A). In comparison with the wild-type complex, the oxygen atom of the negatively charged pS560, which is in the first position after the hydrophobic core region of the PML phosphoSIM, is positioned slightly further from the nitrogen atom of the positively charged K46 (3.6 vs 2.9 A˚; variant/wild-type) and the oxygen atom of pS562 is positioned slightly further from the nitrogen atom of H43 (3.2 vs 2.8 A˚) of SUMO1. However, the major difference between the two complexes involves the role of pS561. In the wild-type complex, the oxygen atom of pS561 is positioned to form an electrostatic interaction with K39 (4.8 A˚), and this interaction is 4 Structure 28, 1–12, February 4, 2020

not possible due to the acetylation of K39. In contrast, the structure of the SUMO1K46Ac:PML-SIM-PO4 complex is quite different when compared with the wild-type SUMO1:PML-SIMPO4 (Figures 3B and S2B). In this complex, the interface is formed only with the hydrophobic residues from the core of the PML-SIM-PO4 peptide and there are no contacts involving the phosphoserine residues and SUMO1. Electron density is only detected for the pS560, which is no longer capable of forming an ionic interaction with the K46Ac and this structure is very similar to the complex containing SUMO1 with the unphosphorylated peptide of PML (Cappadocia et al., 2015). Taken together, the structures of the two complexes clearly indicate how

Please cite this article in press as: Mascle et al., Acetylation of SUMO1 Alters Interactions with the SIMs of PML and Daxx in a Protein-Specific Manner, Structure (2019), https://doi.org/10.1016/j.str.2019.11.019

Figure 2. Structures of PML-SIM in Complex with the Four Acetylated Variants of SUMO1 Structures of the SUMO1K37Ac:PML-SIM (A), SUMO1K39Ac:PML-SIM (B), SUMO1K45Ac:PMLSIM (C), and :PML-SIM (D) complexes. In all complexes, the acetylated variants of SUMO1 (carbon atoms gray) are shown in cartoon representation. The PML-SIM is shown in cartoon in green. See also Table S1.

acetylation at either K39 or K46 of SUMO1 significantly alters its ability to interact with the phosphoSIM of PML. Structures of the K37Ac and K45Ac SUMO1 Variants in Complex with PML-SIM-PO4 Because relatively small differences in affinity were observed for the binding of the K37Ac and K45Ac variants binding to the phosphomimetic PML-SIM in comparison with the wildtype SUMO1, we structurally characterized these variants with the PML-SIM-PO4 peptide to verify that there are only minor alterations in these complexes The crystals of the SUMO1K37Ac:PML-SIM-PO4 and the SUMO1K45Ac:PMLSIM-PO4 complexes diffract to a resolution of 1.58 and 1.65 A˚, respectively (Table 3). In addition, they both contain two copies of the complex in the asymmetric unit, where the peptide is either found close to the interface between two SUMO proteins or exposed to the solvent. Therefore, for each structure, only the complex where the peptide is exposed to the solvent is discussed to minimize potential artifacts that may be due to crystal packing. In the case of the SUMO1K45Ac:PML-SIMPO4 complex, the structure is very similar to the wild-type structure (Figures 4A and S3A), which is consistent with the binding constants observed by ITC. In the case of the SUMO1K37Ac:PML-SIM-PO4 complex, the comparison with the wild-type structure indicates many similarities, but also

some subtle differences (Figures 4B and S3B). For example, the position of the side chain of K37Ac is virtually the same as that of K37 (Figure 4B). This is consistent with the fact that the aliphatic portion of its side chain of K37 forms van der Waals contacts with the methyl groups of V556 and V558 from the core region and that these contacts are not altered significantly by acetylation. Likewise, the nitrogen atom from the side chain of K46 is the same distance away from the oxygen atom of the phosphate group of pS560 (2.9 A˚) in both complexes. However, notable differences in the relative locations of the side chains of K39, H43, and K46 are observed when comparing the two complexes. In the K37Ac complex, the nitrogen atom from the side chain of K39 is 0.9 A˚ farther away from the oxygen atom of the phosphate group of pS561 (4.7 vs 5.6 A˚; unmodified vs K37Ac) and the nitrogen atom of the side chain of H43 is 1.7 A˚ further away from the oxygen atom of the phosphate group of pS562 (4.2 vs 5.9 A˚). However, given the similar binding observed in the ITC studies this seems to be compensated by the fact that the oxygen atom of the phosphate group of pS562 is now 2.5 A˚ closer to the nitrogen atom from the side chain of K46 (6.4 vs 3.9 A˚), where it can now form an electrostatic interaction. Thus, it appears that there is built-in flexibility in the negatively charged region of the phosphoSIM, which allows it to adopt different conformations for binding to SUMO1 depending on the acetylation status and this is particularly evident in the structure with the K37Ac variant. K37, K39, and K46 Acetylated Variants Alter SUMO1 Binding to the Daxx phosphoSIM Next, we attempted to determine whether the differences in binding observed with the acetylated variants are specific for the PML phosphoSIM or similar for all phosphoSIMs that bind SUMO1 in a parallel orientation using additional ITC experiments. The phosphoSIM of Daxx was chosen for these studies because, despite its high sequence similarities to the phosphoSIM of PML, the structures of these phosphoSIMs complexed with SUMO1 presented noticeable differences in the pattern of recognition involving the negatively charged/phosphorylated residues contacting the SUMO1 (Cappadocia et al., 2015). For the phosphoSIM of Daxx, a peptide (Daxx-SIM-PO4) containing Structure 28, 1–12, February 4, 2020 5

Please cite this article in press as: Mascle et al., Acetylation of SUMO1 Alters Interactions with the SIMs of PML and Daxx in a Protein-Specific Manner, Structure (2019), https://doi.org/10.1016/j.str.2019.11.019

Table 3. Data Collection and Refinement Statistics for Structures with PML-SIM-PO4 Dataset

DN-SUMO1K37Ac: PML-SIM-PO4

DN-SUMO1K39Ac: PML-SIM-PO4

DN-SUMO1K45Ac: PML-SIM-PO4

DN-SUMO1K46Ac: PML-SIM-PO4

Data Collection Beamline

F1, CHESS

F1, CHESS

08ID-1, CLS

F1, CHESS

Wavelength (A˚)

0.97680

0.97750

0.97949

0.97680

Space group

P212121

P212121

P212121

P212121

Unit cell parameters a, b, c (A˚) a, b, g ( ) Resolution range (A˚)

38.29, 38.51, 144.06

38.45, 46.94, 62.38

38.53, 63.13, 91.37

38.20, 46.91, 63.62

90, 90, 90

90, 90, 90

90, 90, 90

90, 90, 90

30.04–1.58 (1.64–1.58)

24.23–1.66 (1.72–1.66)

37.01–1.65 (1.71–1.65)

29.63–1.40 (145–1.40)

No. of unique reflections

29,508 (2,839)

13,661 (1,279)

25,859 (1,821)

23,072 (2,229)

Multiplicity

5.8 (5.6)

6.0 (4.0)

6.2 (4.0)

6.2 (5.2)

Completeness (%)

99.35 (97.43)

98.79 (95.52)

95.13 (68.46)

99.74 (98.67)

Rmerge

0.074 (0.983)

0.089 (1.049)

0.076 (0.955)

0.087 (1.12)

CC½

0.999 (0.620)

0.998 (0.391)

0.999 (0.519)

0.999 (0.525)

I/s(I)

14.78 (1.47)

10.28 (1.03)

12.68 (1.15)

12.31 (1.26)

Refinement Statistics Resolution (A˚)

30.04–1.58

24.23–1.66

37.01–1.65

29.63–1.40

Reflections (total/test)a

29,500/2,013

13,661/1,367

25,853/2,000

23,071/2,000

Rwork/Rfree (%)

17.32/20.12

17.44/19.97

16.88/18.46

15.73/17.95

CCwork

0.955 (0.877)

0.968 (0.730)

0.963 (0.764)

0.958 (0.820)

CCfree

0.960 (0.820)

0.967 (0.585)

0.963 (0.662)

0.964 (0.831)

No. of atoms (excluding hydrogens) Protein

1,453

761

1,475

782

Water

198

95

206

113

Protein

28.53

28.42

27.82

16.36

Water

37.96

38.07

38.02

30.83

B factors

Root-mean-square deviation Bond length (A˚) Bond angle ( )

0.010

0.012

0.011

0.009

1.33

1.49

1.33

1.27

100

100

100

Ramachandran (%)b Favored

100

Outliers

0

0 P

0 P Values in parentheses are for highest-resolution shell. Rsym = hkl i|Ihkl,i , where Ihkl,i is the intensity of an individual measurement of the P P reflection with Miller indices hkl and Ihkl is the mean intensity of the reflection. Rwork = hkl||Fo| |Fc|| / hkl |Fo|, where |Fo| is the observed structurefactor amplitude and |Fc| is the calculated structure-factor amplitude. Rfree is the R factor based on at least 500 test reflections that were excluded from the refinements. CHESS, Cornell High Energy Synchrotron Source; CLS, Canadian Light Source. a Reflection for Fo > 0. b MolProbity analysis.

the C-terminal SIM of Daxx (residues 721–740 of human Daxx) with phosphoserine at both S737 and S739 was prepared. In the ITC experiments (Table 1), the K37Ac (KD = 1.0 mM), the K39Ac (KD = 1.9 mM), and the K46Ac (KD = 4.4 mM) variants of SUMO1 all bind with significantly reduced affinity to the DaxxSIM-PO4 peptide by 10.0-, 19.0-, and 44.0-fold, respectively, in comparison with the unmodified SUMO1 (KD = 0.10 mM). In contrast, only a modest 3.9-fold decrease in binding affinity is observed with the K45Ac (KD = 0.39 mM) SUMO1 variant. As observed with the phosphoSIM of PML, acetylation of either K39 or K46 in SUMO1 significantly decreases the binding affinity for the phosphoSIM of Daxx. However, acetylation at K37 also 6 Structure 28, 1–12, February 4, 2020

0

significantly lowers binding toward the phosphoSIM of Daxx, which is in contrast to what was observed with the phosphoSIM of PML, where only a modest reduction was observed. Consistent with what was observed with the unphosphorylated PMLSIM peptide, there were no significant differences in the binding of the unphosphorylated Daxx-SIM peptide binding to the four acetylated variants of SUMO1 (Table S2). Structures of Acetylated Variants of SUMO1 in Complex with the phosphoSIM of Daxx To understand how acetylation of SUMO1 at either K37, K39, or K46 alters the binding to the phosphoSIM of Daxx at the atomic

Please cite this article in press as: Mascle et al., Acetylation of SUMO1 Alters Interactions with the SIMs of PML and Daxx in a Protein-Specific Manner, Structure (2019), https://doi.org/10.1016/j.str.2019.11.019

Figure 3. Structures of PML-SIM-PO4 in Complex with the K39Ac and K46Ac Variants of SUMO1 Structures of the (A) SUMO1K39Ac:PML-SIMPO4 and (B) SUMO1K46Ac:PML-SIM-PO4 complexes. In both the full (A and B, left panels) and zoom (A and B, right panels) views, the K39Ac and K46Ac SUMO1 variants are shown in cartoon representation in gray. The PML-SIM-PO4 peptide is shown in cartoon in yellow. The zoom views (A and B, right panels) highlight the pSer residues at the binding interface. See also Figure S2.

level, we crystallized the three acetylated SUMO1 proteins with a modified peptide corresponding to the phosphorylated form of the Daxx-SIM (mDaxx-SIM-PO4), as previously described (Cappadocia et al., 2015). The SUMO1K37Ac:mDaxx-SIMPO4, SUMO1K39Ac:mDaxx-SIM-PO4, and SUMO1K46Ac:mDaxx-SIM-PO4 crystals diffracted to 1.70, 1.59, and 1.40 A˚, respectively (Table 4). As seen with the PML-SIM-PO4, the complexes of the mDaxx-SIM-PO4 peptide with the different acetylated variants displayed similarities in the hydrophobic core region, but striking differences in the positioning of the negatively charged/phosphorylated residues of Daxx-SIM-PO4 peptide when compared with the complex with wild-type SUMO1. In the SUMO1K46Ac:mDaxx-SIM-PO4 complex (Figure S4), acetylation of K46 prevents all interactions with the negatively charged residues and this is similar to that observed with the PML-SIM-PO4 complexed to the K46Ac variant. In the case of the SUMO1K39Ac:mDaxx-SIM-PO4 complex (Figures 5A and S5A), pS737 forms an ionic interaction with K46 as seen in the complex with unmodified SUMO1 (Cappadocia et al., 2015). However, there is no longer an interaction between pS739 and H43 of SUMO1 and this appears to be caused by a repositioning of D738. In the complex with unmodified SUMO1, the two side

chain oxygens of D738 from the mDaxxSIM-PO4 peptide are stabilized by interactions with the hydroxyl group of T42 from SUMO1 and a bound water molecule. In complex with K39Ac SUMO1, the acetylation of K39 appears to generate a slight torsion in the mDaxx-SIM-PO4 backbone, which leads to a repositioning of the D738 side chain. Ultimately, this results in the loss of the interaction between D738 and the key water molecule as well as a loss of the interaction between pS739 and H43 of SUMO1. In the case of the SUMO1K37Ac:mDaxx-SIM-PO4 complex (Figures 5B and S5B), the observed interactions are remarkably similar to those seen in the SUMO1K39Ac:mDaxx-SIMPO4 complex. As seen with acetylation at K39, acetylation of K37 of SUMO1 also appears to alter the relative positioning of the phosphorylated/negatively charged region of the phosphoSIM of Daxx. In particular, the interaction between the side chain oxygens of D738 from Daxx with the side chain hydroxyl of T42 from SUMO1 and the interaction between the bound water molecule are again absent in this complex. In addition, there is no longer an interaction between pS739 of Daxx and H43 of SUMO1 as a result of the repositioning of D738 in this complex. However, these changes appear to be partially compensated by the formation of a new electrostatic interactions between the side chain oxygen from D738 of Daxx and the side chain nitrogen from K39 of SUMO1. Taken together, the results demonstrate that there is built-in plasticity in SUMO-SIM interactions so that different acetylation events on SUMO proteins can have varying effects. This plasticity depends on the relative composition of negatively charged/phosphorylated residues of the phosphoSIM. PML Interactions with K-to-Q Variants of SUMO1 in Human Cells Analyzed by BRET To assess the role that acetylation of SUMO1 has on its binding to full-length PML in human cells, the interactions between PML and acetyl-lysine mimetics of SUMO1 (K-to-Q variants) were assessed using previously described BRET studies (Cappadocia et al., 2015; Mascle et al., 2013). For these experiments, Structure 28, 1–12, February 4, 2020 7

Please cite this article in press as: Mascle et al., Acetylation of SUMO1 Alters Interactions with the SIMs of PML and Daxx in a Protein-Specific Manner, Structure (2019), https://doi.org/10.1016/j.str.2019.11.019

Figure 4. Structures of PML-SIM-PO4 in Complex with the K45Ac and K37Ac Variants of SUMO1 Structures of the (A) SUMO1K45Ac:PML-SIM-PO4 and (B) SUMO1K37Ac:PML-SIM-PO4 complexes. In both the full (A and B, left panels) and zoom (A and B, right panels) views, the K45Ac and K37Ac SUMO1 variants are shown in cartoon representation in gray. The PML-SIM-PO4 peptide is shown in cartoon in yellow. The zoom views (A and B, right panels) highlight the pSer residues at the binding interface. See also Figure S3.

contrast to that observed with identical substitutions in cellular studies with the C-terminal SIM of Daxx (Ullmann et al., 2012). DISCUSSION

HEK293T cells were co-transfected with a fixed quantity of DNA coding for the wild-type PML fused to Renilla luciferase (PMLRluc) along with increasing amounts of a DNA coding for nonconjugable versions of either wild-type SUMO1 or K-to-Q variants at either K37 (K37Q) or K46 (K46Q) fused to GFP (GFP-SUMO1) (Percherancier et al., 2009). The wild-type PML was chosen as it has been shown that a significant percentage of the PML-Rluc protein is in the phosphorylated form under these experimental conditions (Cappadocia et al., 2015; Mascle et al., 2013; Rabellino et al., 2012). Consistent with these previous studies, the BRET ratio increases as a function of the GFP-SUMO1 concentration and reaches a maximum when GFP-SUMO1 expression levels are no longer limiting relative to the PML-Rluc expression levels (Figure 6). In experiments with GFP-SUMO1K37Q variant, the BRET curves are very similar and there is only a very slight decrease in the saturation level (Figures 6A and 6B). In contrast, a significantly lower BRET saturation signal is obtained for the association between PML-Rluc and the GFP-SUMO1K46Q variant (Figures 6A and 6B). These BRET results are consistent with the ITC results, where a dramatic decrease in binding affinity to the phosphomimetic PMLSIM-4SD is observed with the K46Ac variant, whereas there is only a subtle change with the K37Ac variant (Table 1). Taken together, the BRET results support the ITC studies and demonstrate that Gln substitution at position K37 of SUMO1 does not significantly alter its interaction with PML in human cells in 8 Structure 28, 1–12, February 4, 2020

Structural characterization of SUMO1 bound to the phosphoSIMs of PML and Daxx highlighted that Lys residues in SUMO1 play crucial roles at the interface in both complexes, but the complexes have distinct recognition patterns that appear dependent on the composition of the negatively charged residues adjacent to the core of the SIM (Cappadocia et al., 2015; Chang et al., 2011). However, very little is known about how acetylation of these Lys residues alters SUMO proteins binding to SIMs at the atomic level. In this work, we have characterized the role that acetylation plays in the binding of the phosphoSIMs of PML and Daxx to SUMO1. ITC experiments establish that SUMO1 binding to the phosphoSIMs of both PML and Daxx is altered by acetylation of SUMO1, with acetylation at either K39 or K46 having a dramatic effect on binding to both phosphoSIMs. In contrast, acetylation at K37 impacts the binding to the phosphoSIM of Daxx more dramatically than to the phosphoSIM of PML, whereas acetylation at K45 has only a minimal effect on binding to either phosphoSIMs. Consistent with the ITC results, structural studies provide insights into how specific interactions at the SUMO-SIM-binding interfaces are either disrupted and/or re-organized with each acetylated SUMO1 variant in complex with the phosphoSIMs. Based on these results, it appears that SUMO-SIM interaction can be fine-tuned by discrete acetylation and/or phosphorylation events to help regulate a number of cellular functions, including protein transit in and out of PML-NBs. The structures of the acetylated SUMO1 variants in complex with the phosphoSIMs of PML and Daxx provide an atomic level description of the interplay occurring between discrete acetylation events targeting SUMO1 and specific phosphorylation of the two SIM modules. Comparison of complexes with the acetylated SUMO1 variants and the unmodified SUMO1 reveal that the structural conformation (b strand) of the hydrophobic core of the phosphoSIMs remains unchanged in all four variants.

Please cite this article in press as: Mascle et al., Acetylation of SUMO1 Alters Interactions with the SIMs of PML and Daxx in a Protein-Specific Manner, Structure (2019), https://doi.org/10.1016/j.str.2019.11.019

Table 4. Data Collection and Refinement Statistics for Structures with Daxx-SIM-PO4 Dataset

DN-SUMO1K37Ac: mDaxx-SIM-PO4

DN-SUMO1K39Ac: mDaxx-SIM-PO4

DN-SUMO1K46Ac: mDaxx-SIM-PO4

Data Collection Beamline

F1, CHESS

F1, CHESS

08ID-1, CLS

Wavelength (A˚)

0.97750

0.97750

0.97949

Space group

P21

C2

C2

Unit cell parameters a, b, c (A˚) a, b, g ( ) Resolution range (A˚)

34.29, 81.45, 34.28

57.52, 38.16, 49.75

100.22, 38.31, 67.72

90, 112.41, 90

90, 119.92, 90

90, 119.90, 90

26.9–1.70 (1.76–1.70)

19.08–1.59 (1.65–1.59)

48.27–1.40 (1.45–1.40)

No. of unique reflections

18,368 (1,554)

11,899 (901)

41,097 (2,602)

Multiplicity

3.3 (2.2)

3.3 (2.3)

3.0 (1.9)

Completeness (%)

95.83 (82.31)

95.17 (72.49)

92.85 (58.29)

Rmerge

0.081 (0.861)

0.056 (0.466)

0.070 (0.689)

CCmerge

0.996 (0.269)

0.998 (0.714)

0.997 (0.428)

I/s(I)

8.60 (0.84)

10.67 (1.57)

10.93 (1.27)

Refinement Statistics Resolution (A˚)

26.9–1.70

19.08–1.59

48.27–1.40

Reflections (total/test)a

18,368/1,833

11,899/1,191

41,052/1,998

Rwork/Rfree (%)

16.41/21.60

15.54/17.42

15.81/18.00

CCwork

0.900 (0.276)

0.973 (0.863)

0.969 (0.688)

CCfree

0.879(0.267)

0.976 (0.730)

0.972 (0.687)

Protein

1,434

743

1,480

Water

136

118

298

Protein

30.35

24.51

17.95

Water

36.43

36.04

31.32

No. of atoms (excluding hydrogens)

B factors

Root-mean-square deviation Bond length (A˚) Bond angle ( )

0.018

0.010

0.009

1.70

1.34

1.38

Ramachandran (%)b Favored

98.10

98.77

99.37

Outliers

0

0

0

P P Values in parentheses are for highest-resolution shell. Rsym = hkl i|Ihkl,i , where Ihkl,i is the intensity of an individual measurement of the P P reflection with Miller indices hkl and Ihkl is the mean intensity of the reflection. Rwork = hkl||Fo| |Fc|| / hkl |Fo|, where |Fo| is the observed structurefactor amplitude and |Fc| is the calculated structure-factor amplitude. Rfree is the R factor based on at least 500 test reflections that were excluded from the refinements. a Reflection for Fo > 0. b MolProbity analysis.

However, the impact of acetylation of SUMO1 on the interactions with the negatively charged and/or phosphorylated residues that follow the core region of the SIM is more complex when comparing interactions with the phosphoSIMs of Daxx and PML, which is consistent with their different recognition patterns for SUMO1 (Cappadocia et al., 2015). Despite having different overall recognition patterns, several sites of acetylation on SUMO1 cause similar effects on the binding to the two SIMs. For both phosphoSIMs, acetylation at K39 and K46 of SUMO1 significantly decreases binding, whereas acetylation at K45 has little effect. The structural studies with the K45Ac variant of SUMO1 are consistent with the fact that, despite being near

the SIM-binding interface, this residue does not form contacts with either of the phosphoSIMs in the complex with the unmodified SUMO1. Likewise, the results with the K46Ac variant are consistent with the fact that this residue forms an important ionic interaction with pS560 from the PML-SIM and pS737 from the Daxx-SIM. These phosphoserine residues are located in the first position after the hydrophobic core in both phosphoSIMs. In the structures of both complexes with the K46Ac variant, it is clear that the inability to form this interaction prevents additional interactions from occurring with the subsequent negatively charged residues after the core, which explains the dramatic drop in affinity for both phosphoSIMs in the ITC experiments. However, the Structure 28, 1–12, February 4, 2020 9

Please cite this article in press as: Mascle et al., Acetylation of SUMO1 Alters Interactions with the SIMs of PML and Daxx in a Protein-Specific Manner, Structure (2019), https://doi.org/10.1016/j.str.2019.11.019

Figure 5. Structures of Daxx-SIM-PO4 in Complex with the K39Ac and K37Ac Variants of SUMO1 Structures of the (A) SUMO1K39Ac:mDaxx-SIMPO4 and (B) SUMO1K46Ac:mDaxx-SIM-PO4 complexes. In both the full (A and B, left panels) and zoom (A and B, left panels) views, the K39Ac and K37Ac SUMO1 variants are shown in cartoon representation in gray. The mDaxx-SIM-PO4 peptide is shown in cartoon in blue. The zoom views (A and B, right panels) highlight the pSer and Asp at the binding interface. See also Figures S4 and S5.

results with the K39Ac variant of SUMO1 are somewhat surprising. Although K39 plays an important role at the binding interface between the phosphoSIM of PML, it does not appear to make crucial contacts with the phosphoSIM of Daxx in the complexes with unmodified SUMO1. Nevertheless, a dramatic decrease in binding to both phosphoSIMs is observed with the K39Ac variant in the ITC experiments. From the structures of the two complexes, it is clear that acetylation at K39 alters the interaction with pSer561 in the phosphoSIM of PML as expected. However, the effect appears to be more indirect with the phosphoSIM of Daxx. In this complex, acetylation at K39 appears to change the positioning of the negatively charged residues in the second (D738) and third (pS739) positions after the core, and this leads to the loss of the interaction between pS739 of Daxx and H43 of SUMO1. The result is that, despite having different recognition patterns for K39 with unmodified SUMO1, acetylation at K39 of SUMO1 significantly reduces the binding affinity for both phosphoSIMs albeit by different mechanisms. In contrast to what is observed with the three other acetylated variants, acetylation at K37 of SUMO1 does not affect binding of the phosphoSIMs of PML and Daxx in the same manner. Whereas acetylation of SUMO1 at K37 causes only a slight decrease in the binding to the phosphoSIM of PML, the binding to the phosphoSIM of Daxx is significantly decreased and these ITC results are supported by the crystal structures. In the struc10 Structure 28, 1–12, February 4, 2020

ture of the SUMO1K37Ac:PML-SIM-PO4 complex, there are only subtle changes in the structure, which is almost identical to the structure with the unmodified SUMO1. Consistent with the ITC results, more drastic changes are evident in the structure of the SUMO1K37Ac:Daxx-SIMPO4 complex. In our previously published SUMO1:mDaxx-SIM-PO4 crystal structure, oxygens from the side chain of D738 of Daxx form hydrogen bonds with the hydroxyl group of the side chain of T42 of SUMO1 as well as with a bound water molecule. However, both of these interactions are absent in the structure of the SUMO1K37Ac:mDaxx-SIM-PO4 complex. Similar to that observed in the complex with the SUMO1K39Ac variant and the phosphoSIM of Daxx, the loss of these interactions appears to impact the positioning of the negatively charged residues in the second (D738) and third (pS739) after the hydrophobic core and we no longer observe an interaction between pS739 of Daxx and H43 of SUMO1 when K37 is acetylated. Overall, these results suggest that acetylation at K37 on SUMO1 has the capacity to impact the binding to phosphoSIMs in different manners and this appears to be controlled by the composition of the negatively charged residues immediately adjacent to the hydrophobic core. Taken together, these results provide important insights into mechanisms for fine-tuning the homeostasis of proteins transiting in and out of PML-NBs in response to changing cellular conditions (Cappadocia et al., 2015). Indeed, a number of studies have highlighted that the recruitment of SIM-containing proteins, such as Daxx, to PML-NBs is governed by SUMO-dependent interactions that in some cases require SUMOylation of PML (Chang et al., 2011; Lin et al., 2006; Stehmeier and Muller, 2009; Ullmann et al., 2012). Previous studies have clearly demonstrated how stress-induced phosphorylation of the Serrich cluster adjacent to the core of the SIM of Daxx can increase its binding affinity to SUMOylated PML, and this is essential for Daxx recruitment to PML-NBs (Cappadocia et al., 2015; Chang et al., 2011; Lin et al., 2006; Mascle et al., 2013; Negorev et al., 2001; Rasheed et al., 2002; Scaglioni et al., 2006, 2008; Stehmeier and Muller, 2009; Sung et al., 2011). In addition, in vivo studies have shown that substitution of either K37 in SUMO1 or the

Please cite this article in press as: Mascle et al., Acetylation of SUMO1 Alters Interactions with the SIMs of PML and Daxx in a Protein-Specific Manner, Structure (2019), https://doi.org/10.1016/j.str.2019.11.019

d d d

d d

LEAD CONTACT AND MATERIALS AVAILABILITY EXPERIMENTAL MODEL AND SUBJECT DETAILS METHOD DETAILS B Expression Vectors B Peptides and Proteins Expression and Purification B Peptide Synthesis B In Vitro Phosphorylation of SIM Peptides B ITC Experiments B Crystallization and Data Collection B Structure Determination and Refinement B BRET Experiments QUANTIFICATION AND STATISTICAL ANALYSIS DATA AND CODE AVAILABILITY

SUPPLEMENTAL INFORMATION Supplemental Information can be found online at https://doi.org/10.1016/j.str. 2019.11.019. ACKNOWLEDGMENTS

Figure 6. Effects of Acetyl-Mimics on Binding of SUMO1 to PML in Human Cells (A) BRET titration curves between PML and either wild-type SUMO1 or K-to-Q variants. The GFP-SUMO1, GFP-SUMO1K37Q, and GFP-SUMO1K46Q are shown with PML-Rluc. (B) Bar graph showing a comparison of the BRET ratios obtained using wildtype PML with either the wild-type SUMO1, the K46Q variant or the K37Q variant at similar GFP acceptor/Luc donor expression ratios. The error bars represent the mean ± standard deviation from a minimum of three independent experiments.

equivalent K33 in SUMO2 with an acetyl-mimetic Gln interfered with Daxx recruitment to PML-NBs by inhibiting interactions with the C-terminal SIM of Daxx (Ullmann et al., 2012). Thus, the interplay between phosphorylation and acetylation events targeting both the SIM-containing proteins and the SUMO proteins could help explain the diversity of interactions observed in these subnuclear structures. Notably, our BRET studies in human cells support the ITC and structural studies in that the PMLSUMO1 interaction is altered by substitution of Gln at K46 of SUMO1, but not at K37. Moreover, the fact that acetylation at K37 significantly compromised the binding to the phosphoSIM of Daxx, but not to the phosphoSIM of PML, demonstrates that discrete acetylation events modifying SUMO1 can specifically regulate its interactions with different SIM-containing proteins that reside in PML-NBs. Overall the plasticity observed in the structures of these complexes would provide a robust mechanism for regulating SUMO-SIM interactions of a multitude of proteins that transit into PML-NB using a combination of signaling mechanisms that function to regulate post-translational modifications, such as phosphorylation and acetylation. STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d

KEY RESOURCES TABLE

We thank Dr. Jason Chin for providing the system for the acetylated protein. This work was supported by the Canadian Institutes of Health Research (CIHR) to J.G.O. (no. 74739 and 130414) and by the Photo-excitonix Project at Hokkaido University (to K.S.). Research was carried out at the MacCHESS, supported by the NSF under grant DMR-1332208, the NIH/NIGMS under grant GM-103485, and the Canadian Light Source (CLS) supported by NSERC of Canada, the National Research Council Canada, CIHR, the Province of Saskatchewan, Western Economic Diversification Canada, and the University of Saskatchewan. AUTHOR CONTRIBUTIONS X.H.M., C.G., H.M.W., M.L.-P., L.C., K.S., and J.G.O. prepared reagents and conducted the experiments. X.M.H., C. G., H.M.W., L.C., K.S., and J.G.O. designed the experiments and wrote the manuscript. DECLARATION OF INTERESTS The authors of this manuscript declare no conflict of interest. Received: September 5, 2019 Revised: November 14, 2019 Accepted: November 27, 2019 Published: December 23, 2019 REFERENCES Adams, P.D., Afonine, P.V., Bunkoczi, G., Chen, V.B., Davis, I.W., Echols, N., Headd, J.J., Hung, L.W., Kapral, G.J., Grosse-Kunstleve, R.W., et al. (2010). PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221. Beauclair, G., Bridier-Nahmias, A., Zagury, J.F., Saib, A., and Zamborlini, A. (2015). JASSA: a comprehensive tool for prediction of SUMOylation sites and SIMs. Bioinformatics 31, 3483–3491. Cappadocia, L., and Lima, C.D. (2018). Ubiquitin-like protein conjugation: structures, chemistry, and mechanism. Chem. Rev. 118, 889–918. Cappadocia, L., Mascle, X.H., Bourdeau, V., Tremblay-Belzile, S., ChakerMargot, 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, 126–138. Chang, C.C., Naik, M.T., Huang, Y.S., Jeng, J.C., Liao, P.H., Kuo, H.Y., Ho, C.C., Hsieh, Y.L., Lin, C.H., Huang, N.J., et al. (2011). Structural and functional roles of Daxx SIM phosphorylation in SUMO paralog-selective binding and apoptosis modulation. Mol. Cell 42, 62–74.

Structure 28, 1–12, February 4, 2020 11

Please cite this article in press as: Mascle et al., Acetylation of SUMO1 Alters Interactions with the SIMs of PML and Daxx in a Protein-Specific Manner, Structure (2019), https://doi.org/10.1016/j.str.2019.11.019

Cheema, A., Knights, C.D., Rao, M., Catania, J., Perez, R., Simons, B., Dakshanamurthy, S., Kolukula, V.K., Tilli, M., Furth, P.A., et al. (2010). Functional mimicry of the acetylated C-terminal tail of p53 by a SUMO-1 acetylated domain, SAD. J. Cell Physiol. 225, 371–384. Chen, V.B., Arendall, W.B., III, Headd, J.J., Keedy, D.A., Immormino, R.M., Kapral, G.J., Murray, L.W., Richardson, J.S., and Richardson, D.C. (2010). MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21. Cho, G., Lim, Y., and Golden, J.A. (2009). SUMO interaction motifs in Sizn1 are required for promyelocytic leukemia protein nuclear body localization and for transcriptional activation. J. Biol. Chem. 284, 19592–19600. Choudhary, C., Kumar, C., Gnad, F., Nielsen, M.L., Rehman, M., Walther, T.C., Olsen, J.V., and Mann, M. (2009). Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science 325, 834–840. Dellaire, G., and Bazett-Jones, D.P. (2007). Beyond repair foci: subnuclear domains and the cellular response to DNA damage. Cell Cycle 6, 1864–1872. Dellaire, G., Ching, R.W., Ahmed, K., Jalali, F., Tse, K.C.K., Bristow, R.G., and Bazett-Jones, D.P. (2006). Promyelocytic leukemia nuclear bodies behave as DNA damage sensors whose response to DNA double-strand breaks is regulated by NBS1 and the kinases ATM, Chk2, and ATR. J. Cell Biol. 175, 55–66. Emsley, P., and Cowtan, K. (2004). Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132. Emsley, P., Lohkamp, B., Scott, W.G., and Cowtan, K. (2010). Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501. Gareau, J.R., and Lima, C.D. (2010). The SUMO pathway: emerging mechanisms that shape specificity, conjugation and recognition. Nat. Rev. Mol. Cell Biol. 11, 861–871. Geiss-Friedlander, R., and Melchior, F. (2007). Concepts in sumoylation: a decade on. Nat. Rev. Mol. Cell Biol. 8, 947–956. Guo, D., Li, M., Zhang, Y., Yang, P., Eckenrode, S., Hopkins, D., Zheng, W., Purohit, S., Podolsky, R.H., Muir, A., et al. (2004). A functional variant of SUMO4, a new I kappa B alpha modifier, is associated with type 1 diabetes. Nat. Genet. 36, 837–841. Haindl, M., Harasim, T., Eick, D., and Muller, S. (2008). The nucleolar SUMOspecific protease SENP3 reverses SUMO modification of nucleophosmin and is required for rRNA processing. EMBO Rep. 9, 273–279. Ishov, A.M., Sotnikov, A.G., Negorev, D., Vladimirova, O.V., Neff, N., Kamitani, T., Yeh, E.T.H., Strauss, J.F., and Maul, G.G. (1999). PML is critical for ND10 formation and recruits the PML-interacting protein Daxx to this nuclear structure when modified by SUMO-1. J. Cell Biol. 147, 221–233.

RNF4-or arsenic trioxide-induced degradation of nuclear PML isoforms. PLoS One 7, https://doi.org/10.1371/journal.pone.0044949. Mascle, X.H., Germain-Desprez, D., Huynh, P., Estephan, P., and Aubry, M. (2007). Sumoylation of the transcriptional intermediary factor 1beta (TIF1beta), the co-repressor of the KRAB multifinger proteins, is required for its transcriptional activity and is modulated by the KRAB domain. J. Biol. Chem. 282, 10190–10202. Mascle, X.H., Lussier-Price, M., Cappadocia, L., Estephan, P., Raiola, L., Omichinski, J.G., and Aubry, M. (2013). Identification of a non-covalent ternary complex formed by PIAS1, SUMO1, and UBC9 proteins involved in transcriptional regulation. J. Biol. Chem. 288, 36312–36327. Muller, S., Ledl, A., and Schmidt, D. (2004). SUMO: a regulator of gene expression and genome integrity. Oncogene 23, 1998–2008. Negorev, D., Ishov, A.M., and Maul, G.G. (2001). Evidence for separate ND10binding and homo-oligomerization domains of Sp100. J. Cell Sci. 114, 59–68. Neumann, H., Peak-Chew, S.Y., and Chin, J.W. (2008). Genetically encoding N(epsilon)-acetyllysine in recombinant proteins. Nat. Chem. Biol. 4, 232–234. Percherancier, Y., Germain-Desprez, D., Galisson, F., Mascle, X.H., Dianoux, L., Estephan, P., Chelbi-Alix, M.K., and Aubry, M. (2009). Role of SUMO in RNF4-mediated promyelocytic leukemia protein (PML) degradation SUMOylation of PML and phospho-switch control of its SUMO binding domain dissected in living cells. J. Biol. Chem. 284, 16595–16608. Rabellino, A., Carter, B., Konstantinidou, G., Wu, S.-Y., Rimessi, A., Byers, L.A., Heymach, J.V., Girard, L., Chiang, C.-M., Teruya-Feldstein, J., et al. (2012). The SUMO E3-ligase PIAS1 regulates the tumor suppressor PML and its oncogenic counterpart PML-RARA. Cancer Res. 72, 2275–2284. Rasheed, Z.A., Saleem, A., Ravee, Y., Pandolfi, P.P., and Rubin, E.H. (2002). The topoisomerase I-binding RING protein, topors, is associated with promyelocytic leukemia nuclear bodies. Exp. Cell Res. 277, 152–160. Salomoni, P., Bernardi, R., Bergmann, S., Changou, A., Tuttle, S., and Pandolfi, P.P. (2005). The promyelocytic leukemia protein PML regulates c-Jun function in response to DNA damage. Blood 105, 3686–3690. Salomoni, P., Dvorkina, M., and Michod, D. (2012). Role of the promyelocytic leukaemia protein in cell death regulation. Cell Death Dis. 3, e247. Scaglioni, P.P., Yung, T.M., Cai, L.F., Erdjument-Bromage, H., Kaufman, A.J., Singh, B., Teruya-Feldstein, J., Tempst, P., and Pandolfi, P.P. (2006). A CK2dependent mechanism for degradation of the PML tumor suppressor. Cell 126, 269–283. Scaglioni, P.P., Yung, T.M., Choi, S.C., Baldini, C., Konstantinidou, G., and Pandolfi, P.P. (2008). CK2 mediates phosphorylation and ubiquitin-mediated degradation of the PML tumor suppressor. Mol. Cell Biochem. 316, 149–154.

Jensen, K., Shiels, C., and Freemont, P.S. (2001). PML protein isoforms and the RBCC/TRIM motif. Oncogene 20, 7223–7233.

Schmidt, D., and Muller, S. (2003). PIAS/SUMO: new partners in transcriptional regulation. Cell Mol. Life Sci. 60, 2561–2574.

Kamitani, T., Kito, K., Nguyen, H.P., Wada, H., Fukuda-Kamitani, T., and Yeh, E.T. (1998). Identification of three major sentrinization sites in PML. J. Biol. Chem. 273, 26675–26682.

Song, J., Durrin, L.K., Wilkinson, T.A., Krontiris, T.G., and Chen, Y. (2004). Identification of a SUMO-binding motif that recognizes SUMO-modified proteins. Proc. Natl. Acad. Sci. U S A 101, 14373–14378.

Kerscher, O. (2007). SUMO junction––what’s your function? New insights through SUMO-interacting motifs. EMBO Rep. 8, 550–555.

Song, J., Zhang, Z., Hu, W., and Chen, Y. (2005). Small ubiquitin-like modifier (SUMO) recognition of a SUMO binding motif: a reversal of the bound orientation. J. Biol. Chem. 280, 40122–40129.

Lallemand-Breitenbach, V., and de The, H. (2010). PML nuclear bodies. Cold Spring Harb. Perspect. Biol. 2, a000661. Liang, Y.C., Lee, C.C., Yao, Y.L., Lai, C.C., Schmitz, M.L., and Yang, W.M. (2016). SUMO5, a novel poly-SUMO isoform, regulates PML nuclear bodies. Sci. Rep. 6, 26509.

Stehmeier, P., and Muller, S. (2009). Phospho-regulated SUMO interaction modules connect the SUMO system to CK2 signaling. Mol. Cell 33, 400–409. Sung, K.S., Lee, Y.A., Kim, E.T., Lee, S.R., Ahn, J.H., and Choi, C.Y. (2011). Role of the SUMO-interacting motif in HIPK2 targeting to the PML nuclear bodies and regulation of p53. Exp. Cell Res. 317, 1060–1070.

Lin, D.-Y., Huang, Y.-S., Jeng, J.-C., Kuo, H.-Y., Chang, C.-C., Chao, T.-T., Ho, C.-C., Chen, Y.-C., Lin, T.-P., Fang, H.-I., et al. (2006). Role of SUMO-interacting motif in Daxx SUMO modification, subnuclear localization, and repression of sumoylated transcription factors. Mol. Cell 24, 341–354.

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12 Structure 28, 1–12, February 4, 2020

Please cite this article in press as: Mascle et al., Acetylation of SUMO1 Alters Interactions with the SIMs of PML and Daxx in a Protein-Specific Manner, Structure (2019), https://doi.org/10.1016/j.str.2019.11.019

STAR+METHODS KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Bacterial Strains TOPP2

Bayou Biolabs

Cat#T-101

BL21(DE3)

Novagen

Cat#69450

Inalco

Cat#1758-9030 ; CAS: 3483-12-3

Chemicals, Peptides, and Recombinant Proteins Dithiothreitol (DTT) Isopropyl-b-D-thiogalactopyranoside (IPTG)

Inalco

Cat#1758-1400; CAS:367-93-1

ApexBio

Cat#A7231; CAS:692-04-6

Coelenterazine 400a

GoldBio

Cat#C-320; CAS:0217-82-2

Coelenterazine H

NanaLight Technology

Cat#301-5 ; CAS:50909-86-9

Glutathione Sepharose 4B

GE Healthcare

Cat#17-0756-05

Chelating Sepharose Fast Flow

GE Healthcare

Cat#17-0575-01

C8 Reverse Phase HPLC column

Vydac

Cat#208TP1022

Trifluoroacetic acid (TFA)

Millipore

Cat#TX1276-6 ; CAS:76-05-1

HPLC Acetonitrile

Fisher

Cat#A998; CAS:75-058

Casein Kinase II

New England Biolabs

Cat#P6010

NEBuffer

New England Biolabs

Cat#P6010

Imidazole

Sigma-Aldrich

Cat#I202; CAS:288-32-4

Cacodylic acid

Bioshop

Cat#CAC701; CAS:6131-99-3

PEG 3350

Sigma-Aldrich

Cat#88276 ; CAS:25322-68-3

Tobacco Etch Virus (TEV) protease

Cappadocia et al., 2015

N/A

PML-SIM

Cappadocia et al., 2015

N/A

PML-SIM-4SD

Cappadocia et al., 2015

N/A

PML-SIM-PO4

Cappadocia et al., 2015

N/A

Daxx-SIM

This study

N/A

Daxx-SIM-PO4

This study

N/A

mDaxx-SIM-PO4

Cappadocia et al., 2015

N/A

DN-SUMO1K37Q

This study

N/A

DN-SUMO1K39Q

This study

N/A

DN-SUMO1K45Q

This study

N/A

DN-SUMO1K46Q

This study

N/A

DN-SUMO1

Cappadocia et al., 2015

N/A

DN-SUMO1K37Ac

This study

N/A

DN-SUMO1K39Ac

This study

N/A

DN-SUMO1K45Ac

This study

N/A

DN-SUMO1K46Ac

This study

N/A

DN-SUMO1K37Ac: PML-SIM complex

This study

PDB: 6UYO

DN-SUMO1K39Ac: PML-SIM complex

This study

PDB: 6UYP

DN-SUMO1K45Ac: PML-SIM complex

This study

PDB: 6UYQ

DN-SUMO1K46Ac: PML-SIM complex

This study

PDB: 6UYR

DN-SUMO1K37Ac: PML-SIM-PO4 complex

This study

PDB: 6UYS

DN-SUMO1K39Ac: PML-SIM-PO4 complex

This study

PDB: 6UYT

DN-SUMO1K45Ac: PML-SIM-PO4 complex

This study

PDB: 6UYU

DN-SUMO1K46Ac: PML-SIM-PO4 complex

This study

PDB: 6UYV

e

N-Acetyl-L-lysine

Deposited Data

(Continued on next page)

Structure 28, 1–12.e1–e5, February 4, 2020 e1

Please cite this article in press as: Mascle et al., Acetylation of SUMO1 Alters Interactions with the SIMs of PML and Daxx in a Protein-Specific Manner, Structure (2019), https://doi.org/10.1016/j.str.2019.11.019

Continued REAGENT or RESOURCE

SOURCE

IDENTIFIER

DN-SUMO1K37Ac: Daxx-SIM-PO4 complex

This study

PDB: 6UYX

DN-SUMO1K39Ac: Daxx-SIM-PO4 complex

This study

PDB: 6UYY

DN-SUMO1K46Ac: Daxx-SIM-PO4 complex

This study

PDB: 6UYZ

DN-SUMO1: PML-SIM complex

Cappadocia et al., 2015

PDB: 4WJO

DN-SUMO1: PML-SIM-PO4 complex

Cappadocia et al., 2015

PDB: 4WJN

DN-SUMO1: Daxx-SIM-PO4 complex

Cappadocia et al., 2015

PDB: 4WJP

ATCC

Cat#CRL-1573; RRID: CVCL_0045

PML-SIM

Invitrogen

N/A

Daxx-SIM

Invitrogen

N/A

Experimental Models: Cell Lines HEK293T (Female) Oligonucleotides

Recombinant DNA pET11b-TEV

This study

N/A

pGEX-TEV

This study

N/A

pCDF PylT

Neumann et al., 2008

N/A

pBK-AcKRS3

Neumann et al., 2008

N/A

pGEX-2T-DN SUMO1 C52A

Cappadocia et al., 2015

N/A

pGEX-TEV-DN SUMO1

This study

N/A

pGEX-TEV-DN SUMO1K37Q

This study

N/A

pGEX-TEV-DN SUMO1K39Q

This study

N/A

pGEX-TEV-DN SUMO1K45Q

This study

N/A

pGEX-TEV-DN SUMO1K46Q

This study

N/A

pGEX-TEV-DN SUMO1K37Ac

This study

N/A

pGEX-TEV-DN SUMO1K39Ac

This study

N/A

pGEX-TEV-DN SUMO1K45Ac

This study

N/A

pGEX-TEV-DN SUMO1K46Ac

This study

N/A

pGEX-2T-PML-SIM

Cappadocia et al., 2015

N/A

pGEX-2T-PML-SIM

Cappadocia et al., 2015

N/A

pGEX-TEV-Daxx-SIM

This study

N/A

GFP-SUMO1

Mascle et al., 2013

N/A

PML-Rluc

Mascle et al., 2013

N/A

GFP-SUMO1-K37Q

This study

N/A

GFP-SUMO1-K46Q

This study

N/A

Coot

Emsley et al., 2010

www2.mrc-lmb.cam.ac.uk/personal/ pemsley/coot/

Phenix

Adams et al., 2010

www.phenix-online.org

HKL-2000

HKL Research

HKL-2000

MolProbibility

Chen et al., 2010

http://molprobity.biochem.duke.edu/

Origin Software

OriginLab Corporation

https://www.originlab.com

GraphPad Prism

GraphPad Software

https://www.graphpad.com/scientificsoftware/prism/

PyMol

Schrodinger, LLC

https://pymol.org

Software and Algorithms

LEAD CONTACT AND MATERIALS AVAILABILITY Further information and requests for either resources or reagents should be directed and will be fulfilled by the lead contact James G. Omichinski ([email protected]). The atomic coordinates of the structures have been deposited in the Protein Data Bank.

e2 Structure 28, 1–12.e1–e5, February 4, 2020

Please cite this article in press as: Mascle et al., Acetylation of SUMO1 Alters Interactions with the SIMs of PML and Daxx in a Protein-Specific Manner, Structure (2019), https://doi.org/10.1016/j.str.2019.11.019

EXPERIMENTAL MODEL AND SUBJECT DETAILS HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 1 mM L-glutamine, 10% fetal bovine serum (Wisent), and 100 mg/ml penicillin and streptomycin. Cells were seeded at a density of 1 3 106 cells/100-mm dish, 24 h before transfection. Transient transfections of plasmids were performed using the calcium phosphate precipitation method. Typically, 1 mg of the PML-Luc construct was transfected either alone or together with increasing quantities of the GFP-SUMO1 constructs (wildtype or K-to-Q variants). The transfection medium was exchanged with the supplemented Dulbecco’s modified Eagle’s medium after 26 h and cells remained in culture for an additional 22 h before being processed for the BRET assay. METHOD DETAILS Expression Vectors The vector for expressing the PML-SIM (residues 547-573; PML Isoform I) and PML-SIM-4SD have been previously described (Cappadocia et al., 2015). Daxx-SIM (residues 723-740 of human Daxx with a C728A substitution) was ordered as an oligonucleotide (Integrated DNA Technologies) with flanking BamHI and EcoRI restriction enzyme sites, 5’-phosphorylated, annealed and cloned into a modified pGEX-2T vector (Amersham) with a Tobacco Etch Virus (TEV) protease cut site replacing the original thrombin cut site. DN-SUMO1 was prepared from a PCR amplification of pGEX-2T-DN-SUMO1 C52A (Cappadocia et al., 2015) and cloned as a BamHI/EcoRI fragment into both a pET-TEV vector (a modified pET-15b vector (Amersham) for expression of a His-tagged protein with a TEV protease cut site) and a pCDF PylT vector (a modified pCDF1 vector with added sequence that encodes for a tRNA specific for the incorporation of eN-Acetyl-L-lysine at amber codon (Neumann et al., 2008). Amber codons and glutamine substitutions at K37, K39, K45 and K46 were incorporated into the vectors using site directed mutagenesis. The PML BRET constructs have been described (Percherancier et al., 2009). The non-conjugable version of human SUMO1 fused to pGFP10 was generated by site directed mutagenesis from pGFP10-SUMO1, mutating the SUMO di-glycine motif to alanine residues (Mascle et al., 2007). All SUMO1 mutants were generated by site directed mutagenesis starting from the non-conjugable form of SUMO1. All constructs were verified by DNA sequencing. Peptides and Proteins Expression and Purification PML-SIM, PML-SIM-4SD, Daxx-SIM peptides as well as DN-SUMO1 and the Gln variants of DN-SUMO1 were expressed as GST fusion in E. coli host strain TOPP2 (Stratagene) and purified as previously described (Cappadocia et al., 2015). Cells were grown in Luria Broth (LB) medium supplemented with 100 mg/ml ampicillin overnight at 37 C. The next day, cells were diluted in LB plus antibiotics and grown until reaching O.D.600 between 0.6-0.8. Protein expression was induced with 1 mM IsoPropyl-b-D-ThioGalactopyranoside (IPTG; Inalco) for 4h at 30 C. Cells were pelleted by centrifugation (10 min, 12,000g) and frozen at -20 C. Pellets were resuspended in lysis buffer (20 mM Tris-HCl pH 7.4, 1M NaCl, 0.2 mM EDTA, 1 mM DTT) and passed twice through a French Press followed by a brief sonication. The resulting suspension was centrifuged (1h, 105,000g, 4 C) and the supernatant incubated (1h, 4 C) with a Glutathione Sepharose 4B (GSH; GE Healthcare) resin. Following the incubation, the resin was centrifuged and washed several times with lysis buffer to remove non-specifically bound molecules. The resin was then rinsed three times with TEV buffer (10 mM NaH2PO4/Na2HPO4 pH 7.4, 125 mM NaCl, 5 mM DTT) and incubated overnight at room temperature with 100 units of TEV protease. The next day, the supernatant was collected, filtered and dialyzed extensively in water. The resulting solution was then dialyzed in 5% acetic acid overnight prior to purification on a C4-reverse phase HPLC column (Vydac) using a 0.05% trifluoroacetic acid (TFA) wateracetonitrile gradient. The organic solvent was removed from the collected fractions by roto-evaporation. The resulting solution was dialyzed in 20 mM Tris-HCl pH 7.5, then in water. The final purified solution was lyophylized and stored at -20 C in solid form. For the expression of the acetylated variants of DN-SUMO1, BL21(DE3) (Stratagene) cells were transformed with modified plasmid pCDF PylT carrying the Open Reading Frame (ORF) for DN-SUMO1 with amber codons at desired sites for incorporation of eNAcetyl-L-Lys as well as with the pBK-AcKRS3 plasmid (courtesy of Dr. Jason Chin, Cambridge University) for the expression of the tRNA synthetase specific for the incorporation of eN-Acetyl-L-Lys at amber codons (Neumann et al., 2008). Cells were grown at 37 C in LB medium supplemented with 50 mg/ml streptomycin and 50 mg/ml kanamycin, diluted 1:4 after overnight incubation and grown until the OD600nm reached 0.4. Cells were then supplemented with 20 mM of nicotinamide (Sigma) and with 10 mM eNAcetyl-L-Lys (ApexBio). Protein expression was induced for 4h at 37 C with 0.7 mM IPTG. The cells were suspended in lysis buffer (20 mM Tris-HCl pH 7.4, 1M NaCl and 50 mM imidazole), lysed and centrifuged at 105, 000g for 1h at 4 C. The supernatant was incubated with a nickel chelating-sepharose resin (GE healthcare) for 1h at 4 C and the protein was eluted with elution buffer (20 mM TrisHCl pH 7.4, 1M NaCl and 500 mM imidazole). The eluted protein was dialyzed against TEV buffer and cleavage of the His-tag was performed by adding 100 units of TEV protease. The samples were purified by HPLC on a C8-reverse phase column (VYDAC, Hesperia, CA) and eluted with a linear gradient of 0.05% TFA and acetonitrile. Their identities were confirmed by MALDI-TOF mass spectrometry. Peptide Synthesis The mDaxx-SIM-PO4 peptide was synthesized as previously described (Cappadocia et al., 2015). Sequences for the chemically synthesized mDaxx-SIM and mDaxx-SIM-PO4 peptides were GSGEAEERIIVLSDSDY and GSGEAEERIIVLpSDpSDY, respectively. The Daxx peptides were synthesized by conventional Fmoc chemistry using the automated ABI433A peptide synthesizer (PE Applied Structure 28, 1–12.e1–e5, February 4, 2020 e3

Please cite this article in press as: Mascle et al., Acetylation of SUMO1 Alters Interactions with the SIMs of PML and Daxx in a Protein-Specific Manner, Structure (2019), https://doi.org/10.1016/j.str.2019.11.019

Biosystems, Foster City, CA) for assembly on NovaPEG rink amide resin (Novabiochem). The phosphopeptides were made with Fmoc-Ser(PO(OBzl)OH)-OH (Novabiochem). Following assembly, peptides were deprotected at the side-chains and cleaved from the solid support with reagent K (TFA/H2O/thioanisole/ethanedithiol/phenol = 82.5:5:5:2.5:5) at room temperature for 4h. Peptides were then isolated by ether precipitation. Crude peptides were purified by reverse-phase high performance liquid chromatography (RP-HPLC) on a C8-reverse phase column (VYDAC, Hsperia, CA) and eluted with a linear gradient of 0.05% TFA and acetonitrile. Their identities were confirmed by MALTI-TOF mass spectrometry on Voyager DE-PRO (Perseptive Biosystems, Framingham, MA). In Vitro Phosphorylation of SIM Peptides For preparation of the PML-SIM-PO4 and Daxx-SIM-PO4 peptides, 200-250 nmol of the unphosphorylated peptides were resuspended in 1mL of 1X NEBuffer for Protein Kinase (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 0.1 mM EDTA, 2 mM DTT, 0.01% Brij 35). 3,000 units of CK2 enzyme (NEB) were added to the solutio. Every 24h for 72 h, the reaction was supplemented twice daily with CK2 (500 units, 1 mL) and ATP (2 mmol). The solution was purified by HPLC as described above for the unphosphorylated peptides (Cappadocia et al., 2015). ITC Experiments Lyophilized proteins were dialysed overnight at room temperature into 20 mM Tris-HCl pH 7.4. Protein concentrations were determined by UV absorbance at 280 nm. ITC measurements were performed at 25 C using a VP-ITC calorimeter (MicroCal). Data were analyzed using Origin Software and all experiments fit the single binding site model with 1:1 stoichiometry. Standard deviations in KD values were estimated from duplicate measurements or more. Crystallization and Data Collection DN-SUMO1 proteins were at a final concentration of 500 mM-1 mM in H20. The PML-SIM, PML-SIM-PO4 or mDaxx-SIM-PO4 peptides were added to a final concentration of 600 mM-1.2 mM, to yield a protein:peptide ratio of 1:1.2. Crystals of each complex were obtained by the hanging drop vapour diffusion method using a precipitant solution containing 100 mM sodium cacodylate pH 6.5, 22 to 34% (w/v) PEG3350 and 10 mM calcium chloride. Crystals were cryoprotected in a mother liquor containing 10% glycerol. Diffraction data were collected using either a Rayonix MX300 or a Pilatus3S 6M detector at beamline 08-ID of the Canadian Light Source (CLS) or a Pilatus3S 6M detector at the beamline F1 of the Macromolecular Cornell High Energy Synchrotron Source (MacCHESS). Datasets were indexed, integrated and scaled using HKL2000 (HKL Research, Inc.). Structure Determination and Refinement Initial phases were obtained by molecular replacement using the crystal structure of DN-SUMO1 in complex with PML-SIM (PDB: 4WJO), PML-SIM-PO4 (PDB: 4WJN), or mDaxx-SIM-PO4 (PDB: 4WJP) as search template. Phases were improved by iterative cycles of model building with Coot (Emsley and Cowtan, 2004; Emsley et al., 2010) and refinement with PHENIX (Adams et al., 2010). Test data sets were randomly selected from the observed reflections prior to refinement. Statistics for the final models (Tables 2, 3, and 4) were obtained with PHENIX (Adams et al., 2010) and Molprobity (Chen et al., 2010). The structure coordinates have been deposited in the RCSB Protein DataBank. The figures were prepared with PyMOL. BRET Experiments The BRET assays were conducted essentially as previously (Mascle et al., 2013; Percherancier et al., 2009). HEK293 cells transiently transfected with the luciferase donor (PML-RLuc) and GFP acceptor (GFP-SUMO1 constructs) were resuspended and distributed in 96-well plates. Upon addition of the cell permeable luciferase substrate (coelenterazine deep blue, PerkinElmer Life Sciences), the bioluminescence signal resulting from its degradation was detected using a 370–450-nm band pass filter (donor emission peak 400 nm). The energy transferred results in a fluorescence signal emitted by the GFP acceptor (excitation peak 400 nm, emission peak 510 nm) that was detected using a 500–530-nm band pass filter. The BRET signal (BRET ratio) was quantified by calculating the acceptor fluorescence/donor (GFP/Luc) bioluminescence ratio. Expression level of each construct was determined by direct measurements of the total fluorescence and luminescence on the same aliquots of transfected cell samples. The GFP total fluorescence was measured using a Fusion alpha FP (Packard) with an excitation filter at 425 nm and an emission filter at 515 nm. The total luminescence was measured using the same cells incubated with coelenterazine H (Molecular Probes; emission peak 485 nm) for 10 min. The BRET ratios were plotted as a function of the GFP/Luc fusion protein expression ratio, the expression of both fusion proteins was assessed with the same cells as described above, to take into account the potential variations in the expression of individual constructs from transfection to transfection. QUANTIFICATION AND STATISTICAL ANALYSIS Data from ITC experiments (Figures 1 and S1 and Tables 1, S1, and S2), were analyzed with Origin Software Version 7(OriginLab Corporation, North Hampton, MA). BRET experiments (Figure 6) were performed in triplets and data points were shown as mean +/- standard deviations for the three replicates. The curves were fit with the Graphpad Prism software (GraphPad Software, San Diego, CA). For the X-ray structures, the model building was perfomed with Coot (Emsley and Cowtan, 2004; Emsley et al., 2010) and structural refinement with PHENIX (Adams et al., 2010). Statistics for the final models (Tables 2, 3, and 4), were obtained e4 Structure 28, 1–12.e1–e5, February 4, 2020

Please cite this article in press as: Mascle et al., Acetylation of SUMO1 Alters Interactions with the SIMs of PML and Daxx in a Protein-Specific Manner, Structure (2019), https://doi.org/10.1016/j.str.2019.11.019

with PHENIX (Adams et al., 2010) and Molprobity (Chen et al., 2010). The structure figures (Figures 3, 4, 5, and S2–S5) were prepared with PyMOL. DATA AND CODE AVAILABILITY The atomic coordinates generated in this study are freely available at the RCSB (https://www.rcsb.org) with the following PDB accession codes : PDB: 6UYO (DN-SUMO1K37Ac: PML-SIM complex), PDB: 6UYP (DN-SUMO1K39Ac: PML-SIM complex), PDB: 6UYQ (DN-SUMO1K45Ac: PML-SIM complex), PDB: 6UYR (DN-SUMO1K46Ac: PML-SIM complex), PDB: 6UYS (DNSUMO1K37Ac: PML-SIM-PO4 complex), PDB: 6UYT (DN-SUMO1K39Ac: PML-SIM-PO4 complex), PDB: 6UYU (DN-SUMO1K45Ac: PML-SIM-PO4 complex), PDB: 6UYV (DN-SUMO1K46Ac: PML-SIM-PO4 complex), PDB: 6UYX (DN-SUMO1K37Ac: Daxx-SIM-PO4 complex), PDB: 6UYY (DN-SUMO1K39Ac: Daxx-SIM-PO4 complex) and PDB: 6UYZ (DN-SUMO1K46Ac: DaxxSIM-PO4 complex).

Structure 28, 1–12.e1–e5, February 4, 2020 e5