Chemical Synthesis of Atomically Tailored SUMO E2 Conjugating

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Chemical Synthesis of Atomically Tailored SUMO E2 Conjugating Enzymes for the Formation of Covalently Linked SUMO–E2–E3 Ligase Ternary Complexes Yinfeng Zhang, Tsuyoshi Hirota, Keiko Kuwata, Shunsuke Oishi, Subramanian G. Gramani, and Jeffrey W. Bode J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b06820 • Publication Date (Web): 22 Aug 2019 Downloaded from pubs.acs.org on August 23, 2019

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Journal of the American Chemical Society

Chemical Synthesis of Atomically Tailored SUMO E2 Conjugating Enzymes for the Formation of Covalently Linked SUMO–E2–E3 Ligase Ternary Complexes

Yinfeng Zhang , Tsuyoshi Hirota , Keiko Kuwata , Shunsuke Oishi , 1

1

1

1

Subramanian G. Gramani , Jeffrey W. Bode * 1

1,2

1. Institute of Transformative Bio-Molecules (WPI–ITbM), Nagoya University, Chikusa Nagoya 464-8602, Japan 2. Laboratorium für Organische Chemie, Department of Chemistry and Applied Biosciences,

ETH Zürich, Zürich 8093, Switzerland

ABSTRACT: E2 conjugating enzymes are the key catalytic actors in the transfer of ubiquitin, SUMO, and other ubiquitin-like modifiers to their substrate proteins. Their high rates of transfer and promiscuous binding complicate studies of their interactions and binding partners. In order to access specific, covalently-linked conjugates of the SUMO E2 conjugating enzyme Ubc9, we prepared synthetic variants bearing site-specific non-native modifications including: 1) replacement of Cys93 to 2,3-diaminopropionic acid to form the amide-linked stable E2–SUMO conjugate, which is known to have high affinity for E3 ligases; 2) a photoreactive group (diazirine) to trap E3 ligases upon UV irradiation; and 3) an N-terminal biotin for purification and detection. To construct these Ubc9 variants in a flexible, convergent manner, we combined the three leading methods – native chemical ligation (NCL), α-ketoacid–hydroxylamine (KAHA) ligation, and serine/threonine ligation (STL). Using the synthetic proteins, we demonstrated the selective formation of Ubc9–SUMO conjugates and the trapping of an E3 ligase (RanBP2) to form the stable, covalently linked SUMO1–Ubc9–RanBP2 ternary complex. The powerful combination of ligation methods – which minimizes challenges of functional group manipulations – will enable chemical probes based on E2 conjugating enzymes to trap E3 ligases and facilitate the synthesis of other protein classes.

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INTRODUCTION Ubiquitination, SUMOylation, and the attachment of other ubiquitin-like (Ubl) modifiers are regulated by a complicated molecular machinery. Three enzymes are involved in this process: E1 activating enzymes, E2 1

conjugating enzymes and E3 ligases. The specific interactions between these partners and substrate proteins are mediated by a network of protein–protein interactions, which are modulated by numerous factors, including posttranslational modifications (PTMs) and the presence of a covalently conjugated Ub or Ubl at the E2 catalytic site. Given the complicated nature of these processes, activity-based protein profiling (ABPP) by installation of functional groups to the C-termini of ubiquitin and ubiquitin-like modifiers (Ub/Ubl) have 2–10

proven to be a powerful approach for interrogating these pathways. Virdee and Ovaa have independently 7,9

6

developed activity-based probes derived from ubiquitin to study HECT (homologous to E6AP) and RBR (RING-in-between-RING) E3 ligases – which have a catalytic Cys that accepts and transfers Ub. The majority of E3 ligases, however, do not utilize a catalytic Cys residue or form covalent bonds to Ubls. Statsyuk has reported photocrosslinking of the E2 UbcH7 to ubiquitin E3 ligase E6AP by introducing the diazirine by sitespecific chemical modification of surface exposed Cys residues incorporated into the E2. To date, this elegant 11

approach has only been applied to trap HECT-type ligases, but not to other classes of E3s including RBR, RING, Cullin-RING and APC/C E3s. The success of photocrosslinking to investigate protein–protein interactions in other systems

12–16

suggests that effective probes for the vast majority of Ub/Ubl-E2–E3

interactions, including ubiquitin RING and U-box E3 ligases – which interact with the substrate proteins and E2 enzymes only by non-covalent interactions – can be constructed but are currently limited by the restriction that inclusion of more than one unnatural residue in expressed proteins remains challenging. Given their moderate size in most cases (150–180 residues), the chemical synthesis of E2 conjugating enzymes appeared to be an attractive alternative to providing a platform for preparing E2-based probe bearing any desired modification or atomic substitution.

In this report, we document the synthesis of atomically tailored variants of the SUMO conjugating enzyme Ubc9. Ubc9 serves as the E2 conjugating enzyme and SUMO is loaded onto the catalytic Cys93 of Ubc9 by a transthiolation reaction from the E1 activating enzyme to form a labile E2–SUMO thioester. The SUMO is 17

transferred from Ubc9–SUMO to a lysine residue in specific protein substrates, usually with the aid of an E3 ligase that non-covalently binds to the Ubc9–SUMO conjugate and the substrate proteins. To prepare synthetic ACS Paragon Plus Environment

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Ubc9 bearing specific non-canonical modifications we employed a combination of three leading ligation chemistries – native chemical ligation (NCL), KAHA ligation, and serine/threonine ligation (STL). The 18

19

20

distinct conditions of each ligation technology allow the direct incorporation of the ligation handles for the subsequent reactions, thereby obviating cumbersome functional group manipulations. This flexible approach allows a number of Ubc9 variants bearing multiple modifications at distinct positions can be prepared in short order. These mutations include: 1) replacement of Cys93 to 2,3-diaminopropionic acid (Dap) to form the amide-linked stable E2–SUMO conjugate, which is known to have high affinity for E3 ligases; 2) a photoreactive group (diazirine) to trap E3 ligases upon UV irradiation; and 3) an N-terminal biotin for purification and detection (Figure 1). We demonstrate that this convergent synthetic route allows rapid access to a variety of Ubc9 variants, including probes that can be covalently linked to SUMO by stable amide-bond formation and selectively trap the SUMO E3 ligase RanBP2 by formation of SUMO– 21

Ubc9–RanBP2 via photoaffinity crosslinking strategy. In addition to providing a path to the semi-synthetic preparation of covalently linked protein ternary complexes, this work establishes that distinct ligation technologies can be effectively combined to facilitate the modular synthesis of protein-based probes. photoaffinity probe

Ubc9(1–33)

H 2N

resin

H N

Fmoc SPPS

N

O

O H 2N

Ubc9(36–73)

O CHO

Segment 1 H 2N

Ubc9(76–106)

H N

HS

O

combination of STL, KAHA ligation and NCL chemical protein synthesis

O H N

OH Me

Segment 3

CO 2H

S

Segment 2

O

O

O

Ubc9(109–158)

CO 2H

Cys93Dap biotin affinity tag

three site-specifically incorporated functionalities

OH

SUMO O

substrate protein

CF3

E3 ligase H N

SUMO

substrate protein

CF3

O

SUMO–E2–E3 ternary complex

affinity purification

H N

SUMO

formation of Ubc9–SUMO conjugate N

N N

N

CF3

CF3

E3 ligase

E3 ligase Ubc9

CF3

Ubc9

H 2N

Segment 4

Me

N

O

H 2N

Me

HO

H N

Ubc9 phototrapping (365 nm)

O

covalently trapped E2–E3 conjugate

H N

SUMO

Ubc9

O

SUMO–E2–E3–substrate quaternary complex

incubate with recombinant proteins

SUMO

H N

Ubc9

O Cys93Dap

E2 (Ubc9)–SUMO amide conjugate

Figure 1. Trifunctional Ubc9 probes are synthesized by the combination of STL, KAHA ligation and NCL to include 1) mutation of Cys93 to Dap (2,3-diaminopropionic acid); 2) site-specific incorporation of diazirines into various sites of Ubc9; and 3) N-terminal attachment of a biotin affinity tag. The synthetic Ubc9 enzymes are semi-synthetically conjugated to recombinant SUMO to form the stable, amidelinked Ubc9–SUMO conjugate. In the presence of a substrate protein and E3 ligase, UV irradiation leads to trapping of the SUMO–E2–E3 ternary complex.

RESULTS



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Chemical synthesis of Ubc9 and Ubc9 variants. Remarkable advances in chemical protein synthesis provide access to small and medium sized synthetic proteins and enable the site-specific incorporation of posttranslational modifications or the preparation of mirror image molecules, including Kent’s and Baker’s synthesis of racemic mixture of Rv1738 to facile protein crystallization and Zhu’s and Liu’s synthesis of D22

amino acid polymerase. Other notable achievements – chosen from a large number of impressive reports – 23

24

include Muir’s and other group’s preparation of modified histones,

25,26

Payne’s sulfated proteins with anti-

thrombotic and anticoagulant activities and the synthesis of homogeneous polyubiquitin chains by Brik and 27

28

Liu.

29,30

While the power and utility of chemical protein synthesis is unquestioned, it is often still a tedious process and the construction of most proteins (>100 residues) demands multi-segment ligation strategies that require orthogonal protecting groups, which can complicate the preparation of the constituent peptide segments or interfere with post-ligation processing steps. For example, it can be challenging to mask the C-terminus of segments employed in the native chemical ligation (NCL) of thioesters and the selection of ligation sites may be limited due to the low abundance of cysteine. The α-ketoacid–hydroxylamine (KAHA) ligation offers a 18

distinct set of ligation parameters, but typically forms a non-canonical homoserine residue at the ligation site; while usually innocuous, the introduction of multiple mutations may be undesirable. The Ser/Thr ligation 19

(STL) of C-terminal salicylaldehyde ester operates at relatively common Gly–Ser/Thr junctions, but requires non-aqueous conditions that are suitable for single segment couplings but are difficult to employ in a convergent synthesis. In this report, we take advantages of the mutual orthogonality of the functional groups 20

involved in these ligations to convergently prepare E2 conjugating enzymes.

As part of our ongoing interest in using synthetic proteins to interrogate SUMOylation pathways, we selected the synthesis of Ubc9, the SUMO E2 conjugating enzyme, as a platform to incorporate photoaffinity tag for the construction of E2 conjugating enzyme based probes. Ubc9 is a 158-residue conjugating enzyme with a well-established mechanism featuring the loading of a SUMO protein by an E1 activating enzyme (a heterodimer of Aos1/Uba2) onto catalytic cysteine93 to form a covalent E2–SUMO complex. Importantly, 1,17

while unloaded E2 enzymes typically have a high binding affinity for the E1 enzymes, the formation of covalent E2–Ub/Ubl modifiers complex induces a conformational change that often leads to higher affinity for ACS Paragon Plus Environment

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E3 ligases. This has important implications for our goal of using synthetic E2 enzymes as chemical probes for 1

the identification of E3 ligases, as it is known that covalent E2–Ub/Ubl complexes are preferred for forming the requisite protein–protein interactions.

7

We prepared Ubc9 by a convergent four-segment, three-ligation strategy using STL, KAHA ligation and NCL. We selected Gly34–Thr35, Lys74–Cys75 and Ile107–Thr108 as ligation sites due to the similar size of the resulting peptides and flexibility for introducing site-specific mutations. The presence of a Lys–Cys junction near the middle of the protein provided an ideal site for NCL. Segment S1 for the STL ligation was prepared by Fmoc-SPPS on 2-Cl Trityl resin. Following selective resin cleavage, direct coupling and global deprotection, the C-terminal acid was converted to a salicylaldehyde ester. Li and co-workers have reported 31

that thioesters are stable under STL conditions, allowing us to use a S2 thioester – itself prepared according 32

to Liu’s procedure on hydrazine loaded 2-Cl trityl resin, followed by oxidation with NaNO and trapping of 2

the acyl azide with 3-mercaptopropionc acid. Segments S1 and S2 were assembled by STL in pyridine/acetic 33

acid (1:4 molar ratio) solution at 37 °C for 70 min to yield the initial ligation product as a N, O-benzylidiene acetal, which was treated with TFA/H O/TIPS (95/2.5/2.5, v/v/v) to give S5, the peptide thioester 2

corresponding to Ubc9(1–74). These steps were carried out sequentially because S5 and S2 exhibited similar retention times and their purification was arduous (Scheme 1).



H N

Ubc9(1–33)

R1HN

Page 6 of 22

O

O

H 2N O

O

Me SAcm

O

HO

Me

S3

SAcm H 2N

OH

tBuSS

CHO

S1

O

H N

Ubc9(76–106)

H N

Ubc9(36–73)

O

O H N

CO 2H

S

Ubc9(109–158)

O

CO 2H

H 2N

Me

S4

S2 1) pyridine/acetic acid (1:4 molar ratio), 37 °C, 42%

Serine/Threonine Ligation (STL)

KAHA Ligation

2) TFA/H2O/TIPS (95/2.5/2.5, v/v/v)

1) DMSO/H 2O/TFE (7/2/1, v/v/v) 50 °C, 6 h, 40% 2) 6 M Gnd·HCl (pH 11), r.t., 2 h OH

SAcm

R1HN

Ubc9(1–33)

H N

Me O N H

O

OH H N

Ubc9(36–73) O

H 2N

O

H 2N S

CO 2H

H N

Ubc9(76–106)

R 2S

N H Me

Me

S5 (R1 = H or biotin linker)

purified S5

SAcm

O Ubc9(109–158) O

S6 (R 2 = StBu)

purified S6

S5 with N, O-benzylidiene acetal

S3+S4 depsi-peptide t=6h

t = 70 min

S1

CO 2H

S2 S3 t = 0 min

S4 t=0h

Scheme 1. Four segments for convergent synthesis of Ubc9 can be assembled by STL and KAHA ligation respectively. Ubc9 is divided into four segments due to the size limitation of SPPS. S1 and S2 can be joined by STL to generate S5 corresponding to wild type Ubc9(1–74) while S3 and S4 can be assembled by KAHA ligation to generate S6 corresponding to wild type Ubc9(75–158).

Segments S3 and S4 were assembled by KAHA ligation. For the preparation of α-ketoacid segment S3 with an N-terminal Cys residue, we employed the previously reported protected isoleucine α-ketoacid on Rink amide resin, which delivered C-terminal α-ketoacid segment S3 upon resin cleavage and global deprotection. 34

Although it was not strictly necessary to protect the Cys residues for KAHA ligation, we employed a S-tertbutyl thiol protected cysteine as the N-terminal cysteine residue of S3 and S-acetamidomethyl-L-cysteine 35

(Fmoc-Cys(Acm)-OH) for other Cys residues to avoid undesired disulfide bond formation during handling and purification. Segment S4 was prepared by standard methods with Boc-(S)-5-oxaproline as the N-terminal residue. The KAHA ligation of S3 with S4 was carried out in DMSO/H O/TFE (7/2/1, v/v/v) at 50 °C – slightly 2


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modified condition from previously reported KAHA ligations – to give the ligated product depsi-peptide S3+S4, with a homoserine (Hse) ester at the ligation site. The Ile107–Hse108 junction was chosen as Thr108 is located in an exposed loop region and the mutation of Thr108Hse is not likely to influence the structure and activity of Ubc9. Although the hindered, beta-branched isoleucine α-ketoacid was employed for the KAHA ligation, it did not negatively affect the reaction, and maximum conversion was reached within 6 h. The O36

to N-acyl shift was performed under basic condition (6 M Gnd·HCl, pH 11) to yield S6, corresponding to Ubc9(75–158) (Scheme 1).

The purified STL and KAHA ligation products S5 and S6 could be employed – without any manipulations or processing – for NCL in 0.2 M Na HPO buffer containing 3% (v/v) PhSH, 6 M Gnd·HCl and 50 mM TCEP 2

4

(pH 7.3), at 37 °C for 21 h to generate the full-length Ubc9 protein. Removal of the Acm groups with 1% (w/v) AgOAc under acidic conditions afforded the full length of synthetic Ubc9(Thr108Hse) protein (Scheme 2).

37,38

The synthetic protein was characterized by mass (Supplementary Information) and sequenced by LC-MS/MS after chymotrypsin digestion; tandem mass spectrometry showed 100% sequence coverage of Ubc9 (Figure S1a). The linear synthetic Ubc9(Thr108Hse) was folded by dialyzing against the folding buffer (Scheme 2) and confirmed by circular dichroism (CD) in comparison to an authentic sample (Figure S1b).

39



a

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Native sequence of Ubc9: 158 residues in total 10 MSGIALSRLA

20 QERKAWRKDH

30 PFGFVAVPTK

40 50 NPDG–TMNLMN WECAIPGKKG

60 TPWEGGLFKL

70 RMLFKDDYPS

80 SPPK–CKFEPP

90 LFHPNVYPSG

100 TVCLSILEED

110 KDWRPAI–TIK

120 QILLGIQELL

140 AEAYTIYCQN

150 RVEYEKRVRA

QAKKFAPS

b

130 NEPNIQDPAQ

OH

SAcm

R1HN

Me O

H N

Ubc9(1–33)

N H

OH Ubc9(36–73) O

O

O

H N

H 2N

CO 2H

S

Ubc9(76–106)

H N

S5

Ubc9(109–158)

N H

R 2S

H 2N

SAcm

O CO 2H

O

Me Me

S6 1) 3% (v/v) PhSH, 50 mM TCEP, 6 M Gnd·HCl, 0.2 M Na 2HPO 4 (pH 7.3), 37 °C , 21 h, 35%

NCL & Acm deprotection

2) 1% (w/v) AgOAc, AcOH/H2O (1/1, v/v), 50 °C, 1 h; DTT, 30 min, 56% OH

SH

R1HN

H N

Ubc9(1–33)

Me O

OH H N

Ubc9(36–73)

N H

OH

HS O

H 2N

H N

Ubc9(76–106)

N H

O

O

Me

R 1HN

105 A5

68 A2 69 A3

22 A1

Ubc9 (Thr108Hse) with Acm t = 21 h

i. 6 M Gnd•HCl, 4 °C, pH 7.3, 5 h ii. dialysis, 4 °C

S5 R2 = H S6-1 t = 2 h

biotin tag =

N NH

H

93 A4

protein folding

O HN

CO 2H

Ubc9(109–158)

N H

Me

linear Ubc9 (1–158)

PhSH

SH

O

folded Ubc9 variants

N

H

H N

S

N

NH 2

CF3

N

O O

O

H 2N H 2N

O

F*

E2 (Ubc9) variant

COOH

N H

COOH

COOH

Dap

P*

N-terminus

93

22

68

69

105

R1

A4

A1

A2

A3

A5

108 A

H

Cys

F

Y

P

P

Hse

Ubc9(Thr108Hse, N-Biotin)

biotin tag

Cys

F

Y

P

P

Hse

Ubc9(Cys93Dap, Thr108Hse, N-Biotin)

biotin tag

Dap

F

Y

P

P

Hse

Ubc9(F22F*, Thr108Hse, N-Biotin)

biotin tag

Cys

F*

Y

P

P

Hse

Ubc9(F22F*, Cys93Dap, Thr108Hse, N-Biotin)

biotin tag

Dap

F*

Y

P

P

Hse

Ubc9(Y68F*, Thr108Hse, N-Biotin)

biotin tag

Cys

F

F*

P

P

Hse

Ubc9(Y68F*, Cys93Dap, Thr108Hse, N-Biotin)

biotin tag

Dap

F

F*

P

P

Hse

Ubc9(P69P*, Cys93Dap, Thr108Hse, N-Biotin)

biotin tag

Dap

F

Y

P*

P

Hse

Ubc9(P105P*, Cys93Dap, Thr108Hse, N-Biotin)

biotin tag

Dap

F

Y

P

P*

Hse

Ubc9(Thr108Hse)

Scheme 2. Final assembly of segment S5 and segment S6 by NCL to prepare wild type Ubc9 and Ubc9-based variants can be prepared by combination of STL, KAHA ligation and NCL. (a) Sequence of wild type Ubc9. The disconnection sites are shown in red. (b) Full length Ubc9(Thr108Hse) can finally be conjoined together by NCL, followed by the removal of Acm protecting groups. The conjugating activity of synthetic Ubc9(Thr108Hse) and Ubc9(Thr108Hse, N-Biotin) were restored after proper folding.


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This flexible, four segment strategy was used to prepare wild type Ubc9 as well as variants bearing combinations of side chain modifications, a task that – at the current state of development – is challenging to achieve by recombinant methods including genetic code expansion. We sought to form stable, semi-synthetic 40,41

Ubc9–SUMO conjugates, as these are known to preferentially interact with E3 ligases. The formation of E2– 1,17

Ub/Ubl complex can sometimes be achieved by replacement of the catalytic Cys with Lys or Ser , however, 41

42

Lys has longer side chain than the native linkage and could interfere with substrate and E3 interactions; Ser mutations generate the labile ester linkage in the presence of E3 ligase. We postulated that the amino acid Dap, 1

which substitutes a primary amine for the cysteine thiol, could form an stable amide bond between Ubc9 and SUMO,

44,45

thereby introducing minimal perturbation of the interactions with E3 ligases. Very recently,

Schmeing and Chin successfully used a masked form of Dap to introduce it into recombinant proteins for the first time by expansion of the genetic code. However, no examples of E2 conjugating enzymes bearing this 46

modification have been reported and our synthetic approach allowed facile incorporation of the Cys93Dap mutation simply by employing the appropriate amino acid 2,3-diaminopropionic acid during the synthesis of Segment 3. We also sought to incorporate amino acids bearing side chains suitable for photocrosslinking protein–protein interactions. To this end, amino acid residues containing diazirines as photo-induced crosslinkers were installed at various sites including Phe22, Tyr68, Pro69, and Pro105 (Scheme 2). Finally, to facilitate affinity purification and biochemical detection, we also installed an N-terminal biotin tag. In all cases, the synthesis of these Ubc9 variants proceeded smoothly without complications from the unnatural amino acids or diazirine moieties (Scheme 2).

Biochemical assay of synthetic Ubc9 and formation of covalent Ubc9–SUMO conjugates. The synthetic Ubc9 variants were evaluated in an ATP-dependent SUMOylation assay using RanGAP1 as the substrate and recombinant E1 activating enzyme (Figure 2a). Commercially available recombinant Ubc9 was used as a positive control. The activity of synthetic Ubc9(Thr108Hse) was indistinguishable from recombinant Ubc9 and RanGAP1 was SUMOylated for all three SUMO isoforms, SUMO1, SUMO2 and SUMO3 (Figure 2b, lanes 3&2, 9&8 and 12&11).

17



a H 2N

OH

SUMO

Ubc9

X

O

X

SUMO

Mg-ATP SUMO E1

RanGAP1 H N

Ubc9

SUMO

(SUMOylation substrate protein)

O

RanGAP1

O

biotin affinity tag

X = S or N

b SUMOylation of RanGAP1 with synthetic Ubc9(Thr108Hse)

kDa 75

+ – – –

SUMO1 + + + + + – – +

1

2

3

marker

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 22

– – + –

– – – +

SUMO2 + + + – + + – + – – – +

+ – – –

4 5

6

7

10 11 12 Ubc9(Thr108Hse)

– + – –

8

9

SUMO3 + + + + + – – +

c construction of Ubc9(Cys93Dap, Thr108Hse, N-Biotin)–SUMO conjugate

SUMO GST-RanGAP1 recombinant Ubc9 Ubc9(Thr108Hse)

SUMOylated GST-RanGAP1

+ + – + + –

+ + – + + +

+ + + – + +

+ + + – + –

+ + – – + +

+ + + – – +

– + + – + +

+ – + – + +

1

2

3

4

5

6

7

8

kDa 37

E1 SUMO1 Ubc9(Cys93Dap, Thr108Hse, N-Biotin) Ubc9(Thr108Hse, N-Biotin) Mg-ATP DTT

single SUMOylated Ubc9(Thr108Hse, N-Biotin)

50

Ubc9(Cys93Dap, Thr108Hse, N-Biotin)–SUMO1 conjugate anti-SUMO1

25

anti-SUMO2/3 SUMOylated GST-RanGAP1

50

20

GST-RanGAP1

37

Ubc9(Thr108Hse, N-Biotin) /Ubc9(Cys93Dap, Thr108Hse, N-Biotin) anti-biotin

anti-GST

Figure 2. The conjugating activity of synthetic Ubc9(Thr108Hse) and construction of Ubc9(Cys93Dap, Thr108Hse, N-biotin)–SUMO conjugate. (a) Schematic diagram of SUMOylation of RanGAP1 and construction of Ubc9–SUMO conjugate through the mutation of Cys93 to Dap. (b) In vitro SUMOylation of RanGAP1 was carried out in a 20 μL reaction mixture with synthetic Ubc9(Thr108Hse) or recombinant Ubc9 in the presence of E1, SUMO1/SUMO2/SUMO3 and ATP at 37 °C for 1 h. The proteins were detected with anti-SUMO antibodies and reprobed with anti-GST. RanGAP1 was SUMOylated by synthetic Ubc9(Thr108Hse) as well as the recombinant Ubc9 with all the SUMO isoforms (lanes 3&2, 9&8 and 12&11, full images are in Figure S8). The conjugating activity of Ubc9(Thr108Hse, N-Biotin) is shown in Figure S2. (c) The conjugation of Ubc9(Cys93Dap, Thr108Hse, N-Biotin) with SUMO1 was performed under in vitro SUMOylation assay using SUMO1 (3 μg) and Ubc9(Cys93Dap, Thr108Hse, NBiotin) (3.94 μg) in a 20 μL reaction mixture in the presence of E1 (0.45 μg) and ATP (5 mM) at 37 °C for 14 h. The reducing reagents (100 mM DTT and 20 mM β-mercaptoethanol) were added and maintained for 10 min at the end of the reaction followed by SDS-PAGE. The extent of conjugation was analyzed by immunoblotting against anti-biotin and anti-SUMO1 antibodies. Full images are in Figure S9. Identification of SUMO conjugation site as Dap93 by proteolytic digestion and tandem mass spectrometry is shown in Figure S3.

The biotinylated Ubc9 variants bearing the Cys93Dap mutation were evaluated for their ability to form a covalent complex with SUMO. We performed the in vitro conjugation reaction by incubating Ubc9(Cys93Dap, Thr108Hse, N-Biotin) with SUMO in the presence of activating enzyme E1 and ATP. As it is known that Ubc9 itself can be SUMOylated at multiple Lys residues under SUMOylation conditions, control reactions using 47–49

synthetic Ubc9(Thr108Hse, N-Biotin) were also conducted. All reactions were quenched with a reducing cocktail containing 100 mM DTT and 20 mM β-mercaptoethanol to avoid formation of Ubc9 homodimer, as 50

well as thioesterification between the catalytic cysteine of Ubc9(Thr108Hse, N-Biotin) and SUMO. The extent of conjugation was determined by immunoblotting with anti-SUMO and anti-biotin antibodies. For Ubc9(Thr108Hse, N-Biotin), the single SUMOylated form was observed (Figure 2c, lanes 1,2), which is


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consistent with a published result.

47–49

SUMO1 was attached to Ubc9(Cys93Dap, Thr108Hse, N-Biotin) (Figure

2c, lanes 3,4) and its conjugation was dependent on ATP and E1 (Figure 2c, lanes 6,7).

To confirm the identity of the conjugate, we treated the protein bands corresponding to Ubc9(Cys93Dap, Thr108Hse, N-Biotin)–SUMO1 (Figure 2c, lanes 3,4) with trypsin and chymotrypsin and analysed the resulting digests by tandem mass spectrometry. A peptide remnant from the C terminus of SUMO1 was found to be conjugated to the target mutation residue Dap93 of Ubc9(Cys93Dap, Thr108Hse, N-Biotin) (Figure S3).

E3 ligase trapping. In order to validate our key hypothesis that appropriately placed diazirines can selectively crosslink an E3 ligase bound to our synthetic Ubc9 probes, we selected RanBP2 as a test case. RanBP2 is one of the best characterized E3 ligases and has been reported to enhance Sp100 SUMOylation with a marked preference for SUMO1 over SUMO2/3. The interaction of RanBP2 with conjugating enzyme Ubc9 has been 21

determined by X-ray crystallography and this structure highlighted that the hydrophobic side chain of Phe22 51

of Ubc9 located in the exposed loop region and presented at the binding surface between RanBP2 and Ubc9 (Figure S4a). Accordingly, we chose Phe22 to incorporate the photoreactive Phe derivative L-(4-Tmd)-Phe (represented as F*) and prepared synthetic Ubc9 variants bearing this F22F* mutation. 52

To test the reactivity of the diazirine moiety incorporated into these Ubc9 variants, we attempted to trap RanBP2 using Sp100 as a substrate protein. When Ubc9(F22F*, Thr108Hse, N-Biotin) – which retained catalytic Cys93 – was incubated with SUMO1, Sp100 and RanBP2 in the presence of E1 and ATP at room temperature for 1 h, followed by UV irradiation, we observed hyperSUMOylated RanBP2 with a broad mass range (Figure 3c, entry 1 and Figure S4b, lanes 1, 5–6), in agreement with a previous report that RanBP2 can be modified by up to 25 molecules of SUMO1. The multiply SUMOylated products 21

made it extremely difficult to distinguish the desired photocrosslinked Ubc9–RanBP2 product from hyperSUMOylated RanBP2 products.

In contrast, when the Cys93Dap mutant Ubc9(F22F*, Cys93Dap, Thr108Hse, N-Biotin) was employed in the same experiment, we observed clean formation of a new band (Figure 3b, lane 6, red triangle, Figure 3c, entry 2) that could be detected by anti-biotin (synthetic Ubc9), anti-SUMO1, and anti-GST (attached to RanBP2) ACS Paragon Plus Environment


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(Figure 3b, lane 6). The identity of ternary complex SUMO1–Ubc9(F22F*, Cys93Dap, Thr108Hse, N-Biotin)– RanBP2 was confirmed by tandem mass spectrometry and isolated by affinity purification with streptavidin beads (Figure S5, lane 2). A series of control experiments were performed to differentiate specific crosslinking from background labelling. In the absence of UV light (Figure 3b, lane 1 and Figure 3c, entry 3) or without 53

the diazirine (Figure 3b, lane 5 and Figure 3c, entry 4), no crosslinked products were observed. To test the site specificity, we carried out RanBP2-trapping with a probe bearing a diazirine at a distal site Tyr68 (Scheme 2). The mutant protein Ubc9(Y68F*, Cys93Dap, Thr108Hse, N-Biotin) did not trap RanBP2 (Figure 3c, entry 5, Figure S6). To gauge whether the probe Ubc9(F22F*, Cys93Dap, Thr108Hse, N-Biotin) has the selectivity for RanBP2, we performed the experiment with a different SUMO E3 ligase, PIAS1. As expected, no specific 54

crosslinked product was detected (Figure 3c, entry 6, Figure S7). This observation supports the conjecture that combination of an E3 ligase and a substrate protein strongly favours the binding of the SUMO1–Ubc9 covalent complex.

1,17

a substrate protein

N CF3

N

substrate protein

N N

CF3

H N

SUMO

incubate with recombinant

Ubc9

O Cys93Dap

H N

SUMO

Ubc9

E2 (Ubc9)–SUMO amide conjugate

H N

SUMO

(365 nm)

O

E3 ligase and substrate proteins

CF3

CF3

E3 ligase

E3 ligase

E3 ligase

isolate from gel or

Ubc9

SUMO–E2–E3–substrate quaternary complex

Ubc9

O

O

phototrapping

H N

SUMO

biotin affinity purification

covalently trapped E2–E3 conjugate

SUMO–E2–E3 ternary complex

b kDa

1

2

3

4

5

6

7

kDa

1

2

3

4

5

6

7

kDa

anti-biotin

1

2

3

4

5

6

7

150

150

150

anti-SUMO1

anti-GST

SUMO1–Ubc9(F22F*, Cys93Dap, Thr108Hse, N-Biotin)–GST-RanBP2

+ + + – + + – 1

– + + – + + + 2

+ – + – + + + 3

+ + – – + + + 4

+ + – + + + + 5

+ + + – + + + 6

+ + + – – + + 7

E1 SUMO1 Ubc9(F22F*, Cys93Dap, Thr108Hse, N-Biotin) Ubc9(Cys93Dap, Thr108Hse, N-Biotin) GST-RanBP2 GST-Sp100 UV

c entry

Ubc9 variants

SUMO

E3

Substrate

Ubc9– SUMO

SUMO– Ubc9–E3

comments

1

Ubc9(F22F*, Thr108Hse, N-Biotin)

SUMO1

RanBP2

Sp100

hyperSUMOylation of RanBP2 (Figure S4b)

2


SUMO1

RanBP2

Sp100

key experiment (lane 6)

3


SUMO1

RanBP2

Sp100

(no UV)

UV dependent (lane1)

4

Ubc9(Cys93Dap, Thr108Hse, N-Biotin)

SUMO1

RanBP2

Sp100

nonspecific binding (lane5)

5

Ubc9(Y68F*, Cys93Dap, Thr108Hse, N-Biotin)

SUMO1

RanBP2

Sp100

diazirine position (Figure S6)

6


SUMO1

PIAS1

Sp100

probe specificity ( Figure S7)

Figure 3. Trapping of RanBP2 with Ubc9(F22F*, Cys93Dap, Thr108Hse, N-Biotin) probe and demonstration of specific photocrosslinking with control reactions. (a) Schematic diagram of one-pot trapping E3 ligases using Ubc9-based probes in the presence of substrate protein. (b) Trapping of RanBP2 with Ubc9–SUMO (lane 6) was performed in a 20 μL reaction mixture by incubating trifunctional Ubc9(F22F*, Cys93Dap, Thr108Hse, N-Biotin) (3.94 μg) with SUMO1 (3 μg), Sp100 (1.0 μg) and GST-tagged RanBP2 (0.9 μg) in the presence of E1 (0.45 μg)


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and ATP (5 mM) at 37 °C for 14 h, followed by UV irradiation at 365 nm for 30 min. Reducing reagents (100 mM DTT and 20 mM βmercaptoethanol) were added and maintained for 10 min at the end of the reaction. The mixture was subjected to 7.5% SDS-PAGE and immunoblotted against anti-biotin, reprobed with anti-SUMO1, and with anti-GST. The band marked with red triangle indicates RanBP2 can be trapped by Ubc9(F22F*, Cys93Dap, Thr108Hse, N-Biotin)–SUMO1 conjugate probe. Full images are in Figure S10. (c) Summary of control experiments. The full images of control reactions can be found in Figure S6, S7. The trapping of E3 ligase (RanBP2) is dependent on the canonical substrate protein Sp100 (entry 2) and the diazirine moiety (entry 4). The photocrosslinking is dependent on the UV (entry 3) and the proper position of photoaffinity moiety (entry 5). The combination of E3 ligase (RanBP2) and substrate protein (Sp100) is critical for the formation of SUMO– Ubc9–E3 ternary complex (entry 6).

Endogenous RanBP2 trapping. To test whether Ubc9(F22F*, Cys93Dap, Thr108Hse, N-Biotin) can trap the endogenous RanBP2 in cell lysate, we prepared the HEK293T cell lysate, performed the construction of Ubc9(F22F*, Cys93Dap, Thr108Hse, N-Biotin)–SUMO1 conjugate probe, and incubated the probe with the cell lysate in the presence of substrate Sp100. Following UV irradiation (60 min), the proteins, which were trapped by this biotinylated E2–SUMO probe, were further enriched and purified by streptavidin beads and subjected to proteomic analysis. To our delight, the endogenous RanBP2 was successfully trapped by Ubc9(F22F*, Cys93Dap, Thr108Hse, N-Biotin). Control experiments (no probe and UV light) indicated that E3 ligase (RanBP2) trapping is dependent on the photoactive moiety and UV light, which was shown in Table 1.


No probe

No UV

Peptide spectrum matches (PSMs)

35

1

0

Unique peptides

30

1

0

Coverage (100%)

13

1

0

Table 1. Trapping of endogenous RanBP2 from HEK293T cell lysate with Ubc9(F22F*, Cys93Dap, Thr108Hse, N-Biotin). Endogenous RanBP2 was trapped by Ubc9(F22F*, Cys93Dap, Thr108Hse, N-Biotin) and the trapping is dependent on the photoactive probe and UV light. PSMs refer to specific peptides derived from RanBP2 identified by proteomic analysis.

Taken together, these results establish that trapping of a SUMO E3 ligase in the presence of our semisynthetic E2–SUMO probe and the specific substrate occurs readily with properly designed probes bearing the site-specifically incorporated diazirine group. This indicates that it has the potential to be used for the trapping of E3 ligases for a specific substrate protein. ACS Paragon Plus Environment


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DISCUSSION Given the essential role of E3 ligases in regulating substrate specificity of PTMs with Ub/Ubl modifiers, including SUMO, methods to identify E3 ligases are of upmost importance. While HECT and RBR ubiquitin 55

E3 ligases have a conserved catalytic cysteine residue that can accept ubiquitin from E2–Ub to form an E3– Ub thioester – are therefore amenable to be trapped by some activity-based probes, the known SUMO E3 4–9

ligases including ubiquitin RING and U-box E3 ligases – which constitute the majority of E3 ligases – do not form covalent intermediates with SUMO or ubiquitin. The interaction of these E3 ligases with E2–SUMO or E2–Ub and their substrates is non-covalent and transient, rending them difficult to trap or capture by activity1

based probes. As noted by Ovaa and Vertegaal, chemical tools for the identification of these catalytic cysteineindependent E3 ligases are still missing. The method presented here may provide a flexible platform for 56

identifying E3 ligases using chemically synthesized E2 probes.

In the case of SUMOylation, the conjugating enzyme Ubc9 – as well as many other E2s – covalent binding of SUMO or other Ub/Ubl modifiers induces a conformational change that enhances E3 binding; therefore, effective probes need to be loaded with Ub/Ubl modifiers. To construct the stable Ubc9–SUMO conjugate (requisite covalent complex) for E3 recognition under in vitro SUMOylation, we mutated catalytic Cys93 to Dap, enabling the facile, semi-synthetic formation of the stable, amide linked Ubc9–SUMO conjugate from recombinant SUMO in the presence of E1 activating enzymes. These probes are further decorated by the facile incorporation of amino acid residues with side chains bearing diazirines as photoactivatable crosslinkers at various surface exposed regions. Finally, these probes are equipped with an N-terminal biotin for detection, enrichment, and affinity purification of trapped SUMO–Ubc9–E3 ternary complexes. A small library of these probes were efficiently prepared by combing three ligation methods: STL, KAHA ligation and NCL, in a synthetic strategy that obviated the need for tedious protection-deprotection steps for the functional ligation partners. This construction of folded, protein-based probes bearing multiple site-specific modifications is not possible by other means, such as recombinant expression, genetic code expansion, or chemical protein modification.

40

CONCLUSIONS ACS Paragon Plus Environment

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The complexity of the ubiquitination system demands new chemical tools to investigate networks of protein– protein interactions featuring both non-covalent interactions and multiple covalent protein–protein conjugations. While recombinant methods excel at producing proteins and facilitate the introduction of a single unnatural amino acid, effective protein-based chemical probes require multiple modifications. Our approach provides a platform for the facile construction of a variety of carefully designed Ubc9 variants for studying their interactions with other proteins. Furthermore, uniting multiple ligation technologies to simplify protein synthesis is a long-standing goal of the field; our studies illustrate the advantages and enable a path to sophisticated protein probes.

In principle, this strategy should be applicable to the synthesis of other E2–Ub/Ubl conjugates, as most E2 ligases are less than 200 residues and have a conserved catalytic Cys that could be mutated to Dap by chemical synthesis. Having now established an effective route, confirmed that the Ubc9–SUMO conjugate can be readily formed under SUMOylation conditions, and demonstrate selective trapping of an E3 ligase with an appropriately placed photoaffinity tag, we will pursue the identification of new E3 ligases.

SUPPORTING INFORMATION Supplementary information is available in the online version of the paper. Further information that supports the findings of this study is available from the corresponding author upon request.

AUTHOR INFORMATION Corresponding Author [email protected] or [email protected]

ORCID Yinfeng Zhang: 0000-0002-4408-0175 ACS Paragon Plus Environment


Page 16 of 22

Tsuyoshi Hirota: 0000-003-4876-3608 Shunsuke Oishi: 0000-0001-8807-9072 Jeffrey W. Bode: 0000-0001-8394-8910

Notes The authors declare that they have no competing interests.

ACKNOWLEDGEMENTS We thank our technicians — Yoko Oishi, Mihoko Kato, Chunju Fu, Emi Saita and Yuko Umezawa for their help with solid phase peptide synthesis of peptide segments. We are grateful to Dr. Kinichi Oyama (the Chemical Instruments Facility) for CD measurements. We thank Raphael Hofmann for providing Ubc9 folding condition. This work was supported by JSPS KAKENHI (Grant No. JP16F16344 and JP17H01211) and the Swiss National Science Foundation (Grant No. 200020_169451). ITbM is supported by the World Premier International Research Center Initiative (WPI), Japan. Y. Zhang thanks the JSPS for a doctoral fellowship for foreign researchers.


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Chemical Synthesis of Atomically Tailored SUMO E2 Conjugating

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