This journal is c The Royal Society of Chemistry 2012 Mol. BioSyst., 2012, 8, 1723–1729 1723
Cite this: Mol. BioSyst., 2012, 8, 1723–1729
Systematic determinations of SUMOylation activation intermediates and
dynamics by a sensitive and quantitative FRET assay
Yang Songaand Jiayu Liao*
abc
Received 10th November 2011, Accepted 12th March 2012
DOI: 10.1039/c2mb05465e
Ubiquitination and SUMOylation are multi-step cascade reactions, in which small protein
modifiers are activated by E1 activating enzyme, transferred to E2 conjugating enzyme, and
conjugated to substrates mediated by the E3 ligase in vivo. The structural and biochemical bases
for the cascade reactions have been elucidated by several studies. However, the reaction
intermediates and dynamics of these peptide modifiers among the enzymes have not been
completely elucidated. Here we report detailed investigations of SUMOylation dynamics and
interaction switches of SUMO1 among its ligases using FRET technology. These studies show
that, while SUMO1 and the E1 subunit Aos1 or Uba2 have no intrinsic affinity for each other,
the adenylation of SUMO1 carried out by Aos1 requires the presence of Uba2, and subsequently
conformational changes trigger the interaction of SUMO1 and Uba2 for a thioester bond
formation. The reaction intermediates among SUMO1 and its ligases are indirectly revealed by
FRET signals generated by each pair. Furthermore, the transfer of SUMO1 from Uba2 to E2
enzyme, Ubc9, depends on the formation of a thioester bond between SUMO1 and Ubc9, and
requires non-covalent interaction between Ubc9 and Uba2, but not between Ubc9 and SUMO1.
These interaction switches provide the physical and biochemical bases for the SUMO activation
and a transfer cascade required for SUMO activation.
Introduction
Post-translational modification by Ubiquitin (Ub) and
Ubiquitin-like (Ubls) proteins, such as SUMO and NEDD8,
plays critical roles in many physiological and pathological
processes in eukaryotes, such as signal transductions, genome
integrity maintenance, protein transport, cell cycles, immunity
and tumorigenesis.1–7 The conjugation of Ubiquitin, SUMO
and NEDD8 to target substrates shares an evolutionary
conserved but distinct enzyme cascade that involves the sequential
actions of E1, E2 and E3 ligases. For example, after maturation
of SUMO from pre-SUMO by the cleavage processing of SENPs,
SUMO is conjugated to its substrate by a multienzyme-catalyzed
cascade involving E1, E2 and E3 ligases (Fig. 1). The heterodimer
E1, Aos1/Uba2, activates Ub/Ubls in two major steps. First,
Aos1 catalyzes adenylation of the SUMO at its C-terminus using
ATP, with the help ofMg2+. Second, a catalytic cysteine of Uba2
forms a covalent thioester bond with the C-terminus of SUMOby
replacing the adenylate group.8,9 Then, a transthiolation reaction
takes place between E1 and E2, resulting in the formation of a
Fig. 1 SUMOylation and deSUMOylation cascade. The pre-SUMO is
processed into a mature form by a cleavage of C-terminal peptide by
SENPs. The matured SUMO is then activated by heterodimer E1 ligase,
Aos1/Uba2, in two steps, adenylation and thioester bond formation. The
E1-activated SUMO is transferred to E2, Ubc9, to form thioester with
Ubc9 in a transthiolation reaction. With the help of E3 ligases, such as
PIAS, the SUMO is conjugated to the substrate at the Lys residue.
aDepartment of Bioengineering, Bourns College of Engineering,University of California at Riverside, 900 University Avenue,Riverside, CA 92521, USA. E-mail: [email protected];Fax: +1 951-827-6416; Tel: +1 951-827-6240
b The Center for Bioengineering Research, Bourns College ofEngineering, University of California at Riverside,900 University Avenue, Riverside, CA 92521, USA
c Institute for Integrative Genome Biology, Bourns College ofEngineering, University of California at Riverside,900 University Avenue, Riverside, CA 92521, USA
MolecularBioSystems
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1724 Mol. BioSyst., 2012, 8, 1723–1729 This journal is c The Royal Society of Chemistry 2012
thioester bond between Ubc9 catalytic cysteine and SUMO.8 In
combination with a variety of E3s, such as PIAS family of
ligase, which are generally considered to determine substrate
specificity in vivo, SUMO is finally conjugated to lysine residues
of substrates.1–3 The SUMOylated substrate can also be
deSUMOylated by the same family of protease, SENPs (Fig. 1).
Several studies have revealed insights into the biochemical
and structural bases of Ub/Ubl with E1 and E2 enzyme
recognition and successive steps in substrate conjugation.10,11
First, biochemical studies show that E1 and E2s have different
affinities for each other in their free or Ub/Ubl thioester-linked
form. Free E1 has low affinity for E2s, while double Ub-loaded
E1 binds E2s at higher affinity.11,12 These differential affinities
between different enzyme forms may contribute to the Ub/Ubl
progression along the E1–E2–E3 cascade. Another study suggests
that there is an intrinsic non-covalent interaction between
SUMO and its E2, Ubc9, and this non-covalent interaction
is required for polySUMOylation of substrates but not for
monoSUMOylation.13 Second, structures of E1 of NEDD8,
SUMO or Ubiquitin alone or in complexes with substrate,
respectively, display similar domain orientations.14–17 Interestingly,
the structure of an activation complex of NEDD8 containing
heterodimeric E1, double-loaded NEDD8 (one bound non-
covalently in the adenylation packet and one linked to an E1
catalytic Cys domain via the thioester bond), and the NEDD8
E2 conjugating enzyme Ubc12 bound to the UFD domain
revealed two E2 binding sites on E1.17 One binding site comes
from conformational change of E1 after binding by NEDD8,
and the other binding site comes from E1 and NEDD8
directly. The speculation that a conformational change of E1
upon the binding of Ubl may provide a mechanism to bring E1
and E2 catalytic cysteine domains to a close proximity has been
further demonstrated by a recent structural study of SUMO–E1
adenylate and tetrahedral intermediate analogues.18 A striking
conformational change of E1 has been observed after the thioester
bond formation between SUMO and E1. This conformational
change may potentially bring two catalytic Cys domains of E1
and E2 within 3.5 A which will facilitate the transfer of SUMO
from E1 to E2.18 Although much knowledge about the Ub/Ubl
handoff among ligases has been gained from these studies, the
reaction intermediates and dynamics in the sequential formation
of transient complexes along the E1–E2–E3 conjugation cascade
have not been observed in a real dynamic mode. In addition,
mechanistic mechanisms of Ubl activation and switches between
E1 and E2 ligases have not been completely determined. Here,
we determined the intermediates and reaction dynamics of
SUMO1 with its E1s and E2 ligases and revealed the mechanistic
mechanisms of SUMO activation and switches among ligases
using the sensitive FRET technology.
Results
Design of the FRET-based system for monitoring the
SUMOylation cascade and FRET index definition
To observe and determine intermediates resulting from non-
covalent or covalent conjugation of SUMO1 with E1 or E2,
dynamics during Ub/Ubl adenylation, thioester bond formations,
and SUMO transfer between E1 and E2 in real time, we used a
Forster Resonance Energy Transfer (FRET) technique, which
involves the FRET pair CyPet- and YPet-labeled SUMO1,
Aos1 (E1 subunit), Uba2 (E1 subunit), Ubc9 (E2 subunit), the
C-terminal domain (420–587) of RanGAP1 (RanGAP1C,
substrate) and their mutant proteins which lose different
activities in the SUMOylation cascade19–21 (Fig. 2A). When
a CyPet-tagged protein is mixed with a YPet-tagged protein
in vitro, interaction between two proteins can draw the FRET
pair close to each other and result in two emission peaks at 475 nm
and 530 nm when the mixture is excited at 414 nm. We recently
developed a new spectrum analysis method for FRET signal
analysis.22 In this method, the emission signal at 530 nm emission
(Emtotal) is divided into three components: direct emission from
the donor CyPet (CyPet [cont]), direct emission from the acceptor
YPet (YPet [cont]), and FRET emission from YPet (EmFRET)
(Fig. 2B). The direct emission of CyPet and YPet at 530 nm
(Ex = 414 nm) is proportional to their emission at 475 nm
(Ex = 414 nm) (FLDD) and 530 nm (Ex = 475 nm) (FLAA)
(Fig. 2C) by a constant ratio, namely x and y, which are 0.378 and
0.026, respectively. Therefore, we can calculate the CyPet (cont)
and YPet (cont), and subtract them from Emtotal to derive
EmFRET. In order to use FRET to monitor the change of
protein–protein interaction status, we define the FRET index r
to be the ratio of FRET emission (EmFRET) to the emission at
530 nm (Ex = 475 nm) (FLAA) (Fig. 2D). Since FLAA is
contributed by direct emission of YPet, the FRET index can
indicate the relative percentage of YPet that receives energy from
excited CyPet, and therefore the FRET index can be used as
quantitative measurement of the FRET signal.22 When we mix
the proteins involved in the SUMOylation process and initiate the
conjugation cascade, the formation of protein complexes will lead
to an increase of the FRET index whereas disruption of protein
complexes will result in a decrease of the FRET index.
Revealing intermediates of SUMO/E1 ligase and dynamics
The structures of E1 identified residues from both Aos1 and
Uba2 that contact Mg2+/ATP and the Ub/Ubl C-terminal
Fig. 2 Design of the FRET assay for protein interactions in SUMOyla-
tion and FRET index definition. (A) CyPet-tagged SUMO1 interaction
YPet-tagged SUMO ligases and substrate. (B) Analysis of fluorescence
emission spectra of the mixtures of CyPet and YPet-tagged proteins when
excited at 414 nm. (C) The emission of FRET acceptor YPet proteins when
excited at 475 nm. (D) Definition of the FRET index.
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glycine.14–16,23 We speculated that there would be intrinsic
interactions between SUMO1 and its E1, Aos1/Uba2. We first
determined the interactions and intermediates of SUMO1 with
its heterodimeric E1, which initiate the SUMOylation cascade
and catalyze two critical reactions, adenylation with the help
of ATP and thioester bond formation with one of the E1
subunits, Uba2. To our surprise, when SUMO1 and Aos1
were fused with CyPet and YPet, respectively, the FRET assay
showed that there was no intrinsic interaction between Aos1
and SUMO1 even in the presence of ATP (Fig. 3A). Only a
weak interaction was observed when only Uba2, but not ATP,
was added. However, a strong interaction was observed after
both Uba2 and ATP were added (Fig. 3A). This suggests that
SUMO recognition and/or adenylation of SUMO peptide by
Aos1 cannot take place by itself. Similar results were observed
when SUMO1 and Uba2 were labeled with the CyPet and
YPet pair, respectively. In consistence with the above results,
none or only weak interaction was observed in the absence or
presence of Aos1 alone. However, a strong interaction and an
interaction intermediate were observed only after Aos1 and
ATP were added to the reaction mixture either at the starting
point or after a period of waiting (Fig. 3B). These results
strongly suggest that there are no intrinsic affinities among
SUMO1 and its E1, and a strong interaction is induced
by Aos1 and Uba2 after they bind to ATP and catalyze
the adenylation reaction and thioester bond formation. Inter-
estingly, the FRET peak between SUMO1 and Uba2 decays
much faster than that between SUMO1 and Aos1, indicating
that either the SUMO1–Uba2 thioester bond may not be very
stable, or there could be a conformation change of the
SUMO1–Uba2 intermediate that increases the distance between
the FRET pair. These results demonstrate that striking
dynamic movements occur between E1 and SUMO1 during
adenylation and thioester bond formation, which are consistent
with the recent results of E1 and SUMO1 intermediate crystal
complexes.18
After adenylation, the cysteine from the catalytic domain of
Uba2 will form a thioester bond with the C-terminal Gly of
SUMO1 peptide, forming a high energy covalent intermediate
before it is transferred to the cysteine catalytic domain of E2.
We then investigated whether the interaction between SUMO1
and Uba2 was due to the covalent thioester intermediate
formation or just non-covalent interactions induced by E1
remodelling. A mutant of Uba2 with its catalytic cysteine
changed to serine (C173S) was generated. The interaction
between Uba2 (C173S) mutant and SUMO1 was strong and
more stable than its wild type counterpart (Fig. 3C), indicating
that the formation of thioester covalent intermediate does not
contribute significantly to the interaction observed above. This is
in major contrast to the SUMO1 interaction with its E2, Ubc9,
where thioester covalent interaction contributes significantly to
the interaction (see below). Compared to the transient interaction
between wild type Uba2 and SUMO1, the more stable
complex between Uba2 (C173S) mutant and SUMO1 suggests
that SUMO1 binds to the Aos1/Uba2 complex after being
activated by Aos1 and will not be released if the thioester bond
is not formed.
Wild-type activation complexes containing an E1, a Ubl and
an E2 are not stable, because thioester-bonding Ubl is readily
passed from the E1’s catalytic cysteine to the catalytic cysteine
of E2, and the intermediate of Ubl and E2 is not stable either,
because it is readily passed to substrates. Therefore, an
activation complex trapping strategy was used to determine
the crystal structure of the NEDD8 pathway: APPBP1–UBA3
two NEDD8s (NEDD8 (T) thioester-linked to UBA3 and
NEDD8 (A) noncovalently associated at the adenylation
active site), Mg2+/ATP, and a catalytic cysteine-to-alanine
(C111A) mutant of Ubc12. It would be interesting to determine
how SUMO1–Uba2 active complex forms in the presence of
Ubc9. Consistent with the previous result, the SUMO1–Uba2
active complex reached a peak concentration in the presence of
Aos1 and ATP, and then decayed to the background fluorescence
of two separated proteins over thirty minutes (Fig. 3D, filled
circles). In the presence of Ubc9, the FRET signal of SUMO1–
Uba2 active complex did not reach the peak in contrast to that
it does in the absence of Ubc9, but gradually increased over
time, suggesting a quick transfer of thioester–SUMO1 to Ubc9
(Fig. 3D, open circles). Interestingly, in the presence of catalytic
deficient mutant of Ubc9 (C93S), the interaction between
SUMO1 and Uba2 became more stable, but eventually decayed
(Fig. 3D, filled squares). These results suggest that Ubc9 stabilizes
the SUMO1–Uba2 active complex and two intermediates are
formed in the presence of wild type or catalytic cysteine mutant
E2, suggesting a critical role of E2’s catalytic cysteine in inducing
the transfer of SUMO1 from Uba2.
Determinations of two levels of interaction between SUMO and
E2 ligase, Ubc9
SUMO1 has been shown to interact strongly with Ubc9 in a
non-covalent manner and form a thioester bond with catalytic
cysteine of Ubc9.13 To determine whether these two interactions
can be distinguished by FRET technology, we analyzed the
interaction between SUMO1 and Ubc9, which were tagged with
CyPet and YPet, respectively. In the absence of E1, SUMO1
Fig. 3 Intermediate formation and stability of SUMO1 with E1
ligase subunits, Aos1 and Uba2. (A) Specific binding of Aos1 to
SUMO1 requires its dimerization with Uba2 and ATP. (B) Specific
binding of SUMO1 to Uba2 requires its adenylation mediated by Aos1
and ATP. (C) Thioester bond formation is not required for the
interaction between SUMO1 and Uba2. (D) Ubc9 stabilizes the
Uba2 and SUMO1 intermediate complex.
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can interact with Ubc9 non-covalently. To demonstrate that
this is a truly non-covalent interaction, we added increasing
amounts of non-tagged SUMO1 to the mixture of CyPet–
SUMO1 and YPet–Ubc9. The FRET signal gradually decreased
with the increasing amounts of SUMO1, indicating that the
complex was disrupted by the competitor (Fig. 4A). In the presence
of E1 (Aos1 and Uba2) and ATP, SUMO1 interacted with and
formed a thioester-mediated covalent complex with Ubc9, shown
as a peak of FRET index which can be clearly distinguished from
the baseline indicating the non-covalent interaction of SUMO1 and
Ubc9 (Fig. 4B, filled circles). This thioester-mediated complex
could not form in the absence of ATP and E1. In contrast, the
catalytic cysteine mutant of Ubc9 (C93S), which could not
form a thioester bond with SUMO1, could only interact with
SUMO1 and form a non-covalent complex (Fig. 4B filled
squares). Furthermore, the thioester-mediated covalent complex
can only be formed in the presence of E1 and E2, but not the
catalytic cysteine mutant Uba2 (Fig. 4C). These results suggest
that the coordination of enzyme catalyzed cascade reaction is
required and two levels of interaction between Ubc9 and
SUMO1 can be distinguished by the FRET assay.
However, it remains unclear whether the intrinsic affinity
between Ubc9 and SUMO1 is required for SUMO1 peptide
transfer from E1 to E2, or Ubc9–SUMO thioester bond
formation, or whether it is required merely to maintain the
stable covalent active complex of SUMO1 and Ubc9. To
assess the roles of non-covalent interaction between Ubc9
and SUMO1, mutations of Ubc9 or SUMO1 that would
abolish the non-covalent interaction with each other could
provide tools addressing this question. The previous studies
have shown that the mutation of R17E on Ubc9 abolished its
interaction activity with both E1 and SUMO1 and the mutation of
E67R orG68Y on SUMO1 either completely or partially abolished
the interaction with Ubc9.13 We therefore generated a mutant
Ubc9 (R17E) and a double mutant of SUMO1 (E67R/G68Y).
The mutant Ubc9 (R17E) and the catalytic cysteine mutant of
Ubc9 were obtained from bacterial cells (Fig. 5A). Both Ubc9
(R17E) and SUMO1 (E67R/G68Y) lost their intrinsic affinity
to its partners, respectively (Fig. 5B). Most strikingly, the
SUMO1 (E67R/G68Y) still could interact with and form a
strong thioester covalent intermediate with Ubc9, as good, if
not better, as wild-type SUMO1 (Fig. 5C, filled triangles). In
comparison, SUMO1 could only form a non-covalent complex
with the catalytic cysteine mutant of Ubc9 (C93S) or in the absence
of ATP (Fig. 5C, filled squares and empty circles). Notably,
SUMO1 (E67R/G68Y) formed the active covalent complex with
Ubc9 directly from free status, while wild-type SUMO1 started
from a non-covalent intermediate stage as evidenced by the FRET
index around 0.1, a signal for the non-covalent complex (Fig. 5C,
filled triangles). Not too surprisingly, Ubc9 (R17E) failed to form a
thioester covalent intermediate with SUMO1 (Fig. 5C, empty
squares). This probably is due to its inability to interact with
Uba2, suggesting a critical role of the interaction between Uba2
and Ubc9 for the transfer of SUMO1 from E1 to E2. These results
indicate that the non-covalent interaction between SUMO1 and
Ubc9 is not required for its switch from E1 to E2 or the formation
of thioester covalent intermediate, while as the interaction of Ubc9
and Uba2 is critical for the switch.
SUMO conjugation requires thioester bond switch and physical
interaction between E1 and E2
We next turned our attention to investigate the significances of
these molecular interactions and intermediates of SUMO1/E1
or SUMO1/E2 in the SUMOylation cascade as substrate
conjugation. We were able to determine the final SUMO
conjugation product by determining the FRET signal from
CyPet–SUMO1 and YPet–RanGAP1c, indicating E1’s and E2’s
substrate tolerance of tagged SUMO in the cascade reaction
(Fig. 6A, filled circles). Interestingly, SUMO1 (E67R/G68Y)
could be conjugated to RanGAP1c in the presence of E1, E2
Fig. 4 Non-covalent and covalent intermediates of SUMO1 with E2
ligase, Ubc9. (A) SUMO1 non-covalently interacts with Ubc9 in the
absence of E1. (B) FRET reveals non-covalent and covalent inter-
actions between SUMO1 and Ubc9 in the presence of E1 and ATP.
(C) Thioester bond-mediated interaction of SUMO1 and Ubc9
requires E1, ATP, and the formation of a high-energy bond between
SUMO1 and Uba2.
Fig. 5 Non-covalent interaction between Ubc9 and SUMO1 is not
required for its transfer from E1 to E2. (A) Protein gel of mutant Ubc9
and wild-type Ubc9 expressed in bacterial cell. (B) Mutation of
SUMO1 at E67R/G68Y or mutation of Ubc9 at R17E disrupts the
non-covalent interaction between SUMO1 and Ubc9. (C) Non-covalent
interaction of Ubc9 with Uba2 but not SUMO1 is required to transfer
SUMO1 from E1 to E2.
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and ATP (Fig. 6A, filled squares). This result further suggests
that the non-covalent interaction between SUMO1 and Ubc9 is
not required for its activation by E2, and thereafter, conjugation
to substrates. The catalytic cysteine mutations of Uba2 (C173S)
and Ubc9 (C93S), respectively, abolish the SUMO1 activation
and substrate conjugation (Fig. 6B), indicating the key roles of
thioester intermediate formation between SUMO1 and Uba2 or
Ubc9 for its final conjugation to the substrate. It has been
suggested that the interaction between E1 and E2 is important
for SUMO–Ubc9 thioester formation.24 We went further to
investigate whether this interaction is required for the SUMO
activation cycle. Several mutations and mutation combinations
in Ubc9, including R17E, R13E/F22A, R13E/H20D/F22A,
were chosen based on their co-crystal structure and previous
studies.13 All these mutations abolished the SUMO conjugation
to RanGAP1c (Fig. 5C). Wild-type of SUMO1 and mutant
SUMO1 (E67R/G68Y) were able to conjugate to the substrate,
while all the mutations in E1 and E2 ligases, including Uba2
(C173S), Ubc9 (C93S), Ubc9 (R17E), Ubc9 (R13E/F22A), and
Ubc9 (R13E/H20D/F22A), failed to conjugate SUMO1 to
RanGAP1c (Fig. 6C). These results suggest that intermediates
of SUMO1/E1 and SUMO1/E2, and interactions between Uba2
and Ubc9, are critical for SUMO1 activation along the E1–E2
pathway and thereafter final substrate conjugation.
Discussion
Our results revealed the thioester intermediates and interaction
affinity switches of SUMO1-E1 and -E2 enzymes using dynamic
FRET technology in real time. In order for the SUMOylation
cascade to proceed, sequential formation of transient complexes
during E1–E2–E3 conjugation cascades is mediated by distinct
transient interactions and catalytic activities by the different
players.25 The reaction dynamics revealed by the FRET signal
have shown marked both conformational changes and interactive
partner switches that accompany adenylation and thioester bond
formation. The FRET signal between SUMO1 and Aos1 is only
observed in the presence of another E1 subunit, Uba2 and ATP,
indicating that heterodimer formation of E1s is absolutely
required for the adenylation reaction to take place. The
interaction of SUMO1 and Aos1 needs a signal directly from
Uba2, suggesting a conformational change of Aos1 in the
presence of Uba2. The gradual decrease of FRET signal
between CyPet–SUMO1 and YPet–Uba2 suggests that the
thioester intermediate of SUMO1 and Uba2, in the presence of
Aos1 and ATP, is not stable (Fig. 2B and C). Interestingly, a
stable complex is observed between SUMO1 and the Uba2
cysteine mutant (C173S), which would not form a thioester
intermediate with SUMO1 (Fig. 2C), indicating the thioester
bond formation is required for the leaving of SUMO1 from
Uba2. This difference between the two SUMO1/Uba2 complexes
also suggests that a conformational change of Uba2 takes place
after the SUMO1/Uba2 thioester intermediate formation and
this conformational change is required for the leaving of SUMO1
in the presence or absence of E2, Ubc9 (Fig. 1C and D). This
conclusion is also supported by the fact that intrinsic inter-
action affinity between SUMO1 and Ubc9 is not required for
the switch of SUMO1 from Uba2 to Ubc9 (Fig. 4B and C).
This switch between E1 and E2 seems solely promoted by
conformational changes of E1 triggered by thioester inter-
mediate formation and proximity of Ubc9 Cysteine to
SUMO1 C-terminus. These results are consistent with recent
co-crystal studies of both SUMO1 with its E1 and NEDD8
with its E1s and E2.17,18
Our studies also demonstrate an interesting example that
the tracking of FRET signals can reveal molecular interactions
and dynamics of the reaction cascade involving multiple
proteins. To the best of our knowledge, this is the first time
that the Ubl-ligase thioester intermediate and dynamics of
transfer can be observed in real time. The quantitative estimation
of different affinities for the transient complexes in the conjugation
cascade is hard to determine using other approaches because
the thioester intermediates are chemically not stable. They
could be captured by co-crystals using non-hydrolysable adenylate
mimics.18 But this approach cannot reveal intermediates and
reaction dynamics in real time. The E1 heterodimer-required
interaction with SUMO1 is uniquely detected by FRET
technology. We also observed that the affinity of SUMO1–
Ubc9 thioester is much higher than that of SUMO1–Uba2
thioester (FRET index in Fig. 2 and 5). This may serve the
mechanism of the SUMO peptide switch from E1 to E2. The
observations of reaction intermediates using FRET technology
provide another novel way to track dynamics/kinetics and
sequential order of an enzymatic reaction, and the FRET-
based reaction analysis technology can also be applied to
multicomponent-involved biochemical reactions.
The intermediate detections and dynamics analysis of the
SUMOylation cascade provides frameworks to understand not
only the SUMO activation cycle but also other Ub/Ubls cascades.26
Whether all the Ubls utilize the same mechanisms for activations or
not needs to be determined. In our studies, we have not included the
E3 ligase because in in vitro studies, E1s and E2 seem sufficient for
substrate conjugation.More complete understanding of the detailed
molecular mechanisms underlying the functional interactions
among Ubls, its E3 ligase and the substrate circuitry awaits
future experimentation.
Fig. 6 The stable substrate conjugate requires thioester bond switch
between E1 and E2. (A) SUMO1 conjugation to RanGAP1 does not
require the non-covalent interaction between SUMO1 and Ubc9.
(B) SUMO1 conjugation to RanGAP1 requires thioester bond
formations between SUMO1 and Uba2 or Ubc9. (C) Western blotting
confirms the inability of mutant Ubc9 to mediate the SUMO conjuga-
tion of RanGAP1.
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Materials and methods
DNA constructs
The open reading frames of CyPet and YPet were amplified by
using primers containing NheI or SalI sites, respectively, and
the open reading frames of SUMO1, Aos1, Uba2, Ubc9 and
the C-terminal domain (420–587) of RanGAP1 (RanGAP1C)
were amplified by PCR using primers containing SalI or NotI
sites, respectively. All PCR products were cloned into the
pCRII-TOPO vector (Invitrogen). The fragments encoding
SUMO1, RanGAP1C and SUMO ligases were digested with
SalI/NotI and the purified fragments were inserted into
pCRII-CyPet or pCRII-YPet plasmids which had been linearized
by SalI and NotI. After the sequences were confirmed, the
cDNAs encoding fusion proteins were digested with NheI/NotI
and cloned into the NheI/NotI sites of the pET28(b) vector
(Novagen). To get non-fluorescent tagged proteins, the open
reading frames of SUMO1 and SUMO enzymes were extracted
by SalI/NotI digestion and cloned into the SalI/NotI sites of the
pET28(b) vector. The mutant forms of SUMO1, Uba2 and Ubc9
were created by PCR site-directed mutagenesis and cloned into
the pET28(b) vector, following the protocol described above.
Protein expression and purification
BL21 (DE3) Escherichia coli (E. coli) cells were transformed
with pET28 vectors encoding SUMO1, Aos1, Uba2, Ubc9,
RanGAP1C, their mutants, and fluorescent protein-labeled
versions. The transformed bacteria were inoculated in 2 � YT
medium and the expression of polyhistidine-tagged recombinant
proteins was induced with 0.1 mM IPTG at 25 1C overnight.
The recombinant proteins were purified by Ni2+-NTA agarose
beads (QIAGEN) followed by gel filtration HPLC using the
Superdex75 10/300 GL column on a HPLC purification system
(GE Healthcare, AKTAt purifier). Protein purity was later
confirmed by SDS-PAGE followed by Coomassie blue staining
and their concentrations were determined by the Coomassie Plus
Protein Assay (Pierce).
FRET measurements
After the proteins labeled by CyPet or YPet are mixed in the
presence or absence of other protein members or cofactors,
their fluorescence was determined by the fluorescent plate
reader Flexstation II384 (Molecular Devices) in a 384-well
plate over a period of time. The emission intensities at three
wavelengths were collected: 475 nm and 530 nm at an excitation
wavelength of 414 nm, and 530 nm at an excitation wavelength
of 475 nm. After the emission intensities were corrected by
subtraction of background fluorescence from the plate, the
FRET indexes (see below) at different time points were calculated
and compared.
Protein interaction and SUMOylation assay
All reagents were purchased from Research Products Interna-
tional Corp. unless specified. For the interaction of SUMO1
with E1, 20 pmole CyPet–SUMO1 and 20 pmole YPet–Aos1
or YPet–Uba2 were mixed in the presence or absence of
20 pmole Uba2 or Aos1, respectively, in a buffered solution
containing 50 mM Tris–HCl (pH 7.4), 1 mM DTT and 4 mM
MgCl2 with a total volume of 36 mL. Samples were incubated
at 37 1C and fluorescence emission was monitored after 4 mL10 mM ATP or H2O was added at time zero. For the mutant
Uba2 interaction assay, 20 pmole CyPet–SUMO1, 20 pmole
wild type or mutant YPet–Uba2 and 10 pmole Aos1 were
mixed in the buffer mentioned above in a total volume of 36 mL.For the interaction of SUMO1 and Uba2 in the presence of
Ubc9, 10 pmole CyPet–SUMO1, 10 pmole YPet–Uba2 and
10 pmole Aos1 were mixed in the presence or absence of
100 pmole wild type or mutant Ubc9 in the buffer mentioned
above. Samples were measured by the FRET index after 4 mL of
10 mM ATP was added.
For the interaction disruption assay of SUMO1 and Ubc9,
0.4 mg CyPet–SUMO1 and 0.5 mg YPet–Ubc9 were mixed in
20 mL PBS. 0 to 2 mg nonfluorescence-labeled SUMO1 was
added to the mixture and the FRET indexes were determined.
In the interaction assay of SUMO1 and Ubc9, 20 pmole
CyPet–SUMO1, 20 pmole wild type or mutant YPet–Ubc9,
10 pmole Aos1 and 20 pmole Uba2 were mixed in a buffered
solution containing 50 mM Tris–HCl (pH 7.4), 1 mM DTT
and 4 mM MgCl2 with a total volume of 36 mL. For the
mutant Uba2 assay, 10 pmole wild type or mutant Uba2 was
added. Samples were incubated at 37 1C and fluorescence
emission was monitored after 4 mL 10 mM ATP or H2O was
added at time zero.
Five mg of purified wild type of Ubc9, Ubc9 (C35S) and
Ubc9 (R21E) proteins were loaded onto 10% SDS-PAGE,
separated by electrophoresis and stained by Coomassie blue
staining.
For the interaction assays of wild type or mutant SUMO1
and mutant or wild type Ubc9, respectively, 20 pmole wild
type or mutant CyPet–SUMO1 and 20 pmole wild type or mutant
YPet–Ubc9 were mixed in the presence or absence of 10 pmole
Aos1 and 10 pmole Uba2 in the buffer mentioned above.
For the SUMO1 conjugation assay of mutant SUMO1
peptide, 0.4 mg wild type or mutant CyPet–SUMO1, 0.5 mgYPet–RanGAP1C, 0.05 mg Aos1, 0.1 mg Uba2 and 0.5 mgUbc9 were mixed in the buffer mentioned above, containing
50 mM Tris–HCl (pH 7.4), 1 mMDTT, and 4 mMMgCl2 with
a total volume of 36 mL. Samples were incubated at 37 1C and
fluorescence emission was monitored after 4 mL 10 mM ATP
or H2O was added at time zero. Samples were measured by
FRET index at different time points.
For the SUMO1 conjugation assay of mutant Ubc9 or
Uba2, SUMO1 peptide 0.4 mg CyPet–SUMO1, 0.5 mgYPet–RanGAP1C, 0.05 mg Aos1 were mixed in the presence
or absence of 0.1 mg wild type or mutant Uba2 and 0.5 mg wildtype or mutant Ubc9 in the buffer mentioned above.
The wild type or mutant form of 0.5 mg SUMO1, 0.05 mgFLAG-tagged RanGAP1C, 0.05 mg Aos1, 0.1 mg Uba2 and
0.5 mg Ubc9 were mixed in a buffered solution containing
50 mM Tris–HCl (pH 7.4), 1 mM DTT, 4 mM MgCl2 and
1 mM ATP with a total volume of 40 mL. Samples were
incubated at 37 1C for 1 hour. After they were separated by
10% SDS-PAGE and transferred to a PVDF membrane, the
RanGAP1C proteins were recognized by incubation with
mouse anti-FLAG antibody (Sigma Aldrich) followed by
HRP-labeled goat anti-mouse IgG antibody (Sigma Aldrich).
The chemiluminescence was developed using the Supersignals
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This journal is c The Royal Society of Chemistry 2012 Mol. BioSyst., 2012, 8, 1723–1729 1729
West Dura Extended Duration Substrate (Thermo Scientific) and
captured by the Biospectrums imaging system (UVP, LLC).
Competing interests statement
The authors declare that they have no competing financial interests.
Acknowledgements
We are very grateful to Prof. Victor Rodgers for the valuable
advices on reaction rates. We would like to thank Prof. Jerome
Schultz and Dr Elizabeth Gillard for careful comments on our
manuscript. We thank Prof. Xuemei Chen for allowing us to
use the HPLC system. We thank the members in Liao’s group
for very close collaborative work and help with the work. This
work was supported by the National Institutes of Health
Grant (AI076504) to J.L.
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