From the Department of Biochemistry and the
Institute for Biomolecular Design,
University of Alberta, Edmonton, Alberta T6G 2H7 and the
Department of Microbiology and Immunology, University of
Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada
Received for publication, December 4, 2002, and in revised form, February 4, 2003
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ABSTRACT |
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A heterodimer composed of the catalytically
active ubiquitin-conjugating enzyme hUbc13 and its catalytically
inactive paralogue, hMms2, forms the catalytic core for the synthesis
of an alternative type of multiubiquitin chain where ubiquitin
molecules are tandemly linked to one another through a Lys-63
isopeptide bond. This type of linkage, as opposed to the more typical
Lys-48-linked chains, serves as a non-proteolytic marker of protein
targets involved in error-free post-replicative DNA repair and NF- The post-translational modification of intracellular proteins by
ubiquitination fulfills an important regulatory function in many
cellular pathways. Protein ubiquitination involves a cascade of
enzymatic steps where ubiquitin
(Ub)1 is passed sequentially
as an activated thiolester from a Ub-activating enzyme (E1) to a
Ub-conjugating enzyme (E2), and finally to the protein target with the
help of a Ub protein ligase (E3) (1, 2).
The assembly of poly-Ub chains onto a targeted protein has proven to be
a hallmark of a variety of processes, such as cell cycle control (3),
DNA repair (4), ribosome biogenesis (5), the inflammatory response (6),
endocytosis of cell surface proteins (7), and
NF- The most prevalent and best-documented examples of protein
ubiquitination use the Lys-48-linked chain configuration to target proteins for degradation by the 26 S proteasome (2). Recently, however,
a non-proteolytic ubiquitination pathway has come to light that results
in the substrate-tethered assembly of multi-Ub chains, where Ub
molecules are tandemly linked to one another through Lys-63 (12,
15-19). This pathway plays a key role in error-free DNA
post-replicative repair (20-22), endocytosis (15), and polysome
stability (17) and is an important component of NF- The error-free repair and NF- Insight into the mechanism of Lys-63 chain assembly and its
relationship to structure recently became possible with the
simultaneous determination of both S. cerevisiae (25) and
human (26) Ubc13·Mms2 heterodimer structures by x-ray crystallography
and an NMR-based approach for mapping the protein-protein interactions
within the Ub-bound complex (27). Using the previously determined
assignments for Ub in 1H-15N HSQC NMR
experiments, we were able to footprint the surface of Ub that
interacted with the human hUbc13·hMms2 heterodimer and each of its
subunits in either the thiolester-linked or unlinked forms (27). The
results of this study were consistent with a two-binding site model in
which an "acceptor" molecule of Ub bound non-covalently to hMms2
was positioned in an orientation such that a second Ub molecule that
was linked to hUbc13 as a thiolester could be transferred to Lys-63 of
the accepting Ub molecule. The NMR assignments of both hMms2 and hUbc13
is an obvious prerequisite for footprinting the surfaces of the
heterodimer that interact with both the covalently linked and unlinked
forms of Ub. In the present work we have determined the footprint that
both Ub molecules make on the surface of the hUbc13·hMms2
heterodimer. Taken together with our previous work, a compelling model
is presented for the tetrameric structure that places Lys-63 of the
accepting Ub molecule in catalytic proximity of the C terminus of the
donor Ub molecule.
Protein Expression--
hUbc13 and hMms2 were expressed and
purified as described previously (27) with the following exceptions.
Proteins were expressed in the Escherichia coli strain
BL21(DE3)-RP (Stratagene), and 2-liter cultures were grown at
25 °C to A590 = 0.3 in minimal media
containing 15NH4Cl as the sole nitrogen source
and induced with isopropyl- NMR Spectroscopy--
All NMR spectra were obtained using a
Varian Unity INOVA 600-MHz spectrometer at 30 °C. The
two-dimensional 1H-15N-HSQC NMR spectra were
acquired using the sensitivity-enhanced gradient pulse scheme developed
by Kay and co-workers (28, 29). The 1H and
15N sweep widths were 8000 and 2200 Hz, respectively. A
minimum of 64 transients was collected for each spectrum. All NMR
samples were prepared to include HEPES (50 mM, pH 7.5),
NaCl (75 mM), EDTA (1 mM), dithiothreitol (1 mM), and 2,2-dimethyl-2-silapentane-5-sulfonate (1 mM) in the presence of 9:1
H2O:D2O.
Spectral processing was accomplished with the NMRPipe program (30). The
NMRview program (31) was employed in the assignment of all
two-dimensional 1H-15N-HSQC NMR cross-peaks. To
calculate the total average change in backbone amide
1HN and 15N chemical shifts for
each resonance, the following equation was applied (32),
15N-hMms2 Chemical Shift Perturbation
Experiments--
An initial two-dimensional
1H-15N-HSQC spectrum was acquired for
15N-hMms2 (250 µM) as a point of reference
for subsequent chemical shift perturbation experiments. The spectrum
also served to confirm the proper folding and lack of aggregation of
15N-hMms2.
The interactions between 15N-hMms2 and hUbc13 were examined
by inclusion of a slight excess of unlabeled hUbc13 (300 µM) to the sample described above for
15N-hMms2 alone. The NMR tube was allowed to equilibrate
for 1 h at 30 °C to ensure heterodimerization would proceed to
completion. A two-dimensional 1H-15N-HSQC
spectrum was then acquired for the sample.
Non-covalent interactions between 15N-hMms2 (250 µM) and Ub were examined by including unlabeled UbK48R
(600 µM) into NMR samples in the presence or absence of
unlabeled hUbc13 (300 µM). A two-dimensional 1H-15N-HSQC spectrum was then acquired for each
sample. Chemical shift assignments in the two-dimensional
1H-15N-HSQC spectra were again completed
assuming that the closest cross-peak represented the correct change in
chemical shift. The two-dimensional 1H-15N-HSQC
NMR reference spectrum used when calculating changes caused by Ub
were either (i) 15N-hMms2 alone to examine the changes
cause in hMms2 by itself or (ii) 15N-hMms2·hUbc13 to
probe the changes in chemical shift in hMms2 in the context of the heterodimer.
15N-hUbc13 Chemical Shift Perturbation
Experiments--
As in the case of hMms2, an initial two-dimensional
1H-15N-HSQC NMR spectrum was acquired as a
point of reference and confirmed the proper folding and lack of
aggregation of 15N-hUbc13 (305 µM).
The interactions between 15N-hUbc13 and hMms2 were examined
by inclusion of a slight excess of unlabeled hMms2 (330 µM) to the sample described above for
15N-hUbc13 alone. Sample equilibration and acquisition were
performed as described for the 15N-hMms2 samples.
Thiolester-linked interactions between 15N-hUbc13 (305 µM) and Ub (330 µM) were examined in
situ by inclusion of S. cerevisiae E1 (0.3 µM), ATP (5 mM), and MgCl2 (5 mM) as described previously (27). Addition of hMms2 (330 µM) to this sample allowed for the examination of the
hMms2·15N-hUbc13-Ub species. Studies described
elsewhere (27) have shown that thiolester formation is rapid (minutes)
whereas the formation of Ub conjugate on hUbc13 is slow (hours).
Furthermore, the onset of conjugate formation can be clearly identified
based on the accumulation of new peaks emanating from the mixed
population of Ub species. The two-dimensional
1H-15N-HSQC NMR experiments were therefore
performed between 10 and 120 min after the addition of E1 to minimize
the impact of possible side-reactions. UbK63R was employed as the
Ub species to eliminate the possibility of chain formation
by the hUbc13·hMms2 heterodimer and, hence, to eliminate further
complication of the spectra (27). The two-dimensional
1H-15N-HSQC NMR reference spectra used when
calculating changes caused by Ub in thiolester complexes were either
(i) 15N-hUbc13 alone to examine the changes caused in
hUbc13 by itself or (ii) 15N-hUbc13·hMms2 to probe the
changes in chemical shift in hUbc13 in the context of the heterodimer.
Non-covalent interactions between 15N-hUbc13 and Ub were
detected by including unlabeled UbK63R into NMR samples in the presence or absence of unlabeled hUbc13 under conditions identical to thiolester formation with the exception that E1, ATP, and MgCl2 were
omitted. A two-dimensional 1H-15N-HSQC NMR
spectrum was then acquired for each sample. However, no changes in
15N-hUbc13 cross-peaks were observed in either case, and
therefore no further analysis was performed.
Molecular Modeling--
Molecular modeling of the surfaces of
interaction was accomplished using the BiGGER soft-docking
algorithm (33, 34) using the unbound structures of Ub (target)
(35) and the hUbc13·hMms2 heterodimer (probe) (26). The BiGGER
algorithm systematically searches the complete six-dimensional binding
spaces of both target and probe and then evaluates these solutions in
terms of a global scoring function consisting of geometric
complementarity, electrostatic interactions, desolvation energy, and
the pairwise propensities of amino acid side chains to interact across
molecular interfaces. Docking parameters in this initial search
included a 15° angular step, 5000 maximum solutions, and 300 minimum
atomic contacts. The top 5000 solutions based on global score were then
filtered using the NMR chemical shift perturbation data in the
following manner. First, surface-exposed residues on hMms2 and Ub,
respectively, which produced significant When engaged in catalysis, the hUbc13·hMms2 heterodimer
necessarily exists as part of a tetramer that is composed of the
heterodimer in association with two Ub molecules. One Ub molecule is
linked as a thiolester to the active site of hUbc13 (the donor) while the other Ub molecule interacts non-covalently with hMms2 (the acceptor). Although a high resolution crystallographic structure for
the heterodimer has been determined (26), a crystallographic structure
for the Ub-bound tetramer is unlikely. This conclusion is based both on
the instability of the hUbc13-Ub thiolester bond (36) and the
relatively weak interaction that exists between the acceptor Ub and
hMms2 (Kd ~ 100 µM).2 Based on
these considerations, we have pursued an alternative NMR-based approach
to determine the structure of the hUbc13·hMms2-Ub2 tetramer. The tetramer has three major protein-protein interfaces: 1)
the hMms2-hUbc13 interface, 2) the hMms2-Ub (acceptor) interface and,
3) the hUbc13-Ub (donor) interface. In this and previous studies
(27), we have used 1H-15N HSQC NMR spectroscopy
to observe the chemical shift perturbations that are induced upon
interaction to define the footprint that each protein makes with its partner.
The method that we have chosen here relies upon the comparison of
1H-15N HSQC NMR spectra for each protein
component in an unbound form and bound to its partner. To simplify the
analysis, only one component of the complex is 15N-labeled
in any given experiment. Backbone amide 1HN and
15N chemical shifts are sensitive to a variety of factors,
including hydrogen bonding, electrostatic interactions, and aromatic
ring current effects, to name a few. Therefore, changes to chemical shifts that can result from differences in chemical environment upon
complex formation can be used to identify residues that are either
directly involved at the binding interface or correspond to long range
structural changes.
A necessary precursor to chemical shift mapping is the complete
assignment of backbone amide
1HN-15N cross-peaks in the
1H-15N HSQC NMR spectra for a given component
of the complex. Recently, we have completed the full backbone chemical
shift assignments for both hUbc13 and hMms2 (available upon request).
Each protein exhibits well dispersed and resolved
1H-15N HSQC NMR spectra at 600 MHz, as is shown
for hMms2 (Fig. 1). Furthermore, the
spectra retain these qualities fairly well upon formation of higher
order complexes of up to 42.5 kDa, although the signal-to-noise ratio
is reduced as expected, due to increased linewidths. Chemical shift
assignments in the two-dimensional 1H-15N-HSQC
NMR spectra were made relative to the appropriate reference spectrum,
assuming that the closest shifted cross-peak represented the correct
one. This approach was required due primarily to the lability of
complexes containing thiolester linkages.
B
signal transduction. Using a two-dimensional
1H-15N NMR approach, we have mapped: 1)
the interaction between the subunits of the human Ubc13·Mms2
heterodimer and 2) the interactions between each of the subunits or
heterodimer with a non-covalently bound acceptor ubiquitin or a
thiolester-linked donor ubiquitin. Using these NMR-derived constraints
and an unbiased docking approach, we have assembled the four components
of this catalytic complex into a three-dimensional model that agrees
well with its catalytic function.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B-dependent signal transduction (8). These chains are
synthesized in an E2-dependent reaction where each Ub
within the chain is covalently bound to its neighbor by an isopeptide
bond that links the C terminus to a surface lysine of its
target-proximal Ub partner. Previous observations have demonstrated
that these chains can exist in different configurations that are
defined by the specific lysine residue that links each Ub molecule
within the chain (9-14).
B signal
transduction (18, 19).
B pathways both catalyze the assembly
of Lys-63 chains using a conserved E2 heterodimer, composed of a
catalytically active hUbc13 subunit and a catalytically inactive E2-like subunit termed ubiquitin-conjugating
enzyme variant (UEV). UEV proteins share
significant sequence similarity with other E2s but lack the
characteristic active-site cysteine residue required for thiolester
formation. In the error-free repair pathway of Saccharomyces
cerevisiae, two chromatin-associated RING finger proteins, Rad5
and Rad18, recruit the Ubc13·Mms2 heterodimer and Ubc2 (Rad6) to DNA
(23). In very recent work, Hoege et al. (24) have
demonstrated that a target of this pathway is the yeast proliferating cell nuclear antigen, which is first mono-ubiquitinated through Rad6
and Rad18 and then poly-ubiquitinated by Ubc13·Mms2 in conjunction with Rad5. In NF-
B signal transduction, Traf6, a RING domain E3
protein, functions together with the hUbc13·UEV heterodimer (containing either hUEV1a or the functionally equivalent hMms2) in the
formation of Lys-63-linked poly-Ub chains that are required for the
activation of I
B kinase, a key signal transducer in the NF-
B
pathway (18, 19).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-thiogalactopyranoside (0.4 mM) for an additional 24 h at 25 °C. S. cerevisiae UbK48R, UbK63R, and Uba1 (E1) were expressed and
purified as described previously (27).
where
(Eq. 1)
15N and
1H are the
chemical shift changes in hertz. The average change in total chemical
shift was then calculated for each identified residue, with the
exception of those whose resonances had broadened past detectability in
the two-dimensional 1H-15N-HSQC NMR spectra.
The standard deviation associated with each dataset was also calculated.
total values
upon non-covalent interaction were determined, and the number of atomic
contacts between these two groups within a 5-Å distance cutoff in each
of the top 5000 solutions as determined by global score was evaluated.
The top solution based on these criteria was then accepted as the
"correct" orientation and subsequently underwent minimization using
the INSIGHTII suite of programs. The thiolester-bound Ub placement upon
the heterodimer was then determined in an identical manner using
total values.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Superposition of
1H-15N HSQC NMR spectra of
15N-labeled hMms2, free and in complex with Ub.
1H-15N HSQC NMR spectra resulting from
either 15N-hMms2 (black) or
15N-hMms2 and Ub (red) are overlaid, and a
number of representative backbone cross-peaks, which were affected by
complex formation, are labeled.
Mapping the Heterodimer Interface--
To map the interface
between hUbc13 and hMms2, two heterodimer complexes were prepared
in situ: one containing 15N-hUbc13 with
unlabeled hMms2, and the other containing 15N-hMms2 with
unlabeled hUbc13. The hUbc13·hMms2 heterodimerization (34 kDa)
proceeds efficiently upon equimolar addition of each protein and
results in the formation of a stable complex that remains associated
during high resolution size-exclusion chromatography (27). Residues
whose backbone amide 1H and 15N chemical shifts
exhibited a perturbation upon complex formation were identified and
quantified in terms of the total change in chemical shift,
total. The major
total values upon
heterodimerization for either 15N-hMms2 or
15N-hUbc13 are associated with residues found at the
heterodimer interface (Figs.
2B and
3B), indicating the similarity
of this interface in both the crystalline and solution phases.
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Residues resulting in the greatest effect on total
for interactions within the heterodimer or between the heterodimer and Ub (see below) have been summarized in Fig.
4 according to sequence and secondary
structure. Also shown are the chemical shift indices for each residue
contained in hMms2 and hUbc13, which provide a measure of the deviation
between the observed chemical shifts and their random coil values, and
are indicative of the type of secondary structure (37, 38). A
comparison between secondary structural elements for hMms2 and hUbc13,
determined by x-ray crystallography to those determined from the
chemical shift indices, demonstrate a close correlation between types
of secondary structure determined in the solution and crystalline
states.
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The Non-covalent Interaction between hMms2 and Ub--
Both hMms2
and hUbc13 have each been observed to exist in a monomeric state and as
the heterodimer (23, 27), whereas homodimerization has not been
observed (see "Experimental Procedures"), and therefore an
examination of the interaction between Ub and the hMms2 subunit is of
interest. The chemical shift perturbations that result from the
interaction of 15N-hMms2 subunit with unlabeled acceptor Ub
are shown in Fig. 2A. The greatest effects on
total upon interaction with Ub are observed at the
N-terminal portion of hMms2. Specifically, the affected residues
are located in helix
1 (Glu-20, Gly-22, Lys-24), sections of strand
1 (Val-31, Ser-32, Leu-35), strand
2 (Thr-47, Gly-48, Met-49),
strand
3 (Tyr-63, Leu-65), helix
2 (Leu-119) as well as the loop
joining helix
1 to strand
1 (Val-26, Thr-30). Intermediate effects on
total are found close in sequence to the
greatest changes and include the C-terminal portion of
1 (Gln-23),
sections of
1 (Trp-33),
2 (Trp-46), L2 prior to
3 (Asn-60,
Arg-61),
3 (Val-67, Gly-70), and the loop joining
1 to
1
(Gly-25, Gly-27, Gly-29). Intermediate changes are also found in
2
(Gln-120, Leu-125, Glu-130) and the C terminus (Gly-140, Gln-141).
As expected, many of the residues in hMms2 that exhibit the greatest
backbone amide chemical shift perturbations are located on the surface
of the protein, and contain surface exposed side chains that may be
involved in non-covalent interactions with Ub (Fig.
5A). These residues cluster
onto one face of hMms2, forming three distinct patches. Interestingly,
no significant changes in chemical shift were observed for residues on
the opposite surface of hMms2. The first patch is perpendicular to the
hUbc13·hMms2 interface, and is composed of residues at the C-terminal
end of 1 and the loop that joins
1 to
1 (Glu-20, Glu-21,
Gly-22, Gln-23, Lys-24, Gly-25, Val-26, Gly-27, Gly-29, and Val-31),
portions of
1 (Ser-32, Trp-33, and Leu-35),
2 (Thr-47, Gly-48,
and Met-49), and
3 (Arg-61, Tyr-63, and Leu-65). The second patch is
found at the C-terminal portion of hMms2. Notably, the
total surface area of both these hMms2 patches corresponds well with
the complementary patch on Ub that has previously been demonstrated to
interact with hMms2 (27). Additionally, the combined electrostatic
surface potential of the hMms2 patches is complementary to that found on Ub (Fig. 5C). Interestingly, the third patch involves
hMms2 residues that would normally interact with hUbc13 in the
heterodimer, and include Val-7, Lys-8 (greatest
total), and other N-terminal amino acids of hMms2
(intermediate
total).
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Our previous findings indicated that the Ub contact surface with hMms2
remained largely the same when alone or in complex with hUbc13 (27).
When we next examined the 15N-hMms2-Ub interaction as a
heterodimer with hUbc13 we similarly found that the hMms2 residues that
undergo change on Ub binding closely parallel those of the individual
subunit with some notable exceptions (Fig. 2C). As with
hMms2 alone, many of the major total are found near
the C terminus of
1 (Glu-20, Gln-23), the loop that joins it to
1
(Val-26, Gly-29, Thr-30),
1 (Val-31),
2 (Gly-48, Met-49), and
3 (Arg-61, Tyr-63, Leu-65). Residues with intermediate values of
total are also similar, including
1 (Leu-19,
Glu-21), the loop joining
1 to
1 (Gly-25),
1 (Trp-33, Gly-34),
2 (Thr-47, Gly-52),
3 (Asn-60, Ile-62, Val-67),
2 (Ser-114,
Ile-115, Val-117, Gln-120, Leu-125, Glu-130), and the C terminus
(Gln-141). The backbone amide
1HN-15N HSQC NMR cross-peaks for
three residues (L1 (Asp-37) and
2 (Arg-45, Ile-50)) either
experienced large changes in chemical shift, rendering identification
difficult, or their intensities were severely diminished due to
line-broadening as a result of complex formation.
In contrast to the hMms2 subunit alone, none of the N-terminal residues situated at the heterodimer interface undergo significant change upon Ub binding, whereas significant change is detected within L1 (Asp-38, Asp-40, Met-41, and Arg-45). Notably, the region surrounding the vestigial active site of hMms2 does not appear to play a role in Ub binding. This result clearly distinguishes the hMms2-Ub interaction from other previously reported E2-Ub interactions. The changes in the surface characteristics of the hMms2 component of the heterodimer upon Ub binding are shown in Fig. 5B.
The Interaction between hUbc13 and Thiolester-linked Ub--
The
major changes to the 15N-hUbc13 subunit that result from
thiolester formation with Ub are found in and around the active-site (Cys-87) (Fig. 3A). These include: the active-site cysteine
itself, L4 (Asn-79, Leu-83, Arg-85) to the N-terminal side of Cys-87, the 3-10 helix C-terminal to Cys-87 (Asp-89, Ile-90), the loop preceding helix 3 (Leu-111, Asn-116, Asp-118, Asp-119), and helix
3 (Asp-124, Val-125, Glu-127, Lys-130). Intermediate perturbations of
total are found around and inter-digitated with
the major changes described above. These include: L4 (Met-72, Ile-75,
Tyr-76, His-77), near the active site (Leu-88), the 3-10 helix
(Lys-92, Trp-95, Ser-96, Ala-98), the loop preceding
3 (Ser-113,
Ala-114), and
3 (Ala-126, Thr-131).
Heterodimerization of 15N-hUbc13 with hMms2 results in
somewhat fewer total upon thiolester formation when
compared with the thiolester formed with 15N-hUbc13 alone
(Fig. 3, when comparing C with A). It is noted, however, that a number of cross-peaks in the
1H-15N HSQC NMR spectra of the heterodimer
thiolester remain unassigned due to line broadening or large changes in
chemical shift upon complex formation. The major and intermediate
changes to
total occur within secondary structural
regions, including L4 (Lys-74, Ile-75, Tyr-76, Asn-79, Leu-83, Gly-84,
and Arg-85), the active-site (Cys-87), the 3-10 helix (Leu-88, Asp-89,
Ile-90, Leu-91, and Asp-93), the loop preceding
3 helix (Asn-116,
Asp-118, Leu-121, Ala-122, and Asp-124), and the
3 helix (Val-125,
Ala-126, Trp-129, K130, and Thr-131).
Surfaces involved in the interaction between hUbc13 and its
thiolester-linked Ub were determined by mapping the major
total for the 15N-hUbc13 subunit alone or
in complex with hMm2s onto a surface projection of the hUbc13 crystal
structure (Fig. 5, D and E). In the absence of
hMms2 (Fig. 5D), the greatest effect is found around the
active site (Cys-87) where the majority of affected residues have
solvent-exposed side chains (L4: Arg-70, Leu-83, Arg-85, Ile-86,
Cys-87, and Asp-89;
2: Leu-106, Gln-109, Ala-110, and Leu-111;
3
and preceding loop: Asn-116, Asp-118, Asp-119, Asp-124, Ala-126,
Glu-127, and Lys-130).
From Fig. 5E, it is apparent that hUbc13 exhibits a similar Ub-dependent pattern of backbone amide chemical shift changes when present with hMms2. Significantly, all of the solvent-exposed residues important in thiolester formation present themselves on only one face of the hUbc13 molecule regardless of dimerization state. We conclude from these results that the hUbc13-Ub thiolester interaction is largely unaffected by the presence or absence of hMms2. These results are consistent with our previous NMR experiments demonstrating that both the C-terminal tail and a slightly basic surface on Ub form contacts with hUbc13 within the hUbc13-Ub thiolester regardless of the presence of hMms2 (27).
Modeling the Tetramer--
The soft-docking algorithm BiGGER (33,
34) was employed to generate models for the
Ub2-hUbc13-hMms2 tetramer based on geometric
complementarity, electrostatic interactions, desolvation energy, and
the pairwise propensities of amino acid side chains to interact across
interfaces. Surface residues from the heterodimer (results presented
herein) and Ub (27), which exhibited the greatest change
tototal upon complex formation, were incorporated as
constraints into the BiGGER docking program (see "Experimental Procedures"). The C terminus of the donor Ub was not covalently linked to the active site of hUbc13. The top ten structures based on
these criteria were subsequently averaged, and the resulting structure
was subjected to energy minimization using the INSIGHTII suite of
programs. The final structure of the model is shown in Fig.
6.
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The non-covalent interaction between acceptor Ub and the heterodimer
involves hydrophobic contacts between Ub and hMms2. The surface-exposed
residues of the -sheet of the acceptor Ub, and the loops connecting
strands within the sheet, constitute the contact interface with hMms2,
whereas hMms2 residues that contact the acceptor Ub are found in
1,
1, and
2 and the loops connecting these secondary structural
elements. The hMms2 surface involved in the interaction is located
opposite to the surface containing the vestigial active site. The donor
Ub makes contacts with hUbc13 through C-terminal residues 70-76, as
well as some residues in
1 and
3. The hUbc13 residues that form
contacts with the C terminus of donor Ub are found within the active
site, the loops preceding it, and residues in
2.
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DISCUSSION |
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Together, the NMR chemical shift perturbation results have been
interpreted to produce a model of the tetramer using a molecular docking strategy that is tailored to this NMR-based approach. The
accepting Ub molecule sits on a concave face of hMms2, a distinctive feature of both E2s and UEVs, with its C-terminal tail far removed from
the vestigial active site of hMms2. In combination with hUbc13, the
concave face of hMms2 narrows to form a channel or funnel as it
approaches the active site of hUbc13. The side-chain Lys-63 for the
acceptor Ub lies within this channel, placing the -nitrogen within 3 Å of the sulfur atom contained within the active-site cysteine of
hUbc13. The interaction between the accepting Ub and the heterodimer
buries 2792 Å2 of surface area, a rather large value in
light of our observation that the interaction between the two is weak
(Kd ~ 100 µM).2 The
model likely overestimates the buried surface area of the acceptor Ub,
because the imposed chemical shift restraints force the contact regions
to be maximized and may include residues that are affected indirectly
through induced structural changes in the proteins.
There are two features of the accepting Ub-heterodimer interface that bear directly on its biochemical function. First, the C-terminal tail of the acceptor is neither constrained nor sterically hindered, raising the likelihood that it can serve as the poly-Ub chain anchor in either the free form or when attached to an appropriate protein target. Second, Lys-48 of the acceptor is buried within the protein-protein interface, thereby excluding this residue as a potential site for chain assembly of the canonical type.
The donor Ub interacts exclusively with a hydrophobic concave surface
that narrows to an acidic cleft on hUbc13 and culminating with the
active site cysteine (Fig. 5F). The tail of the donor Ub
lies within the active site cleft of the E2 placing the C-terminal carboxyl carbon of Gly-76, the active site sulfur and the -nitrogen of Lys-63 for the acceptor Ub molecule within 3.5 Å of each other.
In terms of the position and orientation of the components, the model
presented here agrees moderately well with that proposed by VanDemark
et al. (25) for the S. cerevisiae complex. It
differs significantly, however, from the model proposed by Pornillos
et al. (39) who examined the non-covalent interaction
between the human Tsg101 UEV domain and Ub by a similar approach to the
one used here. The structural differences between the Ub-hMms2
interaction and Ub-Tsg101 interaction results from the presence of an
extended -hairpin that links strands 1 and 2 in Tsg101 that
sequester Ub. The fact that this motif is absent in hMms2 illustrates
that UEVs have evolved different strategies for Ub binding.
Our high confidence in this model stems from the NMR-constrained
docking approach used here. The docking algorithm BiGGER is
particularly well suited for these analyses because of its ability to
use NMR chemical shift perturbation results as information to filter
suitable models (33, 34). The BiGGER docking algorithm requires no
information that constrains the orientation of the docking partners
and, therefore, represents a fairly unbiased approach for using NMR
data to model the tetramer interactions. The validation of this
approach lies in the predicted positions of the three atoms involved in
linking the C terminus of the donor Ub molecule to Lys-63 of the
accepting Ub molecule: 1) the cysteine sulfur atom of the hUbc13 active
site, 2) the Gly-76 carboxyl group of the Ub donor molecule, and 3) the
Lys-63 -nitrogen of the accepting Ub molecule. Each of these atoms
is positioned within 3.5 Å of each other (Fig. 6).
The model presented here also agrees well with the findings of a
previous mutagenesis study that used the S. cerevisiae
Ubc13·Mms2 heterodimer (25). A Ubc13 substitution (A110R) located on
the surface of 3, near the center of the predicted interaction
between Ubc13 and the donor Ub, resulted in a 4-fold reduction in the rate of isopeptide bond formation. A Ubc13 substitution (D81A) situated
nearby the predicted position of Lys-63 of the accepting Ub resulted in
a diminished affinity of the acceptor Ub for the heterodimer in
vitro. A Ub substitution (I44A) located in the NMR-derived surface
for the acceptor but not donor, results in reduced binding of Ub to the
acceptor site on Mms2, whereas the interaction with Ubc13 remains
unaffected. Conversely, an Mms2 substitution (E12R) situated near the
heterodimer interface but not predicted by the model to play a role in
acceptor Ub binding does not weaken the interaction of the acceptor Ub
with the heterodimer in vitro (25).
The structure of the hUbc13-Ub thiolester presented here holds features
in common with the models for the Ubc1-Ub thiolester from S. cerevisiae (40) and the human Ubc2b-Ub serine ester (36), each
derived by similar NMR-based approaches. All three E2s employ a common
thiolester-binding motif (L4 around the active site, regions of 2,
and the loop that joins
2 to
3) that constrains the C-terminal
tail similarly among models. In contrast, the folded domain of Ub is
positioned slightly differently on the each of the three E2s (Fig. 4).
These differences are likely explained by properties associated
with catalysis. The tail of the Ub donor must be bound to
the E2 strongly enough to secure its alignment during isopeptide bond
formation with the target, yet weakly enough to assure efficient
transfer and subsequent turnover of the E2. E2 interactions with the
rest of the Ub globular domain are therefore likely to be even weaker
and can be imagined to vary significantly by differences of a few key
surface residues from one E2 to the next.
An examination of high resolution E2 structures has revealed that the active site is part of an unstructured loop (41-47). Our previous and present findings suggest that the interaction of hMms2 with hUbc13 alters the activity of hUbc13 by altering the conformation of the hUbc13 active site. We have previously shown that when hMms2 binds to hUbc13, both the rate of Ub thiolester formation with hUbc13 (reduced 2-fold in the presence of hMms2) and the stability of the resulting thiolester are measurably affected in vitro (27). This observation raises the intriguing possibility that the interaction of an E2 with other proteins could order the loop in a particular conformation, thereby modulating its catalytic activity.
An examination of the chemical shift perturbation data reveals that
there is communication between the acceptor and donor Ub binding sites.
This is reflected by a change in chemical environment at residues that
are known to play a key role in the active-site loop. For instance,
residues in the active-site cleft of hUbc13 (Leu-83, Gly-84, Arg-85,
Leu-88, and Ile-90) show significant values of total
upon dimerization with hMms2. Three of these residues (Leu-83, Gly-84,
and Arg-85) are directly involved in the heterodimer interface, whereas
two of these residues (Leu-88 and Ile-90) are remote from the
interface. In addition, Ub thiolester formation within the heterodimer
results in a significant shift of
total for the
interfacial residues Leu-83 and Arg-85. This observation suggests that
the communication between the heterodimer interface and the active site
is in fact occurring, that is, altering the interface alters the active
site and vice versa. These results appear to be in contrast
with those previously reported for S. cerevisiae
Ubc13·Mms2, for which there appears to be little communication between the dimer interface and the active site. An r.m.s.d. of 0.8 Å for superimposition of all backbone C
atoms between free and
Mms2-bound Ubc13 was reported, with the active site cleft little
changed (25). However, as chemical shift changes cannot be directly
converted into three-dimensional structural changes, further analyses
will be required to establish the extent of similarities and
differences between the human and S. cerevisiae protein complexes.
The arrangement of the four molecules within the tetramer poses no obvious steric problem for the interaction of hUbc13 with its functionally specific E3, Traf6. The interface between hUbc13 and Traf6 can be predicted on the basis of the x-ray crystallographic structure for the E2·E3 complex UbcH7·c-Cbl (48). Both c-Cbl and Traf6 contain E2-binding RING finger domains that share significant sequence identity. Traf6 likely sits on an 11-residue patch of hUbc13, with six residues identical to those employed by UbcH7 in its interaction with c-Cbl (Fig. 6). Notably, none of these residues are involved in forming contacts between Ub and hUbc13.
Despite its small size and highly conserved fold, the E2 core domain
family is apparently the centerpiece for several distinct biochemical
functions that hinge on isopeptide bond formation. These functions
include both target ubiquitination and the synthesis of multi-Ub chains
that differ from one another in configuration. As a consequence of
unknown evolutionary pressure, these proteins have apparently modeled
and remodeled their surfaces with great economy and creativity. The
functional repertoire of protein ubiquitination has been expanded by
the ability of these proteins to interact with common or related
partners in fundamentally different ways. This point is underscored in
part by the present work. The E2 core fold has evolved at least three
relevant and fundamentally different modes of Ub binding. Furthermore,
the juxtaposition of two of these modes, through the interaction of a
catalytically active fold with an inactive fold, provides the
structural basis for Lys-63 multi-Ub chain synthesis.
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ACKNOWLEDGEMENTS |
---|
We thank Susan Smith for secretarial assistance, Linda Saltibus for technical assistance and all of the members of the Ellison and Spyracopoulos laboratories as well as Pascal Mercier and Prof. Brian Sykes for valuable input and assistance. We also thank Prof. Lewis E. Kay for pulse sequences, Deryck Webb for spectrometer maintenance, and Yanni Batsiolas and Robert Boyko for computer expertise.
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FOOTNOTES |
---|
* This work was supported in part by research grants from the National Cancer Institute of Canada (to M. J. E.) and the Alberta Heritage Foundation for Medical Research (AHFMR) (to L. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The online version of this article (available at
http://www.jbc.org) contains Supplemental Figs. 1 and 2.
§ A Natural Sciences and Engineering Research Council of Canada scholar.
¶ A Canadian Institutes of Health Research (CIHR) scholar.
** Funded by CIHR (Grant MOP-53240).
§§ An AHFMR medical scholar. To whom correspondence may be addressed: Dept. of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada. Tel.: 780-492-2417; Fax: 780-492-0886; E-mail: leo.spyracopoulos@ualberta.ca.
¶¶ To whom correspondence may be addressed: Dept. of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada. Tel.: 780-492-6352; Fax: 780-492-0886; E-mail: mike.ellison@ualberta.ca.
Published, JBC Papers in Press, February 4, 2003, DOI 10.1074/jbc.M212353200
2 S. McKenna, J. Hu, T. Moraes, W. Xiao, L. Spyracopoulos, and M. J. Ellison, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: Ub, ubiquitin; UEV, ubiquitin-conjugating enzyme variant; HSQC, heteronuclear single quantum coherence; E1, Ub-activating enzyme; E2, Ub-conjugating enzyme; E3, Ub protein ligase.
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