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Molecular Cell
Article
Allosteric Activation of E2-RING Finger-MediatedUbiquitylation by a Structurally DefinedSpecific E2-Binding Region of gp78Ranabir Das,1 Jennifer Mariano,2 Yien Che Tsai,2,4 Ravi C. Kalathur,3,4 Zlatka Kostova,2 Jess Li,1 Sergey G. Tarasov,1
Robert L. McFeeters,1 Amanda S. Altieri,1 Xinhua Ji,3 R. Andrew Byrd,1,* and Allan M. Weissman2,*1Structural Biophysics Laboratory2Laboratory of Protein Dynamics and Signaling3Macromolecular Crystallography Laboratory
Center for Cancer Research, National Cancer Institute, Frederick, MD 21702-1201, USA4These authors contributed equally to this work
*Correspondence: [email protected] (R.A.B.), [email protected] (A.M.W.)DOI 10.1016/j.molcel.2009.05.010
SUMMARY
The activity of RING finger ubiquitin ligases (E3) isdependent on their ability to facilitate transfer ofubiquitin from ubiquitin-conjugating enzymes (E2)to substrates. The G2BR domain within the E3 gp78binds selectively and with high affinity to the E2Ube2g2. Through structural and functional analyses,we determine that this occurs on a region of Ube2g2distinct from binding sites for ubiquitin-activatingenzyme (E1) and RING fingers. Binding to the G2BRresults in conformational changes in Ube2g2 thataffect ubiquitin loading. The Ube2g2:G2BR interac-tion also causes an �50-fold increase in affinitybetween the E2 and RING finger. This results in mark-edly increased ubiquitylation by Ube2g2 and thegp78 RING finger. The significance of this G2BReffect is underscored by enhanced ubiquitylationobserved when Ube2g2 is paired with other RINGfinger E3s. These findings uncover a mechanismwhereby allosteric effects on an E2 enhance E2-RING finger interactions and, consequently, ubiqui-tylation.
INTRODUCTION
Ubiquitylation occurs as the result of a hierarchical multienzyme
process. Ubiquitin-activating enzyme (E1) activates ubiquitin,
forming a thiolester linkage between the active site Cys of E1
and the C terminus of ubiquitin. Ubiquitin is transferred to the
conserved active site Cys of ubiquitin-conjugating enzymes
(E2), of which there are more than 30 in mammals. E2s bind to
specific ubiquitin-protein ligases (E3s), which mediate the trans-
fer of ubiquitin to primary amines on substrates or to growing
chains of ubiquitin (polyubiquitin or multiubiquitin). In many
cases, the E3s also undergo auto- or self-ubiquitylation.
There are more than 500 E3s in mammals that can be divided
into two major classes. The HECT E3s include �30 E3s and are
674 Molecular Cell 34, 674–685, June 26, 2009 ª2009 Elsevier Inc.
characterized by a conserved 350 amino acid catalytic domain.
HECT E3s are catalytic intermediates in substrate ubiquitylation
as a consequence of the transthiolation of ubiquitin from bound
E2 to their conserved catalytic Cys (Fang and Weissman, 2004).
RING finger and RING finger-like E3s collectively represent the
large majority of E3s. The RING finger is a compact Zn-binding
domain of 40 to 100 amino acids. These domains generally
bind E2s with low affinity and do not form catalytic intermediates
with ubiquitin. It is generally believed that RING fingers either
position E2�Ub to facilitate transfer to substrates or function
as allosteric activators of E2�Ub (Lorick et al., 2005; Ozkan
et al., 2005). Binding sites on E2s for RING fingers, HECT
domains, and E1 all overlap. Therefore, E2s must dissociate
from ligase domains to reload with ubiquitin (Huang et al.,
2005; Eletr et al., 2005). Interestingly, there are a few examples
in which other regions, either within a multisubunit ubiquitin
ligase complex or a single subunit E3, bind specific E2s through
generally uncharacterized interactions (Madura et al., 1993; Ha-
takeyama et al., 1997; Wu et al., 2002; Biederer et al., 1997; Chen
et al., 2006). This could increase the availability of E2�Ub and
theoretically allow for reloading of E2 with ubiquitin without
dissociation from the E3.
Ubiquitylation and proteasomal degradation perform critical
functions in degradation of misfolded, unassembled, and highly
regulated proteins from the endoplasmic reticulum (ER). ER-
associated degradation (ERAD) is a multistep, highly coordi-
nated process (Nakatsukasa and Brodsky, 2008). In mammals,
there are at least five known ER membrane-spanning ERAD
E3s. Among these is gp78, also known as the human tumor au-
tocrine motility factor receptor (AMFR). gp78 is implicated in
degradation of T cell antigen receptor subunits, regulatory
proteins in lipid metabolism (Kostova et al., 2007), CFTRD508
(Morito et al., 2008), and the metastasis suppressor KAI1
(CD82) (Tsai et al., 2007).
gp78 has a complex domain structure for a single subunit E3.
In addition to its RING finger, it has at least three more C-terminal
domains in its extended cytoplasmic tail (Figure 1A). Each of
these is implicated in ubiquitylation and degradation of ERAD
substrates. They include a ubiquitin-binding CUE (coupling of
ubiquitin conjugation to ERAD) domain and a C-terminal binding
Molecular Cell
gp78 Ube2g2-Binding Region: Structure and Function
Figure 1. The gp78 G2BR and Its Interactions with Ube2g2 by NMR
(A) Schematic representation of gp78 in the ER membrane (left). To the right is a linear representation of gp78 cytoplasmic tail with amino acids (corresponding to
the entire human gp78) indicated. Peptides used in this study corresponding to the G2BR, specific mutations/truncations, and a scrambled (Scr) peptide control
are shown below. Mutations are indicated in lowercase.
(B) Overlay of 15N-HSQC NMR spectra of Ube2g2 in free (red) and G2BR-bound (blue) form.
(C) The contact residues observed in an intermolecular NOESY spectrum of Ube2g2:G2BR are painted blue on the free Ube2g2 structure (PDB entry 2CYX).
Region of E1 and E3 binding based on other E2-E3 pairs is indicated by the bracket. The active site Cys (C89) is in red.
(D) 15N-HSQC of isotopically labeled G2BR in free form (red) and bound to Ube2g2 (blue).
site for p97. Unique to gp78 is a high-affinity binding site for its
cognate E2, Ube2g2. This Ube2g2-binding region (G2BR) is
required for the function of gp78 in cells (Chen et al., 2006).
We now report that the G2BR binds Ube2g2 through an
extended interface distinct from sites of RING finger and E1
binding. This results in subtle changes in the Ube2g2 core that
are manifested in functional alterations in loading with ubiquitin
and a marked increase in affinity for the gp78 RING finger, which
is reflected in enhanced ubiquitylation.
RESULTS
The G2BR Is a High-Affinity Binding Site for Ube2g2To evaluate the interaction between the gp78 G2BR (Figure 1A)
and Ube2g2, we employed isothermal titration calorimetry (ITC)
(Figure S1 available online). This confirmed the direct, high-
affinity interaction of Ube2g2 with a 27 amino acid G2BR
peptide. The 1:1 complex of E2 and G2BR has a dissociation
constant (Kd) of 21 (± 4) nM (Table 1). The reaction is exothermic,
and the significant free energy change (DG = �43.72 kJ mol�1)
implies a very stable complex. The decrease in entropy reveals
that part of the complex is losing some degree of freedom and
is likely folding into a regular structure.
‘‘Backside Binding’’ to Ube2g2 Induces G2BR FoldingTo identify the binding surface between Ube2g2 and G2BR, we
examined the interaction by monitoring the NMR spectra of each
molecule independently. First, the Ube2g2 preparation was vali-
dated by confirming the resonance assignments for Ube2g2
compared to a previous study (Briggman et al., 2005). A
secondary structure analysis based on chemical shifts (Wishart
and Sykes, 1994) combined with distance information from
a 15N-edited NOESY-HSQC spectrum confirmed that our
Ube2g2 preparation matched the reported crystal structure
(Arai et al., 2006). Titration of G2BR into isotopically labeled
Ube2g2 (Figure 1B) yielded slow-exchange NMR spectra and
indicated a Kd << 1 mM, consistent with the ITC data. An unex-
pectedly large number of the Ube2g2 HN-N peaks shifted in
the presence of G2BR, requiring reassignment. The 13C-edited
HSQC spectra of the methyl resonances of Ile, Leu, and Val resi-
dues (data not shown) indicated a localized binding site, which
correlates with the largest shifts observed in Figure 1B. The
Molecular Cell 34, 674–685, June 26, 2009 ª2009 Elsevier Inc. 675
Molecular Cell
gp78 Ube2g2-Binding Region: Structure and Function
Table 1. Dissociation Constants of E2-E3 Interactions
Complex Kd DH (kJ mol�1) DS (J K�1 mol�1T�1) Method NMR Exchange Timescale
Ube2g2:G2BR 21 (± 4) nM �56.3 (± 0.01) �41.8 (± 0.04) ITC slow
Ube2g2:G2BRDN 740 (± 110) nM �9.80 (± 0.40) 83.6 (± 1.43) ITC slow
Ube2g2:G2BRDC 192 (± 22) mM n.d.a n.d. NMR fast
Ube2g2:G2BRM4-1 55 (± 18) mM n.d. n.d. NMR fast
Ube2g2:G2BRM4-2 9.5 (± 4) mM n.d. n.d. NMR fast
Ube2g2:gp78-RING 144 (± 10) mM n.d. n.d. NMR fast
(Ube2g2:G2BRDN):gp78-RING 29 (± 5) mM n.d. n.d. NMR fast
(Ube2g2:G2BR):gp78-RING 3 (± 1) mM n.d. n.d. NMR fasta n.d. = not determined.
secondary structure based on chemical shifts of Ube2g2:G2BR
was identical to free Ube2g2, and NOESY spectra of
Ube2g2:G2BR exhibited equivalent patterns of connectivities
between HN-HN, HN-methyl, and methyl-methyl protons
compared to free Ube2g2, implying that the overall structure of
Ube2g2 did not change significantly.
The binding surface was determined by chemical shift
mapping on Ube2g2 and by intermolecular NOESY experiments.
Chemical shift mapping for Ube2g2 indicates the primary binding
site is on b strands b1–b3 (Figure S2). In addition, the C-terminal
regions of helices a1 and a4 are perturbed by G2BR binding.
Intermolecular NOESY cross-peaks were observed between
G2BR and the methyl and amide protons of Ube2g2 (Figure S3)
that correspond to the surface indicated in blue on unbound
Ube2g2 in Figure 1C. These NOEs refined the contact surface
to residues V25, A26, E31, E38, L40–M42, E45, E50, F51–V53,
V159, L163, and L165. This surface includes the hydrophobic
patch formed by V25, A26, L40–M42, and F51–V53 that is mostly
conserved in other E2s (Hamilton et al., 2001; Moraes et al., 2001;
Brzovic et al., 2006). The G2BR-binding site also includes nega-
tively charged residues flanking the hydrophobic core. From the
perspective of the G2BR, 15N-HSQC spectra (Figure 1D) of
a biosynthetically prepared G2BR exhibited poor chemical shift
dispersion, consistent with a random coil conformation. The
backbone chemical shifts were assigned in the free G2BR
peptide, and, except for indications of a nascent 4 residue helical
turn at V583–K586, the peptide lacks any regular secondary
structure. The spectrum of G2BR in the presence of Ube2g2
exhibits a dramatic increase in spectral dispersion and reso-
nance linewidths consistent with a 1:1 complex of Ube2g2:G2BR
(Figure 1D). This suggests that the G2BR must fold into a compact
regular secondary structure utilizing all 27 residues to fit on the
defined E2-binding site. Based on this, we redefine the G2BR
as amino acids 574–600 of gp78 instead of 579–600 as originally
defined (Chen et al., 2006).
The Ube2g2:G2BR Crystal Structure Reveals DistantChanges that Impact E2 Loading of UbiquitinThe Ube2g2:G2BR complex was crystallized, forming rod-
shaped crystals (0.1 mm 3 0.3 mm 3 0.1 mm). These diffracted
to 1.8 A resolution (Table 2). The structure shows that the
Ube2g2 backbone is largely unchanged in the presence of
G2BR (Figure 2A), as predicted by NMR measurements. The
676 Molecular Cell 34, 674–685, June 26, 2009 ª2009 Elsevier Inc.
conserved secondary structural elements include a1 = T4–L18,
b1 = G23–P28, b2 = E36–M42, b3 = V53–S59, b4 = K70–F73,
310 helix = S91–L93, a2 = V116–A128, a3 = V138–D146, and
a4 = R148–L163. Strands b1–b4 form an antiparallel b sheet
(Figures 2A and 2B). A dynamic region in the b4a2 loop is present
between residues H94 and W110. This corresponds to an acidic
extension (aa 96–108) that, in mammals, is limited to Ube2g2 and
two other E2s (Ube2g1 and Ube2r1 [Cdc34 in yeast]). The back-
bone root-mean-square deviation (rmsd) between the free and
bound Ube2g2 is 1.8 A over all residues and decreases to 0.9 A
if residues 96–108 in the b4a2 loop are excluded.
Key structuralchanges wereobserved inUbe2g2near the active
site upon binding the G2BR. Access to the active site Cys (C89) is
via a channel flanked on either side by b4a2 loop and the a2a3 loop
(Figure 2A). The b4a2 loop was observed to exhibit dynamic
behavior for aa 96–108 in the crystal structure of free Ube2g2
(Arai et al., 2006). In fact, there were three molecules in the asym-
metric unit, and the major difference between the molecules was
the conformation of the b4a2 and a2a3 loops (Figure 3A). These
conformations are ordered; however, they exhibit higher B factors
than the rest of the structure (Figure S2). The dynamic region of the
b4a2 loop contains a short 310 helix and generally extends away
from the protein in the three conformations in the free form;
however, in the Ube2g2:G2BR complex presented here, the 310
helix is gone, and a region of the loop (P100–Y103) orients back
toward C89. The multiple conformations of these loops, which
appear to shift to a new average state in the Ube2g2:G2BR
complex,and thehighB factors in the crystal structuresareconsis-
tentwithdynamicaveraging assuggested bychemical shift effects
observed in solution (Figure S2). The present structure may repre-
senta low-energypopulation of the average, inwhich the proximity
of Y103 and C89 changes from 20.8 A in unbound Ube2g2 to 3.8 A
in the complex. Inaddition, the a2a3 loop (comprising N131–G135)
approachesC89 in the complex.This inwardconformation isstabi-
lized in the 1.8 A crystal structure by a new network of water-medi-
ated hydrogen bonding, including Y103, C89, and Y83, as well as
a network including E133 and S91. The structural modifications of
these two loops decrease accessibility around the active site
(compare Figure 1C to 4B and Figure 3B to 3C). These changes
are accompanied by an altered orientation of the C89 side chain
from pointing toward the b4a2 loop in the unbound formtopointing
away from the loop (�80% probability) with bound G2BR
(Figure 2A and compare Figure 3B to 3C).
Molecular Cell
gp78 Ube2g2-Binding Region: Structure and Function
The multiple states surrounding the active site seen in the
crystal structures and the shift in conformational averaging in
the Ube2g2:G2BR complex in solution suggest that these
changes may have an impact on interactions with E1 or ubiquitin
bound to C89. To further evaluate this, we compared the struc-
tures of Ube2g2 and Ube2g2:G2BR with two reported E2-Ub
structures (Hamilton et al., 2001; Eddins et al., 2006). Superpo-
sition of free Ube2g2 with the E2 component of Ubc1-Ub, which
forms K48 ubiquitin chains (Chen and Pickart, 1990), and
Ubc13-MMs2-Ub, which forms K63 chains (Deng et al., 2000)
(Figure 3B), reveals that the C termini of either of the two bound
ubiquitins can readily be accommodated in the channel sur-
rounding C89. However, superposition of Ube2g2:G2BR with
the same two structures (Figure 3C) indicates that the area
around the active site becomes occluded due to rearrange-
ments of residues M101–Y103 and E133–G135 (within the
b4a2 loop and a2a3 loop, respectively), resulting in steric
clashes with the C termini of the two ubiquitins. In Figure 3C,
Table 2. Data Collection and Refinement Statistics
Data Collection
Space group P212121
Cell dimensions
a, b, c (A) 48.92, 60.15, 61.64
a, b, g (�) 90, 90, 90
Resolution (A) 50–1.8 (1.86–1.8)a
Number of observations 105668 (5851)
Number of unique reflections 16657 (1272)bRmerge 7.8 (43.7)
I/s 22.4 (2.7)
Completeness (%) 95.4 (74.2)
Redundancy 6.4 (4.6)
Refinement
Resolution (A) 43–1.8 (1.86–1.8)cRworking (%) 19.3 (23.1)dRfree (%) 24.1 (27.4)
Number of atoms/B factors (A2)
Protein 1318/33.1
Peptide 260/34.4
Water 164/40.7
Rmsd
Bond lengths (A) 0.005
Bond angles (�) 0.92
Ramachandran plot
Most favored region (%) 87.7
Additional allowed region (%) 12.3
Generously allowed region (%) 0
Disallowed region (%) 0a Values in parentheses are for the highest resolution cell.b Rmerge = Sj(I�<I>)j/s(I), wherein I is the observed intensity.c R factor = ShkljjFoj � jFcjj / ShkljFoj, calculated from working data set.d Rfree is calculated from 5% of data randomly chosen and not included in
refinement.
Y103 is buried and is not visible. These structure-based models
predict direct consequences for loading of G2BR-bound
Ube2g2 with ubiquitin from E1. This was tested by comparing
ubiquitin loading of Ube2g2 in the presence of either a control
scrambled peptide (Scr) (Figure 1A) or the G2BR. The G2BR
resulted in a marked decrease in the rate of E2 loading at
30�C (Figure 3D) and almost no detectable loading at 12�C
(Figure S4). Deletion of the extended acidic portion of the
b4a2 loop (aa 96–108; Ube2g2D96–108), which is the major
contributor to steric crowding around the active site, decreased
the G2BR effect by almost 75%, and G2BR binding was retained
(Figures 3D and S5).
Ube2g2-Bound G2BR Forms an a Helix with ExtensiveContacts across the Backside of the E2The crystal structure of Ube2g2:G2BR demonstrates that the
G2BR assumes a completely a-helical fold in the 1:1 complex
and binds to a surface comprising the b sheet (b1–b3) and the
C termini of a1 and a4 (Figure 2A), which is consistent with the
solution NMR data (Figure 1C). The buried surface at the inter-
face of Ube2g2 and G2BR is �1950 A2. The entire peptide is
well ordered, and there is an extensive network of contacts
between the G2BR and Ube2g2, comprising both hydrophobic
and ionic or hydrogen-bonding contacts (Figures 2C, 2D, and
2E and Table S1). The contact network is consistent with the
thermodynamic data for formation of this complex and suggests
that the recognition is highly specific. In the G2BR a helix, there is
evidence for 24 hydrogen bonds between all i and i+4 pairs,
beginning with the Ser 574 carbonyl oxygen to Arg 578 NH and
running through Arg 596 carbonyl oxygen to Lys 600 NH. As
amino acids 574–600 extend completely across the backside
of Ube2g2, this suggests that extensions beyond this region
do not interact with Ube2g2. This is in agreement with previous
data (Chen et al., 2006) and NMR studies of aa 574–643 of
gp78 (unpublished data).
The presence of extensive contacts between Ube2g2 and
G2BR suggests that single-point mutations will not disrupt
binding or activity. The initial characterization of the G2BR
(Chen et al., 2006) indicated that the N- and C-terminal ends of
the G2BR were each critical both for binding Ube2g2 and for
the cellular function of gp78. To examine this observation, we as-
sessed binding of G2BRDN and G2BRDC (Figure 1A) to Ube2g2.
G2BRDC binds Ube2g2 with relatively weak affinity (Kd = 192
[± 22] mM) (Table 1). The affinity between G2BRDN and Ube2g2
is significantly stronger (Kd = 740 [± 110] nM) but nonetheless
exhibits an �35-fold decrease in affinity compared to wild-type
G2BR. This confirms that the N- and C-terminal regions of
G2BR both contribute directly to the high-affinity binding
observed with the intact domain. Binding of either G2BRDN or
G2BRDC to Ube2g2 causes a subset of the observed chemical
shift changes seen with the G2BR, consistent with their pre-
dicted contacts (data not shown).
On the basis of the Ube2g2:G2BR structure, we designed two
peptides with four mutations each to assess the most important
interactions, G2BRM4-1 and G2BRM4-2 (Figure 1A). These
peptides were 15N labeled and examined by NMR (Figure S6).
The 15N-HSQC spectra of both peptides indicated random coil
conformations characteristic of wild-type G2BR. Titration of
Molecular Cell 34, 674–685, June 26, 2009 ª2009 Elsevier Inc. 677
Molecular Cell
gp78 Ube2g2-Binding Region: Structure and Function
G2BRM4-1 and G2BRM4-2 with unlabeled Ube2g2 yielded Kds of
55 (± 18) mM and 9.5 (± 4) mM, respectively (Table 1). These
findings, combined with the data for G2BRDN and G2BRDC,
underscore the highly distributed nature of the contacts in
Ube2g2:G2BR binding.
The G2BR Enhances the Affinity of Ube2g2for the gp78 RING FingerThe positioning of the Ube2g2:G2BR interface on the backside
of Ube2g2 suggests that this interaction should not present
a direct steric impediment to the interaction of Ube2g2 with
Figure 2. Crystal Structure of the
Ube2g2:G2BR Complex
(A) Ribbon representation of the superimposed
Ube2g2:G2BR complex with free Ube2g2 (PDB
entry 2CYX). G2BR is cyan, and Ube2g2 is light
green in the complex. Free Ube2g2 is shown in
orange.
(B) Linear representation of the secondary struc-
ture and binding regions of Ube2g2. a helices are
represented by open rectangles; b strands, by
filled arrows; and the active site Cys, by a red
dot. Binding regions are indicated below the
sequence line as RING finger (magenta) and
G2BR (green). The position of Ube2g2’s extended
dynamic loop is indicated in orange.
(C) Hydrophobic side chains of G2BR (light blue,
residues in black) lock into the hydrophobic
surface of Ube2g2 (dark green, residues in yellow).
(D) Intermolecular hydrogen bonds and salt
bridges between G2BR and Ube2g2 are shown.
Side chains of G2BR are shown in blue; residues,
in black; the Ube2g2 contact side chains and resi-
dues, in red. The P21 and I24 side chains of
Ubeg2g2 were not displayed because the interac-
tion is through backbone hydrogen bonds.
(E) Contacts between Ube2g2 (green) and G2BR
(blue) indicating residues involved in hydrogen
bonds, salt bridges, and hydrophobic interactions.
Black lines link each residue to its reciprocal
contact, and X denotes G2BR residues that do
not have direct contacts in the interface.
the gp78 RING finger-binding site (Zheng
et al., 2000; Brzovic et al., 2003; Domi-
nguez et al., 2004) (Figure 2A). To eval-
uate possible allosteric effects of G2BR
on RING finger binding, the interaction
of isotopically labeled Ube2g2 with
gp78 RING finger was monitored by
NMR (Figure 4). The gp78 RING finger-
binding interface on Ube2g2 consists of
the N-terminal end of a1, the b3b4 loop
(F62–P69), and part of the b4a2 loop
(W110–S115) (see Figures 2A and 2B
for secondary structure elements). The
solution structure of the gp78 RING
finger is similar to other RING finger
structures (R.D. and R.A.B., unpublished
data), and a reverse labeling experiment using isotopically
labeled gp78 RING finger confirmed that the gp78 RING finger
side of the binding surface is similar to other RING finger:E2
interactions. The interaction exhibited fast exchange
(Figure 4A) and a Kd of 144 (± 10) mM (Table 1 and Figure S7).
This is consistent with the low affinity of many RING finger:E2
interactions (Lorick et al., 2005; Christensen et al., 2007). The
affinity of the gp78 RING finger for the Ube2g2:G2BR complex
was also determined. While the binding interface was
unchanged and remained in fast exchange, strikingly, the Kd
decreased by �48-fold to 3 (± 1) mM (Table 1 and Figure S7).
678 Molecular Cell 34, 674–685, June 26, 2009 ª2009 Elsevier Inc.
Molecular Cell
gp78 Ube2g2-Binding Region: Structure and Function
Separate binding experiments confirmed that the G2BR and
RING finger do not directly interact in the gp78 RING finger:
Ube2g2:G2BR complex (data not shown). The allosteric rela-
tionship of Ube2g2:gp78 RING finger affinity to Ube2g2:G2BR
association was tested by measuring the affinity of gp78 RING
finger for Ube2g2 in the presence of G2BRDN, which exhibits
an affinity for Ube2g2 intermediate between the full G2BR and
G2BRDC. The measured Kd was 29 (± 5) mM (Table 1), which
indicates that even a smaller fragment of G2BR has effects
that translate through Ube2g2 and increase its affinity for the
gp78 RING finger.
Figure 3. G2BR-Induced Structural Changes
in Ube2g2 Occlude the Area around the
Active Site and Correlate with Ubiquitin
Loading
(A) Superposition of active site region (full struc-
tures shown in small inset) for four Ube2g2 mole-
cules (yellow, cyan, and magenta from the ligand-
free Ube2g2 in PDB entry 2CYX and green in
Ube2g2:G2BR) showing the conformational flexi-
bility of the b4a2 loop.
(B and C) Images showing the surface rendering of
Ube2g2 and Ube2g2:G2BR combined with the
potential orientation of ubiquitin chains, based on
known E2-Ub structures. Ube2g2 was superim-
posed onto the E2 coordinates of two published
E2-Ub structures: Ubc1-Ub (PDB entry 1FTX,
rmsd = 1.8A) and Ubc13-MMs2-Ub (PDB entry
2GMI, rmsd = 1.4A). We display only the surface
of Ube2g2 and the C-terminal end of the ubiquitin
molecule (in ball and stick form) for Ubc1-Ub
(blue) and Ubc13-MMs2-Ub (magenta). In (B), the
free Ube2g2 structure is rotated relative to the
orientation of Figure 1C by 180� about the vertical
axis and zoomed in on the active site. The active
site Cys (C89) backbone is yellow, and its side
chain is red. Some residues of Ube2g2 that play
a role in the allosteric change are labeled in green.
R74 of both ubiquitin tails are indicated; the
C-terminal diglycine approaches C89. In (C), the
G2BR-bound form of Ube2g2 is shown in an iden-
tical orientation as in (B).
(D) 35S-labeled Ube2g2 or Ube2g2D96–108 gener-
ated by in vitro translation in E. coli lysate was incu-
bated with 100 nM E1 and ubiquitin-lacking lysines
(Ub K0), so as to avoid formation of polyubiquitin
chains. The formation of thiolester-linked Ube2g2
(E2�Ub) was assessed at 30�C with saturating
(4 mM) G2BR peptide or a control ‘‘scrambled’’
peptide (Scr). Shown is the average of two experi-
ments for each condition. Rate constant, K, and
95% confidence index are shown for each condi-
tion.
G2BR Enhances RINGFinger-Dependent Ubiquitylationby Ube2g2gp78 is a substrate for its own ubiquityla-
tion in cells (Fang et al., 2001; Chen et al.,
2006). To begin to assess the effect of the
G2BR on ubiquitylation, we employed
a GST fusion of the cytoplasmic domain of gp78 (GST-gp78C)
in an autoubiquitylation assay (Lorick et al., 1999). Ube2g2 was
provided in�5-fold molar excess relative to glutathione Sephar-
ose-immobilized GST-gp78C, and ubiquitylation of bead-bound
material was assessed. Ubiquitylation was totally dependent on
an intact gp78 RING finger (Figure 5A), and mutations of two
key residues in the G2BR (L582S/L589S; gp78CL582,589S) signif-
icantly decreased ubiquitylation. A truncated form of GST-gp78C
lacking the G2BR (GST-gp78CD577–643) also demonstrated
markedly diminished ubiquitylation (Figure 5B), consistent with
G2BR-dependent increased affinity of Ube2g2 for the gp78
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gp78 Ube2g2-Binding Region: Structure and Function
RING finger. However, when G2BR peptide was provided in
trans, there was a marked increase in ubiquitylation of truncated
gp78 (Figure 5B). This rescue of activity was not observed with
the G2BRDN, G2BRDC, G2BRM4-1, or G2BRM4-2 peptides (Figures
5C and 5D). To determine whether the G2BR peptide also
enhances ubiquitylation of heterologous proteins, we took
advantage of gp78’s function as an E4 in ubiquitylating proteins
already modified with a single ubiquitin (Morito et al., 2008). A
fusion protein of ubiquitin and GFP with a C-terminal His6 tag
(Ub-GFP-His6) was ubiquitylated by GST-gp78CD577–643
as assessed using Flag-tagged ubiquitin (Figure 5E). As with
gp78 autoubiquitylation, ubiquitylation of Ub-GFP-His6 by GST-
gp78CD577–643 was substantially increased in the presence of
G2BR. Thus, the effect of the G2BR applies to ubiquitylation of
a heterologous substrate, as well as to autoubiquitylation.
To evaluate the effect of G2BR on substrate ubiquitylation
using the full-length gp78 cytoplasmic tail, gp78C and
gp78CL582,589S were compared. gp78C resulted in easily detect-
able, high-molecular weight ubiquitylated Ub-GFP-His6, which
was not evident with gp78CL582,589S (Figure 5F). As
gp78CL582,589S showed some persistent autoubiquitylation in
Figure 5A, these reactions were carried out by combining
G2BR mutants with double mutations of Ube2g2 in key contact
points (Figure 5G; see Figure 2 and Table 1 for Ube2g2:G2BR
contacts). Though none of the double mutants of Ube2g2
substantially decreased ubiquitylation with either gp78C or
a single mutation (gp78CL582S), combining any of these E2
mutants with gp78CL582,589S resulted in a complete loss of ubiq-
uitylation. Collectively, these findings demonstrate the functional
importance of structurally defined key contact residues in the
Ube2g2-G2BR interface.
G2BR Enhances Ubiquitylation by Ube2g2with Other RING FingersUbe2g2 also functions with other RING finger ERAD E3s (unpub-
lished data). Two of these, HsHRD1 and Trc8, are implicated in
human disease (Kostova et al., 2007). These E3s also function,
at least in vitro, with members of the UbcH5 (Ube2d1–3) E2
family (Lorick et al., 1999; Nadav et al., 2003; Kikkert et al.,
2004). HsHRD1 and Trc8 lack identifiable regions that are anal-
ogous to the G2BR. Therefore, we asked whether the enhanced
ubiquitylation observed with the G2BR was unique to the gp78
RING finger. Addition of the G2BR to ubiquitylation reactions
utilizing GST fusions of the C-terminal RING finger-containing
regions of either HsHRD1 or Trc8 resulted in a marked increase
in RING finger-dependent autoubiquitylation (Figure 5H, top,
lanes 5 and 8, and Figure S8). Ubiquitylation mediated by these
E3s, as well as by GST-gp78CD577–643, was not increased by the
G2BR when UbcH5B was used as the E2 (Figure 5H, bottom).
Similarly, critical mutations in the G2BR do not decrease ubiqui-
tylation by UbcH5B (Figure S9). Thus, changes in the ERAD E2,
Ube2g2, induced by binding of the G2BR at a site distant from
where this E2 interacts with RING fingers, enhances ubiquityla-
tion with at least three different mammalian E3s. This indicates
both the specificity of the G2BR for Ube2g2 and its apparent
effect on increasing productive interactions between Ube2g2
and RING fingers.
Increased Affinity of Ube2g2 for the gp78 RING FingerAccounts for the Enhanced UbiquitylationTo further evaluate the effect of the G2BR, we assessed the rate
of discharge of ubiquitin from Ube2g2, based on a previously
used approach (Petroski and Deshaies, 2005), in the presence
of saturating amounts of G2BR or an equal concentration of
Scr. In these reactions, acceptors for ubiquitin are other proteins
in the reaction mix, including bacterial proteins and free ubiquitin
added in excess. In the absence of the RING finger, discharge of
ubiquitin from Ube2g2�Ub was slow and unaffected by G2BR
peptide (Figure 5I). In the presence of 8 mM gp78 RING finger,
loss of Ub-bound Ube2g2 was markedly accelerated. This was
significantly increased by saturating amounts of G2BR
(Figure 5I). We wished to determine whether this apparent
increased rate of discharge can be accounted for by an
increased population of Ube2g2:RING finger due to the
Figure 4. Interactions between gp78 RING
Finger and Ube2g2:G2BR Complex
(A) Ube2g2:G2BR complex was titrated with gp78
RING finger followed by acquisition of 15N-HSQC
spectra of isotopically labeled Ubeg2g2 at each
titration point. 15N chemical shifts in parts per
million (ppm) are indicated on the y axis, and 1H
chemical shifts in ppm are on the x axis. Three of
the affected residues of Ube2g2 (N19, L66, and
V113) are shown. The peaks shift from the free
(blue) form toward the bound form (magenta)
with addition of gp78 RING finger. The dissocia-
tion constant was determined by fitting the peak
positions against ligand: protein concentrations.
(B) The surface representation of Ube2g2:G2BR
shows the G2BR interface (blue), RING interface
(magenta), and active site cysteine (red).
680 Molecular Cell 34, 674–685, June 26, 2009 ª2009 Elsevier Inc.
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gp78 Ube2g2-Binding Region: Structure and Function
presence of G2BR (<5% Ube2g2 bound with Scr; >50% bound
with G2BR) or by as yet unknown allosteric effects on the
Ube2g2:gp78 RING finger complex mediated by the G2BR.
Therefore, experiments were carried out with concentrations of
gp78 RING finger at which approximately equal amounts of
RING finger are bound to Ube2g2. Under these conditions, no
significant increase in discharge rate was observed in the pres-
ence of G2BR (Figure 5J). Thus, the G2BR-mediated increase
in affinity of Ube2g2 for the RING finger provides a molecular
basis for the accelerated discharge of ubiquitin from
Ube2g2�Ub and, by extension, for the observed enhancement
of ubiquitylation.
DISCUSSION
Structural Basis for the High-Affinity G2BR Bindingto Ube2g2The NMR and X-ray crystallographic determination of the G2BR-
binding site on Ube2g2 provides an explanation for its high-
affinity interaction with gp78. The G2BR binds Ube2g2 through
an extended region of the core UBC domain of Ube2g2 that
includes the C-terminal ends of both the a1 and a4 helices and
extensive contacts with the intervening b sheets (b1–3). This
backside binding substantially overlaps the noncovalent binding
site for ubiquitin on UbcH5C (Brzovic et al., 2006). Whereas ubiq-
uitin binds UbcH5C with a Kd of �300 mM, which is similar to the
analogous binding of ubiquitin to Ube2g2 (R.D. and R.A.B.,
unpublished data), the binding of the G2BR to Ube2g2 is of
higher affinity by > 10,000-fold. The basis for this difference is
explained by the interacting interfaces. Ubiquitin, characterized
by a stable, ordered structure, interacts with E2s through its
hydrophobic face centered on I44. The G2BR, on the other
hand, folds from an unstructured state into a well-ordered a helix
upon interacting with Ube2g2. At their interface, hydrophobic
side chains along the spine of the G2BR a helix interlock with
Ube2g2 in a fashion similar to the UbcH5C:Ub complex. Addi-
tionally, charged and polar residues distributed along the
G2BR a helix on either side of the hydrophobic spine (Figures
2C and 2D) form salt bridges and hydrogen bonds with
Ube2g2, resulting in a high-affinity complex with a Kd of�21 nM.
Implications of Ube2g2:G2BR for Ubiquitylationand ERADUntil now, the existence of the G2BR as a high-affinity binding
site for Ube2g2 had been assumed to primarily provide a means
to increase the level of Ube2g2�Ub in proximity to gp78 and to
enhance ubiquitylation (Chen et al., 2006). We have now uncov-
ered additional effects of the G2BR. This highly specific 27 aa
binding site for Ube2g2 has the striking effect of increasing the
affinity of Ube2g2 for the gp78 RING finger as a consequence
of conformational/allosteric changes in Ube2g2. The basis for
the dramatic change in Kd, from 144 mM to 3 mM, is not yet fully
understood and awaits further structural studies. A striking
consequence of this increased affinity is the increased associa-
tion of Ube2g2 with the gp78 RING finger, thereby facilitating the
discharge of ubiquitin from Ube2g2�Ub through as yet unde-
fined RING finger-mediated effects (Ozkan et al., 2005; Petroski
and Deshaies, 2005). This is reflected in enhanced ubiquitylation,
even when the G2BR peptide is provided in trans to Ube2g2
together with the cytoplasmic domain of gp78 lacking the
G2BR sequence. Significantly, at least in vitro, the G2BR also
increases ubiquitylation by heterologous ERAD RING finger
E3s. In the cell, this potential for cross-talk between different
ERAD ubiquitin ligases has the potential to contribute to associ-
ations between ERAD RING finger E3s and, more importantly,
to promote synergistic interactions among these proteins
(Ye et al., 2005; Morito et al., 2008). Similarly, if the reported
gp78-oligomerization-dependent formation of K48 ubiquitin
chains on C89 of Ube2g2 occurs in cells (Li et al., 2009), this
will be facilitated by the enhanced affinity of Ube2g2 for the
gp78 RING finger with G2BRs. This domain can serve both as
platforms for Ube2g2 bearing nascent ubiquitin chains and as
a means to enhance transfer of ubiquitin from heterologous
Ube2g2s.
In addition to the increase in RING finger binding, the
Ube2g2:G2BR structure, compared to apo-Ube2g2, reveals
a conformation in which there is narrowing of the channel projec-
ting from the active site Cys89. This is largely due to reposition-
ing of the extended mobile acidic portion of the b4a2 loop,
including a dramatic alteration in orientation of side chains of
M101 and Y103. All of the available structural data indicate
that this region is dynamically disordered (Arai et al., 2006; Li
et al., 2009; this study). One consequence of this repositioning
is a substantial delay in formation of Ube2g2�Ub in the presence
of G2BR. Depending on the rate limiting step(s) in vivo, slowing of
E2 loading with ubiquitin in the presence of the G2BR could allow
for ‘‘proofreading’’ by deubiquitylating enzymes in either sub-
strate selection or chain linkage. The region analogous to the
b4a2 loop is important for interactions between other E2-E3
pairs (Lorick et al., 2005), as well as for Ube2g2:gp78 RING finger
(this study). The acidic extension in this loop is also found in
Cdc34. Mutations in acidic residues in this region in both
Ube2g2 and Cdc34 translate into decreased RING finger-depen-
dent formation of K48-linked polyubiquitin chains (Li et al., 2007;
Petroski and Deshaies, 2005). Moreover, Cdc34-Ub recently has
been shown to manifest �2-fold lower Kd for the core SCF E3
than for Cdc34 (Saha and Deshaies, 2008). As the net effect of
the G2BR, either in cells or in vitro, is to enhance ubiquitylation,
potential effects of this dynamic loop on modulating transfer of
ubiquitin from Ube2g2�Ub, formation of K48 ubiquitin chains,
or in differential RING finger binding of Ube2g2�Ub versus
Ube2g2 may be of equal or greater significance than the
pronounced inhibition of loading with ubiquitin that we observe.
Addressing these possibilities and the unresolved question of
whether ubiquitin can be transferred from E1 to Ube2g2 bound
either to the full cytoplasmic tail of gp78 or the isolated G2BR
are important questions in ERAD and in E2 function that will
require additional in vitro and cellular approaches.
Existence and Implications of E2-Binding Sites Distinctfrom Ligase DomainsE2s interact with E1, as well as with HECT, RING, and RING
finger-like domains on E3s. The question now arises as to the
prevalence of other functionally significant E2 interactions. A
well-characterized example is the E2-like molecule Mms2
(UEV1a), which binds Ubc13 through a region distinct from the
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gp78 Ube2g2-Binding Region: Structure and Function
G2BR-Ube2g2 interface and facilitates formation of K63-linked
ubiquitin chains (Eddins et al., 2006). Another example,
mentioned above, is ubiquitin, which plays a role in the proces-
sivity of BRCA1-mediated ubiquitin chain formation, presumably
through alignment of multiple UbcH5C�Ub complexes (Brzovic
et al., 2006; Christensen et al., 2007). The high-affinity interaction
of Ube2g2:G2BR precludes backside ubiquitin binding in the
context of Ube2g2 bound to gp78. However, it does not exclude
that alignment of multiple E2�Ub complexes (not necessarily
Ube2g2) in vivo could occur in the context of a G2BR-bound
Ube2g2�Ub and play a role in chain formation.
There are several examples in which regions of E3s, which are
not included in their canonical ligase domains, bind E2s, e.g.,
Nedd4 and Ubr1p. For these, neither the sites of interaction on
their respective E2s nor the functional effects of this binding
are known (Madura et al., 1993; Hatakeyama et al., 1997). There
is evidence that SCF E3s recruit Cdc34 through part of the
C-terminal extension of this E2 (Wu et al., 2002). Yeast Ubc7p,
which is the ortholog of Ube2g2, is recruited by Cue1p to the
ER membrane to function with the ERAD E3s Hrd1p and
Doa10p (Kostova et al., 2007). Recent findings suggest that
the 180 amino acid cytoplasmic domain of Cue1p activates
Ubc7p in vitro in a RING finger-independent manner through
unknown mechanisms (Bazirgan and Hampton, 2008; Kostova
et al., 2009). Moreover, an �50 aa domain in Cue1p, with
some sequence homology to G2BR, directly binds Ubc7p. This
domain is sufficient to activate ERAD by Hrd1p in vivo and stim-
ulates ubiquitylation in vitro, analogous to the G2BR (Kostova
et al., 2009), and thus may represent a yeast equivalent of the
G2BR.
A possible parallel to gp78 and Ube2g2 comes from the
SUMO E3 Nup358/RanBP2. This E3 binds the SUMO E2
(Ubc9) through regions on Ubc9 analogous to interactions
between ubiquitin E3s and E2s. However, this E3 also con-
tacts Ubc9 through a second region with similarity to the
Ube2g2:G2BR interface (Reverter and Lima, 2005). As pointed
out by Reverter and Lima, mutations in this interface impact
Ubc9 binding and Nup358/RanBP2 function (Pichler et al.,
2004; Tatham et al., 2005). It will be of interest to know whether
this second site of E2:E3 interaction has analogous effects to
those observed in this study.
It seems reasonable to postulate that E2-binding sites within
E3 complexes but distinct from canonical ligase domains may
be a property of a substantial fraction of E3s. To what extent
these result in functionally significant allosteric effects on E2s,
as demonstrated herein, now becomes an exciting area for
future research.
EXPERIMENTAL PROCEDURES
NMR Spectroscopy
NMR samples were prepared in 50 mM Tris, 2 mM TCEP (pH 7.5) buffer, and
experiments were performed at 25�C. NMR spectra were acquired on 600 and
800 MHz Varian INOVA spectrometers equipped with triple-resonance
gradient cryoprobes. Details of resonance assignments, binding titrations,
and intermolecular NOESY experiments are provided in the Supplemental
Experimental Procedures available online.
X-Ray Diffraction
Crystallization screens were carried out with a Phoenix robot (Art Robbins
Instruments). The crystals were flash-frozen in liquid N2 after a short soak in
a cryoprotection solution. A native data set was collected at beamline 22-ID
of the Advanced Photon Source, Chicago. The structure was solved by molec-
ular replacement. The initial Fo – Fc map revealed the electron density for the
G2BR. Details of crystallization, data acquisition, structure solution, model
building, and structure refinement are provided in Supplemental Experimental
Procedures.
Isothermal Titration Calorimetry
ITC was carried out using a VP-ITC microcalorimeter (MicroCal LLC, North-
ampton, MA) at 25�C. The typical experiment included injection of 25–27
aliquots (10 ml each) of 0.1–0.5 mM peptide solution into a 0.01–0.10 mM
protein solution in the ITC cell (volume �1.4 ml), stirring at 300 rpm. Additional
details are provided in Supplemental Experimental Procedures.
In Vitro Ubiquitylation, E2 Loading, and Discharge
Autoubiquitylation was carried out as described (Kostova et al., 2009; Lorick
et al., 1999). Ubiquitylation of bacterially expressed purified Ub-GFP-His6
Figure 5. G2BR-Mediated Increase in Ube2g2:gp78 RING Finger Affinity Results in Enhanced Ubiquitylation
(A) Glutathione Sepharose-bound GST fusions of the entire gp78 cytoplasmic tail (aa 309–643; GST-gp78C), an inactivating mutation in the RING finger
(GST-gp78CRM) or in the G2BR (GST-gp78CL582,589S), or GST alone were incubated for 90 min in the presence of Ube2g2 and E1. After washing, ubiquitylated
bead-bound material was assessed by SDS-PAGE and immunoblotting with antiubiquitin. See Figure S10 for Coomassie blue stain of fusion proteins.
(B) Glutathione Sepharose-bound GST-gp78C, a truncation at amino acid 577 at the beginning of the G2BR (GST-gp78CD577–643), or GST alone was incubated
with or without G2BR peptide as indicated and assessed in (A).
(C and D) (C) and (D) were carried out as in (A) using GST-gp78CD577–643 and the indicated synthetic or recombinant peptides. Recombinant wild-type G2BR
peptide is indicated by an asterisk to distinguish from the synthetic wild-type peptide.
(E) Ubiquitylation of Ub-GFP-His6 was carried out under the indicated conditions. The asterisk denotes two control samples in which Ub-GFP-His6 was added
after the reaction was first terminated by the addition of 10% SDS for 10 min followed by 8 M urea. Following addition of urea, Ub-GFP-His6 was purified on Ni+
Sepharose beads, and samples were resolved by SDS-PAGE.
(F) Ubiquitylation of Ub-GFP-His6 was carried out as in (E) for 60 min.
(G) Ubiquitylation reactions were carried out with GST-gp78C or the indicated Leu to Ser mutations together with wild-type Ube2g2 or the indicated double muta-
tions of Ube2g2.
(H) GST fusions of the cytoplasmic tails of HsHRD1, Trc8, or truncated gp78 were incubated as in (A) with the indicated peptides and E2s.
(I) 35S-labeled Ube2g2 generated as in Figure 3D was loaded with wild-type ubiquitin for 10 min followed by inactivation of E1 by NEM (5 mM). Discharge of ubiq-
uitin from Ube2g2 (<100 nM), in the presence of either G2BR or Scr (4 mM) with or without gp78 RING finger (8 mM), was monitored by measuring the fraction of
Ube2g2�Ub remaining at each time point. Shown on the left is a representative experiment using gp78 RING finger. Plotted on the right is the average of three
independent experiments; error bars represent SD.
(J) Discharge experiments as in (I) were carried out for 2 or 5 min at the indicated concentrations of gp78 RING finger. Shown on the left are images demonstrating
loss of ubiquitin from Ube2g2. The insert on the right summarizes data from discharge experiments from both (I) and (J). Discharge rate constants, K, are shown
for each experiment.
Molecular Cell 34, 674–685, June 26, 2009 ª2009 Elsevier Inc. 683
Molecular Cell
gp78 Ube2g2-Binding Region: Structure and Function
(�500 nM) was carried out using glutathione Sepharose 4B bound GST-
gp78CD577–643. Reactions were terminated by 2% SDS (final). After 10 min,
samples were diluted with 8 M urea in 50 mM Tris (pH 7.4) to 0.1% SDS. Ub-
GFP-His6 was isolated on Ni2+ beads and eluted with SDS-PAGE sample buffer
after washing. 35S-labeled Ube2g2 was translated in the S30 T7 bacterial TnT
system (Promega) and used at < 100 nM. In loading experiments, Ub K0
was used. For discharge of ubiquitin from E2, E2 was loaded with wild-type
ubiquitin for 10 min. Reactions were quenched with 5 mM NEM, and the buffer
was exchanged to 50 mM Tris (pH 7.4) with Ub (80 mM) and peptide (4 mM) with
or without purified gp78 RING finger (aa 313–393); discharge was monitored at
25�C. Additional details are provided in Supplemental Experimental Proce-
dures.
ACCESSION NUMBERS
Coordinates and structure factors of the Ube2g2:G2BR complex were depos-
ited in the Protein Data Bank under accession code 3H8K.
SUPPLEMENTAL DATA
Supplemental Data include Supplemental Experimental Procedures, 10 fig-
ures, and 1 table and can be found with this article online at http://www.cell.
com/molecular-cell/supplemental/S1097-2765(09)00341-4.
ACKNOWLEDGMENTS
We thank Stanley Lipkowitz and Philip Ryan for critical reading of this manu-
script, Mei Yang and Prasenjit Bhawmik for technical assistance, Kevin Lorick
for assistance in initial studies, and Robert Gemmill, Kazuhiro Iwai, Amy Lam,
and Emmanuel Wiertz for reagents. X-ray diffraction data were collected at the
22-ID beamline of SER-CAT, Advanced Photon Source, Argonne National
Laboratory. Characterization of all proteins (CD and mass spectroscopy)
and calorimetry were performed at the Biophysics Resource in the Structural
Biophysics Laboratory. This work was supported by the National Institutes
of Health Intramural Research Program and by a grant to A.M.W. from the
Multiple Myeloma Research Foundation.
Received: November 20, 2008
Revised: March 12, 2009
Accepted: May 12, 2009
Published: June 25, 2009
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