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INTRODUCTION |
The ubiquitin pathway is crucial in cellular regulation (1-3). It
is now clear that this pathway regulates the life span of many
important proteins, including some cyclins,
cyclin-dependent kinase inhibitors, histones, oncoproteins,
and tumor suppressors. Therefore, this pathway regulates many cellular
processes, including cell cycle progression and apoptosis,
transcription regulation, and antigen presentation (4-6). Ubiquitin is
a protein with 76 amino acids. Its C-terminal Gly residue is involved
in covalent conjugation to a Lys residue of other proteins. The
C-terminal Gly residue of one ubiquitin molecule can also conjugate to
Lys48 of another ubiquitin molecule in
multi-ubiquitination. In the ubiquitination pathway, the ubiquitin
activation enzyme E11
activates ubiquitin by hydrolyzing ATP to form a high energy bond with
ubiquitin. Ubiquitin is then transferred to a ubiquitin conjugation
enzyme (UBC) also known as E2. In this step, the C-terminal Gly is
conjugated to the SH group of the active-site Cys residue of E2. Then
E2 interacts with substrate proteins to transfer ubiquitin to the
substrate proteins. In some cases, this process requires the
participation of ubiquitin-protein ligase (E3). Proteins with high
sequence homology to ubiquitin and to the E1 and E2 enzymes have
been found.
Human UBC9, composed of 158 amino acid residues, is a member of the E2
protein family. Among the conserved residues in the E2 family of
proteins, the fragment containing the ubiquitin-accepting Cys residue
is highly conserved. UBC9 conjugates with a ubiquitin homologue, UBL1,
instead of ubiquitin. The interaction between UBC9 and UBL1 is
specific, because UBC9 does not interact well with ubiquitin, and UBL1
does not interact with UBC2 in a yeast two-hybrid system (7-9). UBC9
in yeast, mouse, and human have been identified (10, 11, 7). Many
proteins interact with UBC9. The human UBC9 has been shown to interact
with several important proteins, such as DNA repair proteins RAD51 and
RAD52, p53, c-JUN, glucocorticoid receptor, the negative regulatory
domain of the Wilms' tumor gene product, and human papillomavirus type
16 E1 replication protein (12-14). Seufert et al. (10) show
that yeast UBC9 may interact with CLB2, an M-phase cyclin, and CLB5, an
S-phase cyclin (10). Hateboer et al. (15) show that murine
UBC9, which has an identical amino acid sequence to the human UBC9,
binds to the adenovirus E1A protein. UBC9 plays critical roles in DNA repair, cell cycle regulation, and p53-dependent processes.
The three-dimensional structures of human UBC9 and several other E2
proteins have been determined (8, 16-19). In addition, conformational
flexibility of UBC9 has been characterized by NMR methods (20). The E2
proteins have highly conserved tertiary structures with root mean
square deviation of C
atoms less than 2 Å, excluding
two surface loops. The two surface loops occur around amino acid
residues 30 and 100 and vary significantly in the lengths of the
sequences and three-dimensional structures among different UBC
proteins. Several regions on UBC9 have higher conformational
flexibility than average. The N-terminal site has the highest mobility
over a wide range of time scales. A region between the C terminus and
the active site also displays higher mobility on the ps~ns and ms
time scales.
The ubiquitin homologue UBL1 is also known as SUMO-1, GMP1, SMTP3,
PIC1, or sentrin (for a review, see Ref. 21). It contains 101 amino
acid residues. Residues 22-97 of UBL1 share approximately 48%
sequence homology with ubiquitin. Residues 98-101 of UBL1 are cleaved
before conjugation with E1. A unique feature of ubiquitin and
ubiquitin-like proteins is that they all contain a diglycine sequence
at the C terminus that is capable of conjugating to other proteins. An
E1 homologue specific for the yeast UBL1 and UBC9 has also been found
(22).
The three-dimensional structures of ubiquitin and UBL1 have been
determined (23, 24). In addition, information on their conformational
flexibilities has been obtained from NMR studies (25, 24). As expected
from sequence conservation, residues 22-97 of UBL1 have a similar
three-dimensional structure as ubiquitin. In addition, the
C-terminal residues of UBL1 are as flexible in solution as those of
ubiquitin. The 21 extra amino acid residues at the N terminus of UBL1
have mainly a random conformation. This segment contains mostly
hydrophilic amino acid residues and is overall negatively charged.
There are only three hydrophobic residues, one Met, Ala, and Leu in
this segment.
Despite the intensive studies on ubiquitination, the mechanism of
protein-protein interactions in ubiquitination is not well understood.
Details of the interaction and conjugation between an E2 and a
ubiquitin are still not clear. One of the major difficulties in
investigating these interactions is that they are not strong and cannot
be easily detected by conventional biochemical approaches. NMR methods
have contributed greatly to the characterization of molecular
interactions. Chemical shift perturbation is one of the most sensitive
methods to monitor specific protein complex formation (26). Chemical
shift changes are capable of mapping the binding interfaces of protein
complexes with dissociation constants in the mM to
nM ranges. This paper describes the identification of the
binding interfaces between UBC9 and UBL1 using NMR chemical shift
perturbation. Because the binding site of UBL1 resides on the ubiquitin
domain and the binding site of UBC9 resides on a structurally conserved
region of E2, the observed interaction may represent a general feature
of E2-ubiquitin interactions.
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MATERIALS AND METHODS |
NMR Studies--
Both human UBC9 and UBL1 were subcloned into
vector PET28 (from Novagen, Inc.). The modified plasmids have an open
reading frame that encodes the His6 tag at the N terminus
followed by the sequence of UBC9 or UBL1. Escherichia coli
containing the expression plasmid were grown at 37 °C in M-9 minimal
media supplemented with trace minerals and basal medium Eagle vitamins
(Life Technologies, Inc.).
(15NH4)2SO4 (1.5 g/liter) was used as the nitrogen source. Both proteins were purified
using nickel nitrilotriacetic acid columns.
Approximately 20~30 mg of UBC9 and 10 mg of UBL1 were purified from 1 liter of the M-9 culture. Both samples were of high purity, because no
impurities were observed on SDS-polyacrylamide gel electrophoresis. All
NMR samples contained 100 mM phosphate buffer (pH 6.0) in
90% H2O, 10% D2O. The concentrations of
protein samples were estimated with the Bio-Rad protein assay.
1H-15N HSQC experiments (27) were performed on
a Varian Unity plus 500 spectrometer equipped with four channels, pulse
shaping, and pulsed field gradient capabilities. The digital
resolutions of the spectra were 0.02 ppm in the proton dimension and
0.05 ppm in the amide 15N dimension. For observing the UBL1
bind site on UBC9, 0.3 mM 15N-labeled UBC9 was
used. Unlabeled UBL1 was titrated into the sample containing UBC9. The
final concentrations of both proteins at the end of the titration were
approximately 0.2 mM. The spectra were acquired at
30 °C, a condition used for obtaining the resonance assignments of
UBC9. For detecting the UBC9 binding site on UBL1, 0.5 mM
15N-labeled UBL1 was used and titrated with unlabeled UBC9.
The final concentrations of both proteins at the end of the titration were approximately 0.3 mM. The HSQC spectra for obtaining
the binding interface on UBL1 were performed at 17 °C, which was
used for the resonance assignments of UBL1. The NMR resonance
assignments for UBC9 have been described previously (28). The NMR
resonance assignments for UBL1 will be described
elsewhere.2
Calculations of Electrostatic Potentials--
We calculated the
surface electrostatic potentials for UBL1, ubiquitin, UBC9, UBC4, and
UBC7 (8, 17-19, 23, 24) using the DelPhi module of INSIGHTII (MSI,
Inc.) with the NMR and crystal structures to better understand our
results. The solvent dielectric constant was set to 80. The radius of
the probe water molecule was 1.4 Å. The grids in the calculation of
the electrostatic potentials were with a spacing of 1.5 Å.
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RESULTS |
We have used 15N-1H HSQC spectra to map
the binding interfaces of the ubiquitin homologue UBL1 and human UBC9.
15N-1H HSQC has been frequently used to map the
binding surfaces of protein-protein, protein-nucleic acid, and
protein-ligand interactions. In this spectrum, resonances are usually
well resolved because of large 15N chemical shift
dispersion. Because each amino acid residue gives one peak in this
spectrum, it is easy to monitor the chemical shift perturbation of each
amino acid. The binding interface on UBC9 was obtained using
15N-labeled UBC9 in complex with unlabeled UBL1.
15N-1H HSQC spectra of UBC9, free and in
complex with UBL1, were compared. Similarly, the binding interface on
UBL1 was mapped using 15N-labeled UBL1 and unlabeled UBC9.
15N-1H HSQC spectra of UBL1, free and in
complex with UBC9, were compared.
The Binding Interface on UBC9--
Specific chemical shift
perturbation and changes in the line widths were observed in the
1H-15N HSQC spectrum of UBC9 upon forming
complex with UBL1. These changes were observed from the beginning of
the titration, when the concentration of UBC9 was approximately 0.3 mM and that of UBL1 was 0.03 mM until the final
concentrations of UBL1 and UBC9 reached approximately 0.2 mM. Superposition of the HSQC spectra of free UBC9 and that
in complex with UBL1 is shown in Fig. 1. The assignments of the resonances in the complex that undergo fast
exchange were made by following resonance shifts during titration. Details of the chemical shift changes of UBC9 at the end of the titration are given in Table I.
Variations in line widths between the resonances of free UBC9 and UBC9
at the end of the titration are indicated by relative changes in peak
heights, also given in Table I. Most peaks of UBC9 were not affected,
indicating that complex formation between the two proteins does not
cause large conformational changes in UBC9. Some residues were
significantly affected by the complex formation. The line widths of
residues Ala10, Ala15, Arg17,
Lys18, Phe22, Gly23, and
Phe24 became so broad in the complex that their cross-peaks
cannot be observed from the beginning of the titration when the ratio of UBC9:UBL1 was 10. These residues are colored in dark blue
in the ribbon diagram of the UBC9 structure (Fig.
2A). Residues
Ser7, Lys14, Trp16,
Glu19, His20, Ala26,
Val27, Met36, Gln37,
Lys59, Leu63, and
Ser158 moved more than 0.2 ppm in the 15N
dimension or more than 0.18 ppm in the 1H dimension at the
end of the titration. These residues are colored in cyan in
Fig. 2A. In addition, residues Ile4,
Thr29, and Gly115 moved more than 0.14 ppm in
the 15N dimension at the end of the titration. The proton
resonances of these residues did not change more than 0.04 ppm. These
residues are shown in green in Fig. 2A. The
chemical shift changes of these residues are linear with the addition
of UBC1. Some residues had larger increases in line widths than average
but had small chemical shift changes. These residues are labeled in the
spectra in Fig. 1. Some of these residues are close to the region that
showed the largest perturbation upon the formation of the complex, and some are located on the main
-sheet. Most of the chemical shift changes on UBC9 occur in the nitrogen dimension. Residues that are most
affected by the complex formation are clustered together in the
three-dimensional structure, suggesting that this region is the binding
interface. A few residues on these surfaces, including Leu9, Asn11, Glu12,
Arg13, Lys30, and Gln31, have
resonances that overlap with other resonances and, therefore, could not
be monitored for their chemical shift perturbations. These residues are
colored in gray in Fig. 2A. Thus, the region of
UBC9 interacting the UBL1, as suggested by the chemical shift perturbations, consists of the first
-helix, the first
-strand, the loop between them, and the second
-strand.

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Fig. 1.
Superposition of
1H-15N HSQC spectra of 15N-labeled
human UBC9, free and in complex with unlabeled human UBL1. The
UBC9/UBL1 ratio in the complex is approximately 1:1. The cross-peaks of
free UBC9 are shown in green, and those in the complex are
shown in red. Only peaks that were affected by the complex
formation are labeled.
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Table I
1HN, 15NH chemical shift changes,
 (ppm), and peak height ratios of hUBC9 in a mixture with UBL1
relative to that of free protein
The chemical shift changes are given as the differences between of the
free hUBC9 resonances and the resonances in the complex. The
resolutions of the 1HN and 15NH
chemical shift are about 0.01 ppm and 0.05 ppm. The peak height ratios
are calculated from the peak heights of the free hUBC9 cross-peaks and
those in the complex. The S.E. of the peak height ratio is about 0.03. Both hUBC9 and UBL1 concentrations are approximately 0.2 mM. Only the residues of hUBC9 that give unoverlap peaks in
two-dimensional 15N-edited HSQC are shown in the table.
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Fig. 2.
A, ribbon diagram of the
three-dimensional structure of UBC9. Chemical shift perturbation upon
UBL1 binding are indicated by the coloring scheme described in the
text. The active-site Cys93 is shown with its side chain in
red. B, surface electrostatic potentials of human
UBC9. The orientation of the molecule is the same as in that in
A. The charge topology was calculated and displayed using
INSIGHTII (MSI, Inc.). The color spectrum from red to
blue corresponds to changes from negative to positive
potentials over a range of 5 to +5
KB/electron.
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The Binding Interface on UBL1--
Specific chemical shift changes
were also observed on UBL1. Unlabeled UBC9 was used to titrate
15N-labeled UBL1. The concentrations of UBL1 and hUBC9 at
the end of the titration are about 0.45 and 0.39 mM,
respectively. The superposition of the HSQC spectra of UBL1, free and
in complex with UBC9, is shown in Fig. 3.
The assignments of the resonances in the complex that undergo fast
exchange were made by following resonance shifts during titration.
Details of the chemical shift changes of UBL1 at the end of the
titration are given in Table II.
Variations in line widths between the resonances of free UBL1 and UBL1
at the end of the titration are indicated by relative changes in peak
heights, given in Table II. The resonances of residues
Ile27, Ser31, Leu65,
Glu67, Asp86, Ile88, and
Val90 were significantly broadened, because their peaks
disappeared in the HSQC spectrum. These residues are shown in
dark blue in the ribbon diagram of the structure of UBL1
(Fig. 4A). Residues Val26, Phe64, Gly68,
Ile71, Gly81, Met82,
Glu83, Glu85, and Val87 showed
chemical shift changes of more than 0.25 ppm in the 15N
dimension and/or more than approximately 0.1 ppm in the 1H
dimension. Many of these residues have large chemical shift changes in
both the proton and nitrogen dimensions. The chemical shift changes of
these residues are linear with the addition of UBC9. These residues are
colored in cyan in Fig. 4A. These residues are
also clustered together in the three-dimensional structure of UBL1,
suggesting that this region is the binding interface with UBC9. This
surface is located on the main
-sheet in the ubiquitin domain of
UBL1. No chemical shift changes were observed for the N-terminal
21-amino acid residues, indicating that this region is not involved in
interaction with UBC9.

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Fig. 3.
Superposition of
1H-15N HSQC spectra of 15N-labeled
human UBL1, free and in complex with UBC9. The UBC9/UBL1 ratio in
the complex is approximately 1:1. The cross-peaks of free UBL1 are
shown in green, and those of UBL1 in complex are shown in
red. Only peaks that were affected by the complex formation
are labeled.
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Table II
1HN, 15NH chemical shift changes,
 (ppm), and peak height ratios of UBL1 in a mixture with hUBC9
relative to that of the free protein
The chemical shift changes are given as the difference between of the
free UBL1 resonances and the UBL1 resonances in the complex. The
resolutions of the 1HN and 15NH
chemical shift are about 0.01 ppm and 0.05 ppm. The peak height ratios
are calculated from the peak heights of the free UBL1 cross-peaks and
those in the complex. The S.E. of the peak height ratio is about 0.03. Both UBL1 and hUBC9 concentrations are approximately 0.4 mM. Only the residues of UBL1 that give unoverlap peaks in
two-dimensional 15N-edited HSQC are shown in the table.
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Fig. 4.
A, ribbon diagram of the
three-dimensional structure of UBL1. Chemical shift perturbation upon
UBC9 binding is indicated with the coloring scheme described in the
text. B displays the surface electrostatic potential of
human UBL1. The orientation of the molecule in B is the same
as that in A. The charge topology was calculated and
displayed using INSIGHTII (MSI, Inc.). The color spectrum from
red to blue corresponds to changes from negtive
to positive potentials over a range of 5 to +5
KB/electron.
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DISCUSSION |
Chemical shift perturbation is extremely effective in mapping the
binding surfaces. The chemical shift of a nuclei is sensitive to the
changes of its local environment including aromatic ring current
effects, peptide bond anisotropy, electrostatic interactions, and
hydrogen bonding. When two proteins form a complex, the interactions between them cause changes in the environment of the amino acids at the
interfaces, resulting in chemical shift changes. Any small additional
conformation changes near the direct contacting surfaces will cause
additional chemical shift perturbation. Thus the surface mapped by
chemical shift perturbation contains but extends beyond the direct
binding surface. However, residues that have the largest chemical shift
changes are usually located within the binding interface.
Sequence Conservation--
The amino acid residues involved in the
recognition between UBC9 and the ubiquitin homologue UBL1 are highly
conserved throughout different species. Residues 10-27 of UBC9 had the
largest changes in resonance line widths and chemical shifts upon
complex formation with UBL1. Twelve of these 17 residues are identical
among UBC9 of human, Saccharomyces cerevisiae and
Schizosaccharomyces pombe (30). Two more residues have
conservative mutations from Arg to Lys and Val to Tyr. Among the
residues that are affected by complexation, four positively charged
residues are conserved between the yeast and human proteins, including
Arg13, Lys14, Arg17, and
Lys18. In addition, two negatively charged residue,
Glu12 and Asp19, are identical between the
yeast and human proteins. Six hydrophobic residues in this region are
highly conserved including Trp16, Pro21,
Phe22, Phe23, Val24, and
Ala25. Five of them are identical between the human and
yeast proteins.
Residues 26, 64-71, and 81-91 of UBL1 had the most significant
changes in line widths and chemical shifts upon complex formation with
UBC9. Nine of these residues are identical between the human and yeast
proteins (24). Four residues are highly conserved, including a Phe to
Tyr mutation, two Glu to Asp mutations, and a Val to Ile mutation.
Among the residues that are affected by complexation, four negatively
charged residues are highly conserved including Glu83,
Glu84, Asp86, and Glu89. In
addition, a positively charged residue, Arg70, is identical
between the yeast and human proteins. Lys25 on this surface
is identical among UBL1s of different species. However, this residue
has overlapping resonances in the 1H-15N HSQC,
and it is not clear whether it had significant chemical shift changes.
Six hydrophobic residues are conserved, including Leu65,
Phe66, Met82, Val87,
Ile88, and Val90. Most of these residues are
identical between the yeast and human proteins, except
Phe66 mutated to a Tyr and Val87 to a Ile in
the S. cerevisiae protein.
Sequence conservation indicates important functions for these residues.
For UBC9 and UBL1, similar numbers of hydrophobic residues are
conserved at the binding interfaces. In addition, similar numbers of
the opposite-charged residues at the binding interfaces are also
conserved. This suggests the compatibility of the binding interfaces.
Mechanism of Recognition and Specificity--
The binding
interfaces of the UBC9-UBL1 complex are highly complementary in their
electrostatic potentials and hydrophobicity. The surface electrostatic
potentials of UBC9 and UBL1 are displayed in Fig. 2B and
4B, respectively. Surface hydrophobic side chains in the
binding interfaces are displayed in yellow along with
electrostatic potentials in Fig. 5. For
UBC9, the surface of residues 10-30 and 35-37 are displayed in Fig.
5A. For UBL1, the surfaces of residues 25-27, 31, 65-71,
and 81-90 are displayed in Fig. 5B. The sizes of the
surface areas that showed significant changes in line widths and
chemical shifts are similar and are approximately 1,500 Å2. The surface of UBC9, which is involved in binding to
UBL1, is mainly positively charged (Fig. 2B and
5A). The surface of UBL1, which is involved in binding to
UBC9, is mainly negatively charged (24) (Fig. 4B and
5B). The opposite surface of UBL1 from the binding interface
has an overall positive potential (24). The N-terminal 21-amino acid
residues of UBL1 are mainly negatively charged and may contribute to
the electrostatic energy when forming a complex with the positively
charged surface of UBC9. However, no specific chemical shift changes
have been observed for residues 1-16. Residue 17 showed some
15N chemical shift perturbation (0.19 ppm). Because this
change is smaller compared with that in the ubiquitin domain, and this residue is close to the region that displays large changes in chemical
shifts and line widths, this perturbation may not result from direct
contacts. Electrostatic interactions can be long range and nonspecific
and, therefore, may not result in specific chemical shift changes.

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Fig. 5.
Connolly surfaces of the binding interfaces
on UBC9 (A) and UBL1 (B), as
identified from chemical shift perturbation. The surfaces are
colored by electrostatic potentials as in Fig. 2B and
4B. In addition, the surface hydrophobic side chains are
indicated in yellow. Other regions of the proteins are shown
as C traces.
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A detailed mechanism for UBL1 and UBC9 recognition is proposed. The
conserved charged residues of UBC9, Arg13,
Lys14, Arg17, and Lys18, are
clustered together at the lower right corner of Fig. 5A. The
conservative residues of UBL1, Glu83, Glu84,
Glu85, and Asp86, are clustered at the lower
left corner in Fig. 5B. Both regions are adjacent to the
surface hydrophobic side chains. These two regions may interact with
each other in the complex. Conserved residues Glu12 of UBC9
and Lys25 of UBL1, located at the inner sides of Fig. 5,
may form a salt bridge. Surface hydrophobic side chains are located
near the outer upper sides on the binding surfaces shown in Fig. 5.
These residues are in complementary positions and can readily form
hydrophobic interactions. These hydrophobic interactions may contribute
significantly to the specificity of the complex, because hydrophobic
interactions are of short range. The outer lower regions, which have
positive and negative potentials shown in Figs. 5, A and
B, respectively, are composed of several polar residues that
may form intermolecular hydrogen bonds. Because the highly conserved
residues on the binding interfaces are charged and hydrophobic
residues, the mechanism of recognition is likely through mainly
hydrophobic and electrostatic interactions.
Our results explain the specificity of UBC9 and UBL1 interactions. It
was reported previously that UBC9 conjugated with UBL1 but not
ubiquitin (9). Although the sequences and three-dimensional structures
are highly conserved between UBL1 and ubiquitin, the surface
electrostatic potentials between the two proteins are very different.
The surface of UBL1 that binds UBC9 has an overall negative
electrostatic potential. However, this surface of ubiquitin is
positively charged (24). Electrostatic repulsion will prevent UBC9 from
interacting with ubiquitin well. Titration of unlabeled ubiquitin to
15N-labeled UBC9 until the concentration ratio of ubiquitin
to UBC9 reached 6~7 did not show any specific chemical shift changes
(data not shown), indicating that the two proteins do not show specific binding. The surface electrostatic potentials were also calculated for
UBC4 and UBC7 (17, 18), which interact with ubiquitin. The regions that
superimpose with the UBL1 binding site of UBC9 on the two E2 proteins
have negative or neutral potentials, which is compatible with the
electrostatic potential of the ubiquitin surface that superimposes with
the UBC9 binding site on UBL1.
Binding of the E1-UBL1 Conjugate to UBC9--
UBL1 and the E1
homolog for this system may bind to different locations on UBC9. It is
likely that UBL1 contributes significantly to the affinity with UBC9 in
the E1-UBL1 conjugate. It has been shown that deletion of the N
terminus of UBL1 affected its interaction with UBC9 (31). This seems
unrelated to E1, because the conjugation of UBL1 to E1 and other
proteins is through its C-terminal Gly residue. Because the UBC9
binding site on UBL1 is not close to the C terminus of UBL1, and the
C-terminal tail is flexible, the UBL1-E1 conjugate may be flexible
relative to each other. From the above-described model of UBC9-UBL1
interaction, the C terminus of UBL1 should extend toward the main
-sheet. Significant chemical shift changes and line broadening have
been observed for residues in the
-sheet (Fig. 2A). In
addition, many residues in the
-sheet show larger than average line
broadening. Residues 91-95 of UBL1 have overlapping resonances, and
therefore, it is not clear whether chemical shift changes and line
broadening occurred to these residues. Simple modeling shows that
although the binding site on UBC9 is not close to the active-site
Cys93, the C-terminal Gly of UBL1 can span the
-sheet to
reach Cys93 of UBC9 for conjugation. A region of high
sequence conservation have been observed on E2 structures (19, 17).
This region is between the conjugation active-site Cys residue and the
second helix and is on the opposite surface from the UBL1 binding
site. This surface has been predicted to bind the E1-ubiquitin
conjugate. It is possible that this surface binds E1 in the E1-UBL1
conjugate to place the C terminus of UBL1 at the conjugation active
site of UBC9. Because the C-terminal residues of ubiquitin and UBL1 are
flexible in solution and the binding site of UBL1 on UBC9 does not
involve the C terminus, UBL1 and E1 may bind to different surfaces on
the E2. E1 should bind to a location close to the active site, because
it catalyzes the conjugation between E2 and ubiquitin.
A General Feature of E2-Ubiquitin Interaction--
The functional
importance of the N terminus of E2 has also been implicated from
previous studies; however, the exact function is still unclear.
Mutating residues 6-8 of Arabidopsis thaliana UBC1
significantly reduces the conjugation of ubiquitin to the E2 (32). As
suggested from the crystal structure of Rad6 (19), the triple mutation
is likely to destabilize the protein structure, because the side chains
of both Arg6 and Arg8 are involved in
intramolecular hydrogen bonding. Therefore, the effect of these
mutations may not be because of the disruption of direct binding
interactions. Deletion of residues 1-22 of Rad6 resulted in failure to
form a complex with Rad18, which is a single-stranded DNA-binding
protein possibly functioning to target Rad6 to sites of DNA damages
(29). Because residues 8-22 have extensive contacts with the main
structure of Rad6, it is not clear whether deletion of these residues
disrupted the structural integrity of the protein.
The UBL1-UBC9 system shares many similarities to other E2-ubiquitin
systems in the mechanism of conjugation with each other and with other
proteins. The binding interfaces between UBL1 and UBC9 are located on
the regions that are highly conserved in the three-dimensional
structures of E2 and ubiquitin. Therefore, the mechanism of recognition
between UBC9 and UBL1 may represent a general feature of E2-ubiquitin
interactions. Interestingly, the surface of the E2s that conjugate with
ubiquitin does not have highly conserved sequences. This may result in
differences in their affinities for the E1-ubiquitin conjugate.