From the Biochemistry Department, National Institute
of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan and the
§ Graduate School of Biological Sciences, Nara Institute of
Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0192, Japan
Received for publication, October 15, 2002, and in revised form, February 4, 2003
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ABSTRACT |
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EL5, a RING-H2 finger protein, is rapidly induced
by N-acetylchitooligosaccharides in rice cell. We
expressed the EL5 RING-H2 finger domain in Escherichia coli
and determined its structure in solution by NMR spectroscopy. The EL5
RING-H2 finger domain consists of two-stranded Upon sensing the invasion of microorganisms, plants evoke a
variety of defense reactions, including the synthesis of antimicrobial compounds (phytoalexins) and proteins. Many of these biochemical reactions are based on the activation of defense-related genes. In some
cases, the level of protein accumulation and the rapidity of gene
induction in the host plant are correlated to the degree of its disease
resistance. Therefore, it might be possible to control disease
resistance by modifying the regulatory factors for the expression of
defense-related genes. Such regulatory factors could be elements of
signal transduction pathways leading from the recognition of invading
pathogens to the activation of defense-related genes. Most of the
defense responses are reproducible in suspension-cultured cells treated
with specific substances called elicitor (1). Chitin fragments
(N-acetylchitooligosaccharides) can act as elicitors (2),
which induce the transient expression of several "early responsive"
genes, such as EL5 (3). EL5 is a RING finger protein, which
is structurally related to proteins of the Arabidopsis ATL family. These proteins are characterized by a transmembrane domain (domain I), basic domain (domain II), conserved domain (domain III),
and RING-H2 finger domain (domain IV) followed by the C-terminal region
with highly diverse amino acid sequences (4). Although some ATL family
genes resemble EL5 in being induced in early stages of the defense
responses (5), their biochemical function is obscure. Recently, it was
shown that the fusion protein of EL5 with maltose-binding protein
(MBP)1 was polyubiquitinated
by incubation with ubiquitin-activating enzyme (E1) and
ubiquitin-conjugating enzyme (E2). Apparently, EL5 acted as a ubiquitin
ligase (E3) and catalyzed the transfer of ubiquitin to the MBP moiety.
Further, it was shown that a rice E2, OsUBC5b was induced by
elicitor (6). Although these results strongly suggest that EL5 and
OsUBC5b have roles in plant defense response through the
turnover of protein(s) via the ubiquitin/proteasome system, their
molecular function has not been clarified at the atomic level.
The RING finger motif, which is known from many functionally distinct
proteins, was first identified in the product of the human gene RING1
(Really Interesting New Gene 1) that is located proximal to the major
histocompatibility region on chromosome 6 (7). The RING finger motif is
defined by the consensus sequence Cys-X2-Cys-X9-39-Cys-X1-3-His-X2-3-(Cys/His)-X2-Cys-X4-48-Cys-X2-Cys, in which X can be any amino acid. It binds two zinc atoms
with its Cys and His residues in a unique "cross-brace"
arrangement. The invariable spacing between the second and third pair
of Cys/His residues indicates conservation of the distance between the
two zinc-binding sites (8). The RING finger motif is widely distributed among proteins that play major roles in cell growth and differentiation (9). Certain types of RING finger domains seem to be required for
multimerization, while others are elements of proteins involved in
ubiquitination (10). Ubiqutination usually results in the formation of
a bond between the C terminus of ubiquitin (Gly76) and the
To elucidate the properties of EL5 at the atomic level, we determined
the three-dimensional structure of its RING-H2 finger domain in
solution by NMR spectroscopy. Furthermore, we characterized its
functional properties by an ubiquitination assay in vitro and by NMR titration experiments. Our results illuminate the function of not only EL5 but also the ATL family proteins in general.
Cloning and Purification of Recombinant Proteins for NMR
Experiments--
EL5 RING-H2 finger domains and OsUBC5b
were cloned as fusion proteins with thioredoxin (Trx) and His tag. The
fusion proteins were constructed in the vector pET32a (Novagen).
EL5(129-181) and EL5(96-181) DNA fragments from EL5 cDNA
(AB045120) were amplified by polymerase chain reaction (PCR).
OsUBC5b was amplified by PCR from OsUBC5b
cDNA (AB074412). Trx-EL5(129-181) was constructed using the
primers 5'-CATGCCATGGACGACGGCGTCGAGTG-3' and
5'-CGAATTCTTACACGACGACGGTGAGGC-3'. Trx-EL5-(96-181) was constructed
using the primers 5'-CATGCCATGGGGGTCGACCCGGAGGTG-3' and
5'-CGGGATCCTTACACGACGACGGTGAGGC-3'. The purified PCR products were
digested with NcoI and EcoRI or BamHI
and were ligated into pET32a. Trx-OsUBC5b was constructed
with the primers 5'-TTCCATGGCGTCCAGCGGATCCTCAAG-3' and
5'-AACTCGAGCTAGCCCATAGCATATTTCTGGGT-3'. The purified PCR product was
digested with NcoI and XhoI and was ligated into pET32a.
All of the clones were transformed into Escherichia coli
BL21(DE3) cells. The bacteria were grown at 37 °C in Luria Bertani for non-labeled protein and in M9 minimal medium for uniformly 15N- and 15N/13C-labeled protein,
with 50 µg/ml ampicillin. Protein expression was induced by addition
of isopropyl-
Trx-EL5(129-181) and Trx-EL5(96-181) were purified by a similar
procedure. Frozen pellets were thawed on ice and resuspended in 50 mM Tris-HCl buffer (pH 7.4), 50 mM NaCl, 50 µM ZnSO4, and 2.5 mM
For purification of Trx-OsUBC5b, frozen pellets were thawed
on ice, resuspended in 20 mM phosphate buffer (pH 7.4) with
500 mM NaCl, and lysed by sonication. Insoluble material
remaining after lysis was removed by centrifugation at 27,000 × g for 30 min. The supernatant was loaded onto a 5-ml
Ni-chelating column. After washing the column with buffer (20 mM phosphate buffer, pH 7.4, 500 mM NaCl),
protein was eluted in buffer using a linear gradient of imidazole
(0-500 mM). Peak fractions were concentrated and applied
to a HiLoad Superdex 75 pg 26/60 column equilibrated in buffer (20 mM phosphate buffer, pH 7.4, 100 mM NaCl). Trx
tag was removed by cleavage with 28 units of thrombin (Novagen) per about 13 mg of protein at 37 °C for 12 h. The solution was
applied to a Ni-chelating column. Remaining tag was removed by cleavage with 130 units of enterokinase per about 5 mg of protein at 37 °C
for 16 h. Then the solution was applied to a HiLoad Superdex 75pg
26/60 column. Peak fractions were dialyzed against NMR buffer (100 mM NaCl, 20 mM Tris-HCl buffer, pH 7.0, 5 mM dithiothreitol) and concentrated using a Centricon spin
dialysis tube (30-kDa cutoff; Amicon, Inc.).
NMR Spectroscopy--
All NMR spectra were recorded at 35 °C
on a Bruker DMX750 spectrometer equipped with a 5-mm inverse triple
resonance probe head with three-axis gradient coils. 1H,
13C, and 15N sequential resonance assignments
were obtained using two-dimensional double resonance and
three-dimensional double and triple resonance through-bond correlation
experiments (16-18): two-dimensional
1H-15N HSQC, two-dimensional
1H-13C CT-HSQC optimized for observation
of either aliphatic or aromatic signals, three-dimensional
15N-separated HOHAHA-HSQC, three-dimensional HNHA,
threedimensional HNHB, three-dimensional HNCO, three-dimensional
CBCA(CO)HN, three-dimensional HBHA(CBCACO)NH,
three-dimensional HNCACB, three-dimensional C(CO)NH, three-dimensional
HCABGCO, and three-dimensional HCCH-TOCSY. Stereospecific assignments
of Structure Calculations--
NOE-derived interproton distance
restraints were classified into four ranges: 1.8-2.7, 1.8-3.3,
1.8-4.3, and 1.8-5.0 Å, corresponding to strong, medium, weak, and
very weak NOEs. The upper limit was corrected for constraints involving
methyl protons and methylene protons that were not assigned
stereospecifically (23). Hydrogen bond distance restraints were applied
to N and O atoms (2.8-3.3 Å) and to HN and O atoms (1.8-2.3 Å), in
regular secondary structures that had small amide exchange rates.
Torsion angle restraints on
A series of two-dimensional 1H-15N HSQC spectra
was recorded for the RING-H2 finger domain in 20 mM sodium
phosphate buffer (pH 7.4) containing 100 mM NaCl and 2 mM dithiothreitol, as a function of OsUBC5b
concentration. Solutions containing 0.25 mM of either 15N-labled RING-H2 finger domain or non-labeled
OsUBC5b were mixed to yield molecular ratios
(OsUBC5b : RING-H2 finger domain) of 0, 0.25, 0.5, 0.75, 1, 1.25, and 2. Assignments of the HSQC signals of EL5 RING finger domain
bound to E2 were made by tracing the peaks during titration and were
confirmed by analyzing three-dimensional triple resonance experiments
carried out on the solution containing 0.6 mM
15N/13C-labeled EL5 RING finger domain and 0.72 mM OsUBC5b.
Cloning and Purification of Recombinant MBP Fusion
Proteins--
The EL5 fragments Gly96-Asn325
and Gly96-Val181 were amplified by PCR using
5'-GAATTCGGAGGAGGGGTCGACCCG-3' and
5'-GGAATTCTCAATCCGGACATGCGC-3' primers or
5'-CGAATTCGGAGGAGGGGTCGACCCG-3' and 5'-GGAATTCTCAATCCGGACATGCGC-3' primers, respectively. The PCR products were digested with
EcoRI and inserted into the EcoRI site of pMAL-p2
(New England BioLabs, Beverly, MA) to express MBP-EL5(96-325) and
MBP-EL5(96-181). To yield MBP for negative control experiments,
pMAL-p2 was digested with EcoRI, blunted, and self-ligated.
All PCR products were verified by DNA sequencing. The plasmids were
introduced into the E. coli strain JM109 to produce the
recombinant proteins and were purified by amylose affinity
chromatography according to the manufacturer's instructions (New
England BioLabs).
In Vitro Ubiquitination Experiments--
The ubiquitination
reaction was performed in 150 µl of ubiquitination buffer containing
300 ng/µl bovine ubiquitin (Sigma), 50 ng of mouse E1, 10 ng
OsUBC5b as E2, and 250 ng of MBP fusion protein. The
reaction buffer was incubated at 35 °C for 1 h, and the
reaction was stopped by the addition of SDS sample buffer. After
boiling for 5 min, the samples were separated by 7.5% SDS-PAGE and
subjected to immunoblotting using anti-MBP antibody (New England BioLabs).
Determination of NMR Experimental Conditions--
EL5 consists of
a transmembrane domain (domain I), a basic domain (domain II), a
conserved domain (domain III), and the RING-H2 finger domain (domain
IV) followed by the C terminus (Fig.
1a). For NMR experiments, two
protein fragments were prepared: EL5 (129-181, domain IV), containing
the RING-H2 finger domain only, and EL5 (96-181, domain III + IV),
which additionally contained the conserved domain. The
1H-15N HSQC spectrum of EL5(129-181) obtained
at 35 °C showed dispersed, intense signals, indicating a
well-defined molecular structure (Fig. 1b). In the spectrum
of EL5(96-181), the strengths of peaks differed considerably (Fig.
1c). The weak signals corresponded to those of
EL5(129-181), that is, to the RING-H2 finger domain. The intense
signals were concentrated in the center of the spectrum, indicating
that domain III is a highly flexible random coil structure.
To investigate the role of Zn2+ in the folding of the EL5
RING-H2 domain, Zn2+ was chelated by EDTA. Removal of zinc
from the purified domain by addition of EDTA led to a loss of the
chemical shift dispersion characteristics of the folded proteins (Fig.
1d). The protein was reversibly refolded upon addition of
excess Zn2+, as indicated by the reappearance of a spectrum
that was identical to that in Fig. 1b. Therefore we decided
to carry out the purification of recombinant EL5(129-181) and its NMR
analysis in the presence Zn2+.
1H, 15N, and 13C Signal
Assignments and Secondary Structure of the EL5 RING-H2 Finger
Domain--
The backbone amide resonances in the
1H-15N HSQC spectrum were sequentially assigned
to all non-proline residues based upon the analysis of correlations
observed in CBCA(CO)NH and HNCACB spectra, respectively. The former
type of spectrum correlates each 15N, HN signal pair to the
C
In order to obtain a high-resolution NMR structure, the resonance
assignments were refined by stereospecific assignments of numerous
methylene and methyl protons of Val and Leu residues. Stereospecific
assignments of Gly
On the basis of essentially complete signal assignments, interproton
distance constraints were derived from short range NOE connectives
obtained from three-dimensional 15N-separated NOESY-HSQC,
13C/15N-separated NOESY-HSQC, and
four-dimensional 13C/13C-separated
HSQC-NOESY-HSQC spectra. Dihedral constraints for Tertiary Structure of RING-H2 Finger Domain of EL5--
The
three-dimensional structure of the RING-H2 finger domain of EL5 was
determined by a hybrid distance geometry/dynamic-simulated annealing
approach (26) based upon 913 experimental restraints derived from NMR
spectroscopy. Structural statistics for the EL5 RING-H2 finger domain
are shown in Table I. The
superposed backbone N, C
The three-dimensional structures of a few RING finger domains have been
determined. The molecular function of two of them, RAG1 and c-Cbl, is
known. The V(D)J recombination-activating protein, RAG1, is a dimer.
Its dimerization domain consists of a zinc finger and a RING finger
(14). It also contains a unique binuclear zinc cluster instead of the
mononuclear zinc site in the RING finger. The determination of the
crystal structure of c-Cbl bound to a specific ubiquitin-conjugating
enzyme (E2), UbcH7, has revealed that the RING finger domain of c-Cbl
recruits the E2 (15). The RING finger domain of EL5 is structurally
similar to those of RAG1 and c-Cbl (Fig.
3a). The backbones of the
secondary structural elements of the proteins are almost
superimposable. The backbone (N, C
Although the overall three-dimensional structures of the RING finger
domains of RAG1 and c-Cbl are similar, their molecular functions are
different. To obtain information on the molecular function of the
RING-H2 finger domain of EL5, we compared the RING finger domains of
the three proteins at the atomic level. The RAG1 RING finger domain has
one additional The EL5 RING-H2 Finger Domain Catalyzes Auto-ubiquitination in
Vitro--
Takai et al. (6) showed that the fusion protein
of EL5(96-325) and maltose-binding protein (MBP-EL5(96-325)) was
polyubiquitinated by incubation with ubiquitin, ubiquitin-activating
enzyme (E1), and UbcH5a or OsUBC5a/b, a rice E2. Thus, the
EL5 domains III, IV, and the C-terminal region are sufficient to
catalyze ubiquitin transfer to the MBP moiety in cooperation with E2.
It was also demonstrated that replacement of Cys153 by Ser
abolished the E3 activity. Our structural analysis suggested that the
RING-H2 finger domain of EL5 binds E2. We expressed the EL5 RING-H2
finger domain as a fusion protein with MBP (MBP-EL5(96-181) and
MBP-EL5(129-181), respectively) and determined its E3 activity. When
MBP-EL5(129-181) was incubated with ubiquitin, E1 and
OsUBC5b in an in vitro ubiquitination assay, only
one ubiquitinated derivative of the fusion protein was detected. When
MBP-EL5(96-181) was used in the same experiment, several ubiquitinated
derivatives of the fusion protein were observed (Fig.
4, lanes 3 and 4).
The latter result was similar to that obtained with MBP-EL5(96-325)
(Fig. 4, lanes 1 and 2). These findings showed
that only the RING-H2 finger domain was sufficient to bind E2, but that
the polyubiqutination chain reaction was disturbed in the small
construct, MBP-EL5(129-181), probably because the MBP moiety
sterically hindered the development of a polyubiquitin chain. Therefore
we decided to carry out the in vitro ubiquitination assay
using MBP-EL5(96-181). Since Zn2+ is needed for the
correct folding of the EL5 RING finger domain, we tested its effect in
the ubiquitination assay. No ubiquitination was detectable in the
presence of EDTA (Fig. 4, lanes 5 and 6), but the
activity was restored by addition of excess ZnSO4 (Fig. 4,
lanes 7 and 8). Thus, Zn2+
facilitates effective EL5-mediated ubiquitination by structurally stabilizing the EL5 RING-H2 finger domain.
Identification of Residues in the EL5 RING-H2 Finger Domain That
Interact with OsUBC5b--
To identify EL5 residues that interact with
E2, we recorded amide 15N and 1H chemical
shifts of the EL5 RING-H2 finger domain as a function of the
concentration of OsUBC5b. Residues located close to
OsUBC5b in the E3/E2 complex were anticipated to exhibit
large changes in their chemical shifts, because the shift depends on
the residue's magnetic environment. The HSQC signal of EL5(96-181)
had indicated that the conserved domain remained unchanged
(i.e. unstructured) in the presence of OsUBC5b,
implying that it was not involved in the EL5/OsUBC5b
interaction. Therefore we used EL5(129-181) in the NMR titration experiments.
In these experiments, the extreme broadening of the signals observed in
several residues indicated intermediate exchange on the NMR time scale.
From the titration curve that was plotted on the basis of the chemical
shift variations observed in the RING-H2 finger domain, the
stoichiometry between RING-H2 finger domain and OsUBC5b
(1:1) and the dissociation constant (Kd ~1 × 10 Intense interest in RING finger proteins has arisen because of
their role in human disease and their widespread occurrence. In higher
plants, numerous genes for RING finger proteins have been identified.
For example, 387 RING finger proteins have been predicted from a search
of the Arabidopsis genome data base (33). Although RING
finger proteins play important roles in plants, there is no information
on their structure and/or the relationship between the structure and function.
The EL5 RING-H2 Finger Domain Interacts with
UbcH4/5a-Type Ubiquitin-conjugating Enzymes--
Our
structural-based analysis suggested that the RING-H2 finger domain of
EL5 could bind E2. NMR titration experiments and an in vitro
ubiquitination assay showed that the RING-H2 finger domain of EL5 could
bind the E2, OsUBC5b, along a groove formed by a cluster of
hydrophobic residues. The primary sequence of the EL5 RING-H2 finger
domain shows high similarity with other RING finger domains that
interact with UbcH4/5a (Fig.
6a). In fact, the fusion
protein of EL5 with MBP was polyubiquitinated by incubation with E1 and
UbcH5a. OsUBC5b, the E2 used in the present study, is highly
similar to UbcH4/5a (6). Thus, available evidence suggests that the EL5
RING-H2 finger domain belongs to the group of UbcH4/5a-type E2 binding
domains. Consequently, EL5 is a UbcH4/5a-type E2 binding E3.
Identification of Residues in the RING Finger Domain That Are
Critical for E2 Interaction--
The primary sequences of various RING
finger domains are compared in Fig. 6a. Four groups can be
distinguished on the basis of RING finger domain interaction with other
proteins. The first group consists of ubiquitin ligases that cooperate
with UbcH4/5a, but not with UbcH7/8; it includes EL5 and AO7 (34).
Members of the second group, including HHARI (35), interact with
UbcH7/8, but not with UbcH1 and UbcH5. c-Cbl forms a group on its own; it is E3-interacting both with UbcH7 (36) and UbcH4 (37). The fourth
group includes KAP-1 (38) and RAG1 (14). Its members appear to function
in the formation of macromolecular assemblages.
The result of the EL5-E2 NMR titration experiment detects an altered
chemical environment for amide groups on EL5. The observed chemical shift changes reveal direct contacts as well as dynamic conformational changes at a particular position or its close proximity. Our NMR titration experiments also shed some light on the structural basis of the functional classification of the four groups of RING finger domains. Seven residues of the EL5 RING-H2 finger domain (Val136, Cys137, Ala147,
Arg148, Glu160, Thr171,
Leu174) displayed significant perturbations of chemical
shift. Five residues (Leu138, Val162,
Asp163, Met164, Trp165) were
undetectable in EL5/OsUBC5b complex (Fig. 5). Among these twelve residues (highlighted in Fig. 6b), six
(Val136, Cys137, Leu138,
Val162, Trp165, and Leu174;
magenta in Fig. 6b) are well conserved in E2
binding RING finger domains (Group 1, 2, and
3; Fig. 6a). All of these residues are hydrophobic and may be the most important ones for hydrophobic interaction with E2. In fact, the spatial position of these residues is
almost identical in c-Cbl and CNOT4 (Fig. 6, b-d).
Interestingly, the pair of hydrophobic and Trp residues located at the
first and fourth position after the third pair of zinc chelating
residues (positions 162 and 165 in EL5) is conserved in almost all RING finger domains that are known or expected to act as E3. Only in CNOT4
and HHARI, the position of the two residues are interchanged as shown
Fig. 6d. This suggests that this pair of hydrophobic and Trp
residues may be important for E2 binding. The residues map to identical
positions on the
In this study, we determined the three-dimensional structure of the
RING-H2 finger domain of EL5, and found that it closely resembles other
RING finger domains. The surface and charge distribution of the
putative E2 binding site, a groove formed by N-loop, C-loop, and
PDB and BMRB Accession Code--
The coordinates of the final
structures and the structural constraints used for the calculations
have been deposited in the RCSB Protein Data Bank (accession code
1IYM). The chemical shift values of the 1H,
13C, and 15N resonances have been deposited in
the BioMagResBank data base (accession number 5459).
-sheets (
1,
Ala147-Phe149;
2,
Gly156-His158), one
-helix
(Cys161-Leu166), and two large N- and
C-terminal loops. It is stabilized by two tetrahedrally coordinated
zinc ions. This structure is similar to that of other RING finger
domains of proteins of known function. From structural analogies, we
inferred that the EL5 RING-H2 finger is a binding domain for
ubiquitin-conjugating enzyme (E2). The binding site is probably formed
by solvent-exposed hydrophobic residues of the N- and C-terminal loops
and the
-helix. We demonstrated that the fusion protein with
EL5-(96-181) and maltose-binding protein (MBP) was polyubiquitinated
by incubation with ubiquitin, ubiquitin-activating enzyme (E1), and a
rice E2 protein, OsUBC5b. This supported the idea that the
EL5 RING finger domain is essential for ubiquitin-ligase activity of
EL5. By NMR titration experiments, we identified residues that
are critical for the interaction between the EL5 RING-H2 finger and
OsUBC5b. We conclude that the RING-H2 finger domain of EL5
is the E2 binding site of EL5.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amino group of a substrate Lys residue. This reaction requires the
sequential action of three enzymes: (i) an activating enzyme (E1) that
forms a thiol ester with the carbonyl group of Gly76 in
ubiquitin, (ii) a conjugating enzyme (E2) that transiently carries the
ubiquitin as a thiol ester, and (iii) a ligase (E3) that transfers the
activated ubiquitin from the E2 to the substrate Lys residue. The
efficiency and high selectivity of ubiquitination reactions depend on
the accuracy of E3 action. All known E3s utilized either of catalytic
domains, the RING finger domain or the HECT domain (11). The structures
of RING finger domains have been determined by NMR (12, 13) and x-ray
(14, 15). However, the relationship between molecular function and high
order structure has not yet been established.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-thiogalactopyranoside (IPTG) at a
concentration of 1 mM. For EL5(129-181) and EL5(96-181), ZnSO4 (final concentration 100 µM) was added
at the same time. After 3 h the bacteria were harvested by
centrifugation, and the pellets were frozen.
-mercaptoethanol, before lysis by sonication. Insoluble material
remaining after lysis was removed by centrifugation at 27,000 × g for 30 min. The supernatant was loaded onto a 5-ml Ni-chelating column (Amersham Biosciences). After washing the column
with buffer (50 mM NaCl, 50 mM Tris-HCl buffer
(pH7.4), 20 µM ZnSO4, 1 mM
-mercaptoethanol, and 15 mM imidazole), protein was
eluted in buffer using 400 mM imidazole. Peak fractions
were concentrated and applied to a HiLoad Superdex 75pg 26/60 column (Amersham Biosciences) that had been equilibrated in buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 20 µM ZnSO4, 2.5 mM
-mercaptoethanol). Trx tag was removed by cleavage with 75 units of
enterokinase (Invitrogen) per 31.5 mg of protein at 37 °C for
16 h. The solution was applied to a HiLoad Superdex 75pg 26/60
column. The peak fraction was 5-fold diluted with 50 mM
Tris-HCl, 20 µM ZnSO4, and 2.5 mM
-mercaptoethanol and was applied to a 5-ml HiTrap Q column (Amersham Biosciences). Peak fractions were dialyzed against NMR buffer (100 mM NaCl, 20 mM Tris-HCl buffer, pH 7.0, 20 µM ZnSO4, 1 mM dithiothreitol),
and were concentrated using a Centricon spin dialysis tube (3-kDa
cutoff; Amicon, Inc.).
- and
-protons and the methyl side chains of Val and Leu
residues were achieved by a combination of quantitative J measurements
and NOESY data. 3J couplings were measured using
quantitative two-dimensional and three-dimensional J correlation
spectroscopy (18). Interproton distance restraints were derived from
multidimensional NOE spectra (16-18): three-dimensional
15N-separated NOESY-HSQC spectrum with a mixing time of 100 ms, three-dimensional 13C/15N-separated
NOESY-HSQC spectrum with a mixing time of 100 ms, and four-dimensional
13C/13C-separated HMQC-NOESY-HMQC spectrum with
a mixing time of 100 ms. Amide-proton exchange rates were determined by
recording a series of two-dimensional 1H-15N
HSQC spectra at different time points immediately after the H2O buffer was changed to D2O buffer. These
spectra were processed using NMRPipe software (19) and were analyzed
using Capp/Pipp/Stapp software (20). 1H, 13C,
and 15N chemical shifts were referenced to HDO (4.68 ppm at
35 °C), indirectly to TSP (13C) (21), and to liquid
ammonia (15N), respectively (22).
and
were derived from
3JHNH
coupling constants (24), short-range
NOEs (
Hi to NHi+1 and
Hi to NHi+1), and a
data base analysis of backbone (13C
, 13C
,
13C', 1H
, 15N) chemical shifts
using the program TALOS (25). Side-chain
1 angle
restraints were derived from HOHAHA connectivities and the distances
between
H and
1H and between
H and
2H, which were estimated
from NOEs and ROEs. Values for 3JH
H
and
3JNH
coupling constants were also taken into
consideration. The structures of the RING-H2 finger domain of EL5 were
calculated using the hybrid distance geometry-dynamical simulated
annealing method (26), as contained in X-PLOR 3.1 (27). For structure calculations, we used 755 interproton distance restraints (comprising 290 intraresidue, 185 sequential (|i
j| = 1), 73 medium
range (1 < |i
j| < 5), and 207 long range (|i
j|
5)
restraints) obtained from heteronuclear three- and four-dimensional NOE
spectra. In addition to the NOE-derived distance restraints, 18 distance restraints for 9 hydrogen bonds and 140 dihedral angle
restraints (46
, 47
, 38
1, and 9
2) were included in the calculation. A final set of 15 lowest energy structures was selected from 100 calculations. None of
them had NOE and dihedral angle violations of >0.5 Å and >5°,
respectively. Structural statistics calculated for the final 20 structures are summarized in Table I. Statistics did not change
significantly when the 40 lowest energy structures were used for
calculation. The average coordinate of the ensembles of the final 15 structures was subjected to 500 cycles of Powell restrained energy
minimization to improve stereochemistry and non-bonded contacts. The
structural statistics for the restrained energy minimized average
structure are also summarized in Table I. Figures were generated using
MOLMOL (28).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
a, structure of EL5. b-d,
two-dimensional 1H-15N HSQC spectra of
EL5-(129-181) (b), EL5-(96-181) (c), and
EL5-(129-181) unfolded upon addition of EDTA (d).
Sequential assignments of backbone amides are shown in
b.
and C
signals of the preceding residue, while the latter
additionally provides the 15N, HN, C
, and
15N, HN, C
intraresidue correlations. The assignments
were checked and extended to backbone H
and CO signals, and to
1H and 13C side-chain signals by correlations
observed in the three-dimensional spectra, 15N-separated
HOHAHA-HSQC, HBHA(CO)NH, HNCO, C(CO)HN, and HCCH-TOCSY. Side-chain
1H and 13C signals in aromatic regions were
assigned to aromatic amino acids according to cross-peak patterns
observed in 1H-13C CT-HSQC and CT-HSQC-relay
spectra that were optimized for aromatic side chains. The aromatic H
and C
signals were then correlated to sequentially assigned
backbone signals through cross-peaks observed in three-dimensional
15N-separated NOESY-HSQC and
13C/15N-separated NOESY-HSQC spectra and a
four-dimensional 13C/13C-separated NOESY spectrum.
-protons were derived from 3JHNH
coupling constants (24), intraresidue
NOEs between HN and
-protons, and sequential NOEs between Gly
-protons (i) and HN protons (i+1).
-protons
were stereospecifically assigned using J couplings obtained from HNHB
(29) and HOHAHA-HSQC spectra, and by intraresidue
-
distances
estimated from NOE/ROE spectra, as described by Clore and Gronenborn
(17). In some cases, information from local secondary structure and
short-range NOEs was used to facilitate stereospecific assignments.
Signals from all Val
-methyl groups were stereospecifically assigned
from estimates of 3JNC
(30),
3JNH
(29), and
3JHNH
coupling constants and from NOEs
between Val
-
and
-
protons. Leu
-methyl group signals
were stereospecifically assigned from NOE patterns observed in
three-dimensional NOESY, ROESY, and four-dimensional NOESY spectra.
angles were
determined from 3JHNH
coupling constants
derived from H
/HN intensity ratios measured in HNHA experiments.
Slowly exchanging amide protons were assigned in
1H-15N HSQC hydrogen exchange experiment and
were identified as protons involved in interresidue hydrogen bonds. The
-strand and helical domains indicated by the NOE and J coupling data
were corroborated by secondary structural elements predicted by the
observed displacements of C
and C
chemical shifts from their
random-coil values (31). In summary, the analysis indicated that the
RING-H2 finger domain of EL5 contained two
-strands and an
-helix
in a
arrangement.
, and C' coordinates of the final 15 structures (Fig. 2a) were well
aligned, except for residues 129-131 at the N terminus and residues
179-181 at the C terminus. For the residues 132-178, the root mean
square deviation (r.m.s.d.) for backbone heavy atoms was 0.34 Å,
compared with 0.72 Å for all heavy atoms. A ribbon diagram (Fig.
2b) representing the backbone conformation of the restrained, energy-minimized mean structure of the RING-H2 finger domain illustrates its
fold (
1,
Ala147-Phe149;
2,
Gly156-His158;
1,
Cys161-Leu166). There is a long flexible loop
at each side of the
structure (N-loop,
Val133-Glu146; C-loop,
Gly167-Val180).
Structural statistics for the RING-H2 finger domain of EL5
View larger version (17K):
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Fig. 2.
a, best-fit superposition of the
backbone (N, C , and C') atoms of the final 15 structures of the
RING-H2 finger domain of EL5. The structures are superimposed on
the energy-minimized average structure using the backbone coordinates
of residues 132-178. b, ribbon diagram of the
energy-minimized average structure of the RING-H2 finger domain of
EL5.
, C', O) atomic r.m.s.d. values
for the
-strand- and
-helix-forming residues between the EL5 RING
finger domain and RAG-1 or c-Cbl are 0.73 and 1.30 Å,
respectively.
View larger version (43K):
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Fig. 3.
a, comparison of the free RING finger
domain of EL5 with the E2-bound form of c-Cbl and the dimerization
domain of RAG1. The additional -helices at the N and C termini
of RAG1 are shown in yellow. b, surface potential
representations of these domains. Positively charged areas are
blue, and negatively charged areas are red. The
hydrophobic grooves in c-Cbl and EL5 are indicated by
arrows. The atomic coordinates for c-Cbl and RAG1 were
downloaded from the Protein Data Bank at Brookhaven National Laboratory
(accession numbers: 1FBV and 1RMD, respectively).
-helix each at its N- and C terminus (Fig.
3a). The RAG1 dimer interface is stabilized by an extensive
hydrophobic core containing two clusters of three Phe residues in these
additional
-helices (14). In accordance with the observation that
c-Cbl is a monomer, its RING finger domain lacks the dimer-stabilizing
N- and C-terminal
-helices. As the EL5 RING-H2 finger domain
resembles c-Cbl in this respect, it is not expected to form dimers. The
c-Cbl RING finger domain binds E2 along a hydrophobic groove (indicated
by arrows in Fig. 3b) that is formed by the
-helix of the
structure and the two zinc-chelating N- and
C- terminal loops (Ref. 15, compare electron potential map in Fig.
3b). On the contrary, the RING finger domain of RAG1, which
is a DNA-binding protein without ubiquitin ligase (E3) activity (32),
does not possess this groove. Its N- and C-terminal loops are closer
together with the remaining space occupied by the side chain of
Arg57 (Fig. 3b). Thus, the groove that enables
the hydrophobic interaction between c-Cbl and E2 is occupied by a basic
group (Arg57) in RAG1, preventing interactions between RAG1
and E2. The residues in c-Cbl that form the hydrophobic contact with E2
(Ile383, Cys404, Ser407,
Trp408, Ser411, and Pro417) (15)
are mostly conserved in the RING-H2 finger domain of EL5
(Val136, Cys161, Trp165,
Ser168, and Pro173) as shown in Fig.
3b. The spatial arrangements of these hydrophobic residues
is quite similar in the two molecules (Fig. 3b), suggesting that EL5 should bind E2 similarly as c-Cbl does.
View larger version (31K):
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Fig. 4.
EL5 RING-H2 finger domain-catalyzed
auto-ubiquitination in vitro. MBP-EL5(96-325)
(lanes 1 and 2), MBP-EL5(96-181) (lanes
3 and 4) and MBP (lanes 9 and 10)
were incubated at 35 °C with ATP, ubiquitin, E1, and
OsUBC5b (E2) for 1 h, and were then subjected to
SDS-PAGE followed by immunoblotting with an anti-MBP antibody.
MBP-EL5(96-181) was incubated with EDTA before starting the assay
(lanes 5-8), in the absence (lanes 5 and
6) or in the presence of an excess amount of
ZnSO4 (lanes 7 and 8). Ladders of
bands at higher molecular weights (lanes 2, 4,
and 8) indicate the occurrence of ubiquitination.
5 M) were calculated. Seven residues
(Val136, Cys137, Ala147,
Arg148, Glu160, Thr171, and
Leu174) displayed significant chemical shift perturbations
upon complex formation with OsUBC5b (Fig.
5a). The amide signals of 5 residues (Leu138, Val162, Asp163,
Met164, and Trp165) were not detectable due to
extreme broadening of the signal. We ascribe this phenomenon to an
exchange between the free and bound forms on the chemical shift time
scale. The residues with the chemical shifts most sensitive (chemical
shift perturbations,
=
1HN + 0.1
15N greater than 0.15 ppm) to complex formation were mapped on the free form structure of the EL5 RING-H2 finger domain (Fig. 5b). These residues are all localized on
one side of the molecule where they form the E2 binding surface. The location of this binding site is in good agreement with that of the
binding site of the c-Cbl RING finger domain for E2 (15).
View larger version (40K):
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Fig. 5.
a, NMR chemical shift perturbation
of the EL5 RING-H2 finger domain upon binding to OsUBC5b.
Changes in the NMR chemical shifts of RING-H2 finger domain ( ) as
induced by complex formation with OsUBC5b, were calculated
by the function
=
HN (pink) + 0.10
15N (magenta). The light blue bars indicate
resonances broadened beyond recognition. b, two views of the
surface of the EL5 RING-H2 finger domain. Residues that showed highly
sensitive backbone amide chemical shift (
> 0.15 ppm) are
colored magenta. Residues marked in red
were not detectable due to extreme broadening of the signal after
binding of OsUBC5b. The left view is in the same
orientation as the models in Fig. 3, while the right view is
from the opposite side.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (49K):
[in a new window]
Fig. 6.
a, alignment of amino acid sequences of
RING finger domains. The Cys and His residues responsible for zinc
binding are marked yellow. Residues that are
conserved or conservatively exchanged with respect to the EL5 RING-H2
finger domain are marked blue and light blue,
respectively. Asterisks indicate residues that are crucial
for the binding of E2 by EL5, c-Cbl and CNOT4, respectively.
b, EL5 RING finger domain. Residues that exhibited a high
sensitive amide chemical shift in NMR titration experiments are
highlighted. Residues that are well and poorly conserved between E2
binding RING finger domains (Group 1, 2, and 3) are shown in
magenta and gray, respectively.
Pro173, which cannot be detected in a 15N HSQC
spectrum, is shown in yellow. c, c-Cbl RING
finger domain. Residues that interact with UbcH7 are colored
magenta (14). d, CNOT4 RING finger domain.
Residues that showed a high sensitive amide chemical shift in NMR
titration experiment, and those that are critical for UbcH5b
interaction are colored magenta (39).
-helix of the E2 binding RING finger domains and
form the center of the hydrophobic groove together with the hydrophobic
residue (Val136 for EL5, Ile383 for c-Cbl and
Leu16 for CNOT4) of loop 1. A detailed comparison of the
free EL5 RING finger domain with the E2-bound form of c-Cbl suggested
that the side-chain conformations (
1) of the Trp
residues in both molecules are different (Fig. 3). This difference of
conformations may not reflect a principle difference between the
structures of EL5 and c-Cbl, but may be caused by E2 binding. In fact,
the Trp at this position is required for E3 activity, because its
replacement by Ala reduced E3 activity in c-Cbl (36). Moreover, E2
binding was completely abolished in a CNOT4 mutant in which
Ile45 was replaced by Ala (13).
-helix, is essentially identical to that of the RING finger domain
of c-Cbl, a UbcH7-binding protein. We demonstrated that the EL5 RING-H2
finger domain is crucial for E3 activity. Moreover, we identified key
residues for EL5 interaction with a rice E2, OsUBC5b. We
clarified for the first time the functional and structural basis of E2
binding by RING finger domain in plants. It is hoped that these
insights will facilitate the understanding of E2 recognition by ATL
family RING finger proteins, which are widespread in higher plants.
![]() |
ACKNOWLEDGEMENTS |
---|
The purified recombinant mouse E1 was kindly provided by Dr. Keiji Tanaka and Dr. Toshiaki Suzuki of the Tokyo Metropolitan Institute of Medical Science.
![]() |
FOOTNOTES |
---|
* This work was supported in part by a grant from the Rice Genome Project PR-2201, MAFF, Japan (to E. K.) and by a grant from the Bio-oriented Technology Research Advancement Institution, Japan (to T. Y.).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 atomic coordinates and the structure factors (code 1IYM) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
The chemical shift values of the 1H, 13C, and 15N resonances have been deposited in the BioMagResBank data base (accession number 5459).
¶ To whom correspondence should be addressed. Tel. and Fax: 81-298-38-7006; E-mail: ekatoh@nias.affrc.go.jp.
Published, JBC Papers in Press, February 14, 2003, DOI 10.1074/jbc.M210531200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: MBP, maltose-binding protein; r.m.s.d., root mean square deviation.
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REFERENCES |
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