From the Bijvoet Center for Biomolecular Research,
Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands, the
§ Biomedical Research Laboratories, Sankyo, Limited, 2-58 Hiromachi 1-chome, Shinagawa-ku, Tokyo 140, Japan, and the
Laboratory for Physiological Chemistry and the Centre for
Biomedical Genetics, University Medical Center Utrecht,
3508 AB Utrecht, The Netherlands
Received for publication, October 11, 2000, and in revised form, November 17, 2000
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ABSTRACT |
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The NOT4 protein is a component of the CCR4·NOT
complex, a global regulator of RNA polymerase II transcription. Human
NOT4 (hNOT4) contains a RING finger motif of the
C4C4 type. We expressed and purified the
N-terminal region of hNOT4 (residues 1-78) encompassing the RING
finger motif and determined the solution structure by heteronuclear
NMR. NMR experiments using a 113Cd-substituted hNOT4 RING
finger showed that two metal ions are bound through cysteine residues
in a cross-brace manner. The three-dimensional structure of the hNOT4
RING finger was refined with root mean square deviation values of
0.58 ± 0.13 Å for the backbone atoms and 1.08 ± 0.12 Å for heavy atoms. The hNOT4 RING finger consists of an The CCR4·NOT complex was first detected in Saccharomyces
cerevisiae as a global transcription regulator, affecting
transcription of multiple functionally unrelated genes positively as
well as negatively (1). The complex consists of CCR4
(carbon catabolite repressor
4), CAF1 (CCR4-associated
factor 1, also known as POP2), the five NOT
proteins (NOT1-5), and several unidentified proteins (1). The yeast
NOT genes have been identified in a screen for elevated
HIS3 expression (2-4). The HIS3 gene contains
two core promoters, TC, a TATA-less element, and
TR, a canonical TATA sequence (5, 6). Mutations in
NOT genes selectively elevate transcription from
TC (2-4). Besides repressing genes involved in histidine biosynthesis (HIS3 and HIS4), NOT proteins also
affect transcription of genes involved in pheromone response
(STE4), nuclear fusion (BIK1), and RNA polymerase
II transcription (TBP) (2, 3). The CCR4 gene
product regulates expression of ADH2 and other genes involved in nonfermentative growth, cell wall integrity, and ion sensitivity (7-9). CCR4 exists in a complex with other proteins (10),
and two-hybrid screening with CCR4 identified CAF1 (11, 12) and DBF2 (a
cell cycle-regulated kinase) (9, 13) as binding partners. Recently, it
was found that CCR4 and CAF1 reside with the NOT proteins in a 1.2-MDa
complex (1). Besides physical interactions between CCR4, CAF1, and NOT
proteins, there is also a functional association. Mutations in the
NOT, CCR4, and CAF1 genes lead to
similar, but not identical, phenotypes (1, 14). Interestingly,
mutations in NOT1, NOT3, NOT5, and
CAF1 genes suppressed a mutation in SRB4, which is an
essential component of the RNA polymerase II holoenzyme and required
for the expression of most protein-coding genes. This suggests that the
yeast CCR4·NOT complex has a very general role in RNA polymerase II
transcription (15).
Recently, the human counterpart of the yeast CCR4·NOT complex has
been identified (16). cDNAs for four subunits,
hNOT2,1 hNOT3, hNOT4, and
human CALIF (CAF1-like factor),
were isolated and characterized. Like yeast NOT4, hNOT4 interacts with
yeast NOT1 and an N-terminally truncated hNOT1 protein, and hNOT4
complements a not4-null mutation in yeast (16). Human NOT4
contains two protein motifs in its N-terminal region, a RING finger and
an RNA recognition motif (16). The N-terminal part of the protein is
evolutionarily conserved, in contrast to the C-terminal part (16). The
RNA recognition motif has been implicated in binding of single-stranded
nucleic acids (reviewed in Ref. 17). The RING finger is found in a
large number of proteins in animals, plants, and viruses involved in
distinct cellular functions (reviewed in Refs. 18 and 19). RING fingers
are thought to mediate protein-protein interactions, and RING finger
proteins are often found in large multiprotein complexes (reviewed in
Refs. 18-20). Recently, an increasing amount of data showed that
several RING finger-containing proteins function as E3 ubiquitin
ligases, which target proteins for degradation (reviewed in Ref. 21).
Examples include the proto-oncogene product c-Cbl (22), which
ubiquitinates receptor protein-tyrosine kinases and the SCF
complex, containing the RING finger Rbx1 protein, which targets several
proteins, including G1 cyclins for degradation (23, 24). Possibly,
NOT4 also functions as an E3 ligase.
The RING finger motif can be 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
using its cysteine and histidine residues (reviewed in Refs. 18 and 19). By primary sequence comparison, RING finger variants have been
identified in which the zinc-coordinating ligands have been replaced
with other residues (25, 26). The structure of three C3HC4 RING fingers has been solved to date
(reviewed in Ref. 27). The solution structures of the immediate-early
EHV-1 protein from equine herpesvirus (IEEHV) and the acute
promyelocytic leukemia proto-oncogene product (PML) have been solved by
NMR methods (28, 29), and the crystal structures of the
immunoglobulin gene recombination enzyme RAG1 (30) and the c-Cbl
RING finger bound to ubiquitin-conjugating enzyme UbcH7 (31) have been
solved by x-ray diffraction. The RING finger structures of RAG1 and
IEEHV are remarkably similar, but differ considerably from the PML RING
finger structure. Despite this, all C3HC4
structures possess some common features, the most obvious being the
coordination of the two zinc atoms in a cross-brace configuration. In
this system, Cys1, Cys2, Cys5, and
Cys6 coordinate the first zinc atom, and Cys3,
His1, Cys7, and Cys8 share the
second zinc atom. The inter-zinc distance in all three structures is 14 Å.
The consensus sequence for the RING finger of NOT4 orthologs is
Cys-X2-Cys-X13-Cys-X-Cys-X4-Cys-X2-Cys-X11-16-Cys-X2-Cys. It constitutes a novel RING finger variant of a
C4C4 type in which His1 is replaced
with cysteine. Also, the spacing between the fourth and fifth
metal-coordinating residues is different. To investigate whether this
motif in NOT4 can adopt a RING finger conformation, we determined its
structure by NMR methods. We found that the overall fold of the NOT4
RING finger resembles that of the C3HC4 RING
fingers. However, important differences especially in the secondary
structure elements are notable.
Plasmids--
pET15b-hNOT4-N78 encodes the first 78 amino acids
of hNOT4 N-terminally fused to a 23-residue His6 tag. The
hNOT4-N78 insert was obtained by polymerase chain reaction using
pET15b-hNOT4-N227 as a template, a T7 primer
(5'-TAATACGACTCACTATAGGG-3'), and a hNOT4-specific primer
(5'-GCGGGATCCTATATCCTTTGCAGCTCTTCCTG-3'). The resulting polymerase
chain reaction fragment was digested with XhoI and
BamHI and ligated into pET15b (Novagen) digested with the
same restriction enzymes. The DNA sequence of this fragment was
verified by sequence analysis using an automated ABI310 sequencer (PerkinElmer Life Sciences).
Purification of hNOT4-N78--
Escherichia coli
BL21(DE3) bacteria containing the pET15b-hNOT4-N78 plasmid were grown
in LB medium containing 0.4% glucose at 37 °C and induced at
A600 = 0.75 with 1 mM
isopropyl-
To overexpress 15N-labeled,
15N/13C-labeled, and 15N/10%
13C-labeled hNOT4-N78, bacteria were grown in synthetic
medium (6.0 g/liter Na2HPO4·2H2O,
3.0 g/liter KH2PO4, 0.5 g/liter NaCl, 1 mM MgSO4, 20 µM
CaCl2, 36 nM
FeSO4·7H2O, 20 µM
ZnCl2, and 5 mg/liter thiamine) containing 0.5 g/liter
15NH4Cl as the sole nitrogen source and either
4.0 g/liter [12C]glucose or 2.5 g/liter
[13C]glucose or a mixture of 2.25 g/liter
[12C]glucose and 0.25 g/liter [13C]glucose
as the only carbon source. Induction took place at
A600 = 0.7-0.75, and proteins were purified as
described above.
Protein concentrations were determined using Bio-Rad protein assays
employing bovine NMR Measurements--
NMR experiments were carried out at 300 K
and pH 7.0 on a Bruker DRX600 apparatus equipped with a
triple-resonance z-gradient probe, unless indicated
otherwise. For the backbone resonance assignments, three-dimensional
HNCO, three-dimensional CBCA(CO)NH, three-dimensional HNHACB,
three-dimensional NOESY-(15N,1H)-HSQC, and
three-dimensional TOCSY-(15N,1H)-HSQC spectra
were recorded; and for side chain resonance assignments, three-dimensional H(C)CH TOCSY and three-dimensional (H)CCH TOCSY spectra were recorded (reviewed in Ref. 32). For the assignments of
proline residues, a CDCA(NCO)CAHA spectrum was recorded (33).
Stereospecific assignments of
3JHN-H
113Cd-1H HSQC spectra were recorded using a
broadband z-gradient probe. The delays for magnetization
transfer were set to 6, 9, and 12 ms. Twenty t1
increments of 1000 scans were recorded. The 113Cd chemical
shifts are reported relative to 1 M CdSO4. NOE
distance restraints were obtained from two-dimensional NOE,
three-dimensional NOESY-(15N,1H)-HSQC, and
three-dimensional NOESY-(13C,1H)-HSQC spectra
with mixing times of 50 and 150 ms. NOE cross-peaks were classified as
strong (1.8-2.7 Å), medium (1.8-3.5 Å), weak (1.8-5.0 Å), and
very weak (1.8-6.0 Å) using secondary structure elements for
calibration. All NMR spectra were processed using the NMRpipe package
(38) and analyzed using the program REGINE (39).
Structure Calculations--
Structure calculations were
performed with the program X-PLOR (40, 41). To correct for multiple
atom selection, we used the sum averaging option as implemented in
X-PLOR. Distance restraints containing diastereotopic groups were
corrected as described by Fletcher et al. (42).
The structures were calculated using the distance geometry protocol,
which is followed by a simulated annealing and refinement protocol
using standard parameters. Until this stage, information about the zinc
coordination was not included. Then, the constraints between metal
ligands (3.6 Å < S
The final set of structures was analyzed using the program PROCHECK-NMR
(44). The Ramachandran plot of Fig. 4 was produced with the program
PROCHECK-NMR (44). Molecular figures were prepared using the program
MOLMOL (45).
Expression and Assignments--
The first 78 residues of hNOT4,
which contain the C4C4 RING finger, were fused
to an N-terminal His6 tag. This protein was overexpressed
and 15N- and 15N/13C-isotopically
labeled in bacteria and purified to homogeneity as described under
"Materials and Methods." The sequence of the first 78 residues of
hNOT4 is shown in Fig. 1 and is compared with the corresponding region in yeast NOT4 and with the
C3HC4 RING fingers of IEEHV and RAG1.
Sequence-specific assignments were obtained by the analysis of
three-dimensional CBCA(CO)NH and three-dimensional HNCACB spectra and
by the application of a three-dimensional CDCA(NCO)CAHA spectrum (33)
for 11 proline residues. The assignments for 10 residues of the
23-residue His6-tagged region were also obtained.
1H-15N correlations for the remaining residues
in the His6-tagged region were not observed, and the
resonances for those residues were therefore left unassigned.
Stereospecific assignments for methyl protons in the prochiral center
of Val12, Leu16, Leu21, and
Leu52 were obtained. In addition to this, 14 out of 64 Metal-binding Sites--
To determine the number of metal ions
present in the hNOT4 RING finger and the coordination system, we
performed 113Cd-1H HSQC experiments using
hNOT4-N78 in which the zinc ions were replaced with cadmium ions.
113Cd-substituted hNOT4-N78 was obtained by adding
113Cd- EDTA to a final concentration of 4 mM to zinc-containing hNOT4-N78, and the exchange of zinc
with 113Cd was monitored using
1H-15N HSQC spectra.
Since most of the resonances were shifted by the substitution, the
assignments of 1H and 15N resonances of
113Cd-hNOT4-N78 had to be confirmed by three-dimensional
NOESY-(1H,15N)-HSQC and three-dimensional
TOCSY-(1H,15N)-HSQC spectra. Fig.
2A shows the chemical shift
differences between 113Cd-hNOT4-N78 and Zn-hNOT4-N78.
Cys17, Cys33, and Cys56 displayed
the largest differences in both the 1H and 15N
chemical shifts, which is correlated with hydrogen bonding from amide
protons to the sulfur atoms of the zinc cluster (see below). Residues
other than cysteines displayed only a slight change in chemical shifts,
showing that exchange took place without breaking the integrity of the
whole structure. Fig. 2B shows the two-dimensional 113Cd-1H HSQC spectrum of
113Cd-hNOT4-N78 displaying the correlation between two
cadmium resonances and the 1H chemical shifts belonging to
the Structure of hNOT4-N78--
The three-dimensional structure was
determined for the zinc-containing form of hNOT4-N78. In total, 397 distance restraints (20 intraresidue, 171 sequential, 74 medium-range,
and 132 long-range) and 32 angle constraints (19
The structure of hNOT4-N78 consists of three long loops, L1 (residues
12-22), L2 (residues 27-38), and L3 (residues 49-61), and an
The Ramachandran plot for residues 12-61 of the 30 calculated
structures is shown in Fig.
4A. In addition to
Gly34 and Gly51, Met18 in L1 and
Arg57 in L3 also have positive
The distance between the two zinc atoms is 14.9 ± 0.3 Å, which
is slightly longer than the well conserved value of 14 Å as found in
the C3HC4 RING finger structures of PML (29),
IEEHV (28), RAG1 (30), and Cbl (31). This could be due to the fact that
there is a 4-residue spacing between Cys33 and
Cys38 in hNOT4-N78, whereas only 2 residues separate the
same zinc-ligating residues in the other three
C3HC4 RING fingers.
Several NH protons surrounding the metal atoms display large upfield
shifts in the amide proton resonance positions upon exchanging zinc
with cadmium, as shown in Fig. 2A. These protons were found within a short distance of the sulfur atoms of the cysteines involved in metal ligation in 30 structures. For example, the distances between
NH and the sulfur atom are 2.90 ± 0.25 Å for
NH(Cys17)-S(Cys14), 2.94 ± 0.38 Å for
NH(Cys33)-S(Cys31), 2.55 ± 0.08 Å for
NH(Cys56)-S(Cys53), 2.45 ± 0.06 Å for
NH(Cys38)-S(Cys14), and 2.54 ± 0.31 Å for NH(Cys41)-S(Cys38). The chemical shift of
the amide proton is sensitive to hydrogen bond length (37), and
substitution of zinc with cadmium causes a small change in hydrogen
bond length due to the increase in the metal-sulfur bond length (48,
49). It is likely that these observed large chemical shift changes upon
exchanging zinc with cadmium reflect the presence of hydrogen bonds
from NH protons to sulfur atoms. These hydrogen bonds could contribute
to stabilize the coordination of the zinc ion.
Comparison with C3HC4 RING Finger
Structures--
Fig. 5 shows a schematic
drawing of the RING finger structures of hNOT4-N78, IEEHV, and RAG1.
The
The root mean square deviation values of C-
In the hydrophobic region of the C3HC4 RING
finger, 2 central residues are well conserved (Phe28 and
Ile33 in IEEHV, Phe309 and Ile314
in RAG1, and Met400 and Leu405 in c-Cbl). In
hNOT4-N78, the backbone atoms of Ile37 and
Trp42 are located in a similar position in space. The
orientation of the side chains, however, is slightly different. The
side chain of Ile37 is still positioned in the core,
similar to the conserved Phe28 of IEEHV and
Phe309 of RAG1; but the side chain of Trp42 is
pointing away from Ile37, although they have limited
contact. Instead, the side chain of Ile45 is pointing
toward the side chain of Ile37 and participating in the
hydrophobic core, taking over the role of Trp42. Consistent
with this, Ile45 is invariant in all known NOT4 orthologs
to date (Fig. 1 and data not shown).
Recently, the crystal structure of the c-Cbl RING finger bound to the
ubiquitin-conjugating enzyme UbcH7 has been reported (31). The
UbcH7-binding site on the c-Cbl RING finger is provided by the shallow
groove formed by the Implication for Function--
So far, the exact role of the hNOT4
RING finger domain in the CCR4·NOT complex is unclear. Yeast
complementation analysis showed that unlike full-length hNOT4, a hNOT4
protein lacking the RING finger motif does not complement its yeast
counterpart.2 Surprisingly,
the RING finger domain of hNOT4 is not required for interaction with
hNOT1, as this is mediated by the nonconserved C-terminal part of hNOT4
(data not shown). Analogous to the RING finger of c-Cbl (22,
31), the hNOT4 RING finger may serve in a role as a E3 ligase in
(poly)ubiquitination of proteins. In accordance with this proposal, we
have identified in yeast two-hybrid screens components of the ubiquitin
pathway as NOT4 RING interaction
partners.3 The structure of
the NOT4 RING finger displays the features that were observed in the
Cbl-UbcH7 interaction. The described structure of the NOT4
C4C4 RING finger allows the rational design of
phenotypic mutations that affect these interactions and subsequent
testing of the effects on the in vivo function of NOT4. This
should provide a better understanding of transcription regulation by
the CCR4·NOT complex.
-helix and
three long loops that are stabilized by zinc coordination. The overall
folding of the hNOT4 RING finger is similar to that of the
C3HC4 RING fingers. The relative orientation of
the two zinc-chelating loops and the
-helix is well conserved. However, for the other regions, the secondary structural elements are distinct.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
-D-thiogalactopyranoside and 10 µM ZnCl2 for 2 h at 37 °C. After
harvesting, cells were washed with phosphate-buffered saline and
suspended in lysis buffer (100 mM Tris-HCl (pH 7.9), 20%
sucrose, 0.1 mM EDTA, 100 µM
ZnCl2, 1 mM imidazole, 0.1% Triton X-100, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM
-mercaptoethanol, 0.2 mM sodium bisulfite, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin). Lysozyme was
added to a final concentration of 200 µg/ml, followed by one freeze-thaw cycle to lyse the cells. After addition of KCl to 300 mM, the lysate was centrifuged for 1 h at 250,000 × g. The cleared lysate was applied to a
Ni2+-nitrilotriacetic acid-agarose column (QIAGEN Inc.)
equilibrated in lysis buffer containing 300 mM KCl. After
washing the column with buffer A (20 mM Tris-HCl (pH 7.9),
10% glycerol, 50 mM KCl, 20 µM
ZnCl2, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM
-mercaptoethanol, 0.2 mM sodium
bisulfite, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml
pepstatin) containing 1 and 16 mM imidazole, protein was
eluted in buffer A using a linear gradient from 16 to 400 mM imidazole. Peak fractions were pooled and dialyzed
against buffer B (20 mM potassium phosphate (pH 7.0), 10 µM ZnCl2, 0.5 mM
phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 0.2 mM sodium bisulfite, 0.1 µg/ml aprotinin, and 0.1 µg/ml
leupeptin) containing 100 mM KCl (buffer B100)
and applied to a MonoQ HR 10/10 column (Amersham Pharmacia Biotech)
equilibrated in buffer B100. After washing with buffer
B100, the column was developed using a linear gradient from
100 to 500 mM KCl. hNOT4-N78 eluted from the column at
~170 mM KCl and was purified to homogeneity as judged by
Coomassie Blue staining of protein gels. hNOT4-N78 was concentrated
using a Centricon spin dialysis tube (10-kDa cutoff; Amicon, Inc.).
-globulin as a standard. Protein yields ranged
between 10 and 15 mg/liter of bacterial culture.
protons and
1 dihedral
angle restraints were obtained using three-dimensional
NOESY-(13C,1H)-HSQC and
TOCSY-(15N,1H)-HSQC spectra (reviewed in Ref.
32) recorded with short mixing times (50 ms and 20 ms, respectively)
and a three-dimensional HNHB spectrum (34). Stereospecific assignments
for the methyl protons in the prochiral center of leucine and valine
were obtained by 10% 13C-labeled hNOT4-N78 (35).
coupling constants were
derived from a three-dimensional HNHA experiment (36) and converted
into
dihedral angle restraints. Hydrogen bond restraints were
obtained by observing the cross-hydrogen bond
15N-13C coupling constants using a long-range
HNCO spectrum (37).
(Cys)-S
(Cys) < 3.9 Å) were added to the restraints list, and an additional refinement
protocol was performed. The zinc atoms were added at the average
position of four metal-ligating atoms. Thereafter, the geometric
restraints to the zinc atoms were added, and additional refinement was performed. The geometric restraints are as
follows: bond lengths for Zn-S
(Cys) are 2.3 Å and
geminal bond angles of S
(Cys)-Zn-S
(Cys)
and Zn-S
(Cys)-C
(Cys) are 109.5°
(43).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Comparison of the first 78 residues of hNOT4,
encompassing the C4C4 RING finger, with the
C4C4 RING finger of yeast NOT4 (residues
20-99) and with the C3HC4 RING fingers of
IEEHV (residues 1-68), RAG1 (residues 277-350), and c-Cbl (residues
368-441). Zinc-coordinating residues are indicated with
asterisks. Sequence alignment was performed using the
ClustalW algorithm (46). yNOT4, yeast NOT4.
-methylene protons were also obtained.
protons of the coordinating cysteine residues. The observed
chemical shift values of cadmium resonances, 687.5 and 714.4 ppm, are
both typical for cadmium(II) coordinated by four sulfur ligands. The
113Cd-1H HSQC spectrum shows that
Cys14, Cys17, Cys38, and
Cys41 (site 1) share one metal ion that resonates at 687.5 ppm and that Cys33, Cys53, and
Cys56 (site 2) share the other metal ion at 714.4 ppm. The
correlation of the remaining Cys31 was not observed in any
of the 113Cd-1H HSQC spectra with magnetization
delays of 6, 9, and 12 ms. However, the last metal-coordinating ligand
of site 2 was assigned to Cys31 since the resonance value
for this metal is characteristic for cadmium(II) coordinated by four
sulfur atoms. These results show that the NOT4 RING finger contains two
zinc ions, which are ligated in a cross-brace manner, similar to the
canonical RING fingers.
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Fig. 2.
A, chemical shift differences of the
amide protons and nitrogens between the zinc and cadmium forms of
hNOT4-N78. The values of were calculated as follows:
=
(Zn form)
(Cd form). B,
113Cd-1H HSQC spectrum of cadmium-substituted
hNOT4-N78 showing the three-bond J-coupling between
113Cd and
protons of the cysteine residues.
and 13
1) obtained from various two- and three-dimensional
spectra were used for the structure calculations. Four hydrogen bond
restraints, which were identified in a long-range HNCO spectrum, were
also included. Finally, 200 structures were calculated, and 30 structures with a low energy were selected. Fig.
3A shows the superposition of
the backbone atoms of these 30 calculated structures, and a summary of
structural statistics is given in Table I
and Fig. 3 (B-D). The region between residues 12 and 61 is
well structured, whereas the N- and C-terminal parts are disordered due
to lack of NOEs. Root mean square deviation values in the structured
region versus the mean coordinates are 0.58 ± 0.13 Å for the backbone atoms and 1.08 ± 0.12 Å for all heavy
atoms.
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Fig. 3.
A, superposition of the backbone
atoms of 30 simulated annealing structures of hNOT4-N78. The three loop
regions (residues 12-22, 27-38, and 49-61) are colored
green; the region containing the -helix (residues 39-48)
is in red; residues 23-26 containing the helical turn are
in orange; and the remaining unstructured region is in
white. Zinc atoms are indicated as magenta balls.
B-D, overview of the structural parameters of residues
10-65 of hNOT4-N78. B, number of distance restraints per
residue; C and D, root mean square deviation
(RMSD) for backbone atoms and all heavy atoms
versus the residue number, respectively.
Structural statistics of hNOT4-N78
-helix (residues 39-48) between the second and third loops. Of the
8 cysteine residues that are involved in zinc coordination, the first
(Cys14, Cys17), second (Cys31,
Cys33), and forth (Cys53, Cys56)
pairs are located in L1, L2, and L3, respectively. The remaining 2 cysteines (Cys38, Cys41) are located at the end
of L2 and in the
-helix. Region 23-26 is recognized as a helical
turn in 15 out of 30 calculated structures using secondary structure
analysis in PROCHECK-NMR. All proline residues were found to have a
trans-configuration on the basis of the observation of NOEs
between the
i
1 and
i
protons. The three loops are stabilized by the coordination with the
zinc ions and by hydrophobic interactions. Leu16 in L1 and
Pro54 in L3 form a hydrophobic area with Ile37
in L2 and Ile45 belonging to the
-helix. The
conformations of both L1 and L3 are remarkably similar.
Val12-Pro20 can be superimposed on
Gly51-Tyr60 with a root mean square deviation
of 0.18 Å versus the mean coordinates. Consistent with
this, hydrogen bonds were also identified for CO(Val12)-NH(Leu21) in L1 and
CO(Gly51)-NH(Tyr60) in L3 on the basis of
hydrogen bond J-couplings (36) in a long-range HNCO spectrum.
angles in all 30 structures, although their
angles were not refined well. These
positive
angles of Met18 and Arg57 were
confirmed by observation of cross-correlated relaxation of HN-N and
HN-H-
dipolar interactions of the multiple lines (47). For this, we
measured the intensity ratio of the 15N-coupled amide
proton resonances in 15N-labeled hNOT4-N78. Fig.
4B shows the NH multiplets of these residues together with
those of Glu49 and Leu52, which have negative
angles. For Met18 and Arg57, the peak
heights of the inner two multiplet components were lower than those of
the outer two multiplets.
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Fig. 4.
A, Ramachandran plot of the 30 structures of hNOT4-N78 (residues 12-61). The shading
indicates the favorable and unfavorable regions of the plot; the
darker the shading, the more favorable the region. The
triangles show the glycine residues, and the
squares show the remaining residues. B,
cross-section along the F2 dimension through selected amide
proton resonances in the 15N-coupled
1H-15N HSQC spectrum.
-helix in hNOT4-N78 is well conserved in IEEHV and RAG1.
However, the
-sheet that exists in both IEEHV and RAG1 is not
present in hNOT4-N78. The region corresponding to the third strand of
the
-sheet in IEEHV, which is absent in RAG1, is unstructured in
hNOT4-N78. The region corresponding to the first and second
-strands
adopts a loop conformation in hNOT4-N78. Although these regions have a
different conformation in hNOT4-N78 and the other two RING fingers,
they are located in a similar position and have a similar
orientation.
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Fig. 5.
Schematic view of the RING finger structures
of NOT4, IEEHV, RAG1, and c-Cbl. Please note that in RAG1, the
first zinc-binding site of the RING finger is part of the binuclear
zinc cluster. The conserved -helix is colored red, and
the remaining helices and helical turns are in yellow. The
-sheet is colored green. Zinc ions are indicated as
balls and colored magenta for conserved zinc and
gray for the remaining zinc in RAG1. Residues that
coordinate zinc ions are shown and colored yellow for
cysteine and blue for histidine.
atoms between the mean
coordinates of the C4C4 RING finger and
C3HC4 RING finger structures are 1.7 Å for
IEEHV (45 C-
atoms; Protein Data Bank code 1CHC), 1.6 Å for
RAG1 (45 C-
atoms; Protein Data Bank code 1RMD), and 1.8 Å for
c-Cbl (45 C-
atoms; Protein Data Bank code 1FBV). Despite the
differences in secondary structural elements, the overall structure of
hNOT4-N78 is quite similar to the structures of IEEHV, RAG1, and
c-Cbl.
-helix and two zinc-chelating loops. It is
interesting to note that this region is well conserved with the hNOT4
C4C4 RING finger in structural but not chemical terms. Also, hNOT4 has a shallow groove that is formed by
Leu16 in L1; Pro54 in L3; and
Arg44, Ile45, and Glu49 in the
-helix. Although L1 and L3 come close to each other, there is a
groove between the two loops and the
-helix. It is important to note
that although the structure is conserved, the side chains are not.
Ile383, Ser407, Trp408, and
Ser411, which form the binding site for UbcH7 in the c-Cbl
RING, are replaced by Leu16, Arg44,
Ile45, and Asp48, respectively, in hNOT4.
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FOOTNOTES |
---|
* This work was supported in part by grants from the European Communities Program "Access to Large Scale Facilities" and by the Netherlands Foundation for Chemical Research.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 1E4U) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
¶ The first two authors contributed equally to this work.
** Supported by the Dutch Cancer Foundation.
Supported by a postdoctoral fellowship from the Netherlands
Organization for Scientific Research-Medical Sciences.
§§ Supported by a Pioneer grant from the Netherlands Organization for Scientific Research-Medical Sciences.
¶¶ To whom correspondence should be addressed. Tel.: 31-30-253-4035; Fax: 31-30-243-7623; E-mail: Boelens@NMR.chem.uu.nl.
Published, JBC Papers in Press, November 21, 2000, DOI 10.1074/jbc.M009298200
2 M. J. de Ruwe, F. A. J. van den Heuvel, M. A. Collart, and H. T. M. Timmers, unpublished observation.
3 T. K. Albert, F. A. J. van den Heuvel, Y. I. A. Legtenberg, and H. T. M. Timmers, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: hNOT, human NOT; E3, ubiquitin-protein isopeptide ligase; NOESY, nuclear Overhauser effect correlation spectroscopy; HSQC, heteronuclear single quantum correlation spectroscopy; TOCSY, total correlation spectroscopy; NOE, nuclear Overhauser effect.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Liu, H. Y.,
Badarinarayana, V.,
Audino, D. C.,
Rappsilber, J.,
Mann, M.,
and Denis, C. L.
(1998)
EMBO J.
17,
1096-1106 |
2. | Collart, M. A., and Struhl, K. (1993) EMBO J. 12, 177-186[Abstract] |
3. | Collart, M. A., and Struhl, K. (1994) Genes Dev. 8, 525-537[Abstract] |
4. | Oberholzer, U., and Collart, M. A. (1998) Gene (Amst.) 207, 61-69[CrossRef][Medline] [Order article via Infotrieve] |
5. | Iyer, V., and Struhl, K. (1995) Mol. Cell. Biol. 15, 7059-7066[Abstract] |
6. | Mahadevan, S., and Struhl, K. (1990) Mol. Cell. Biol. 10, 4447-4455[Medline] [Order article via Infotrieve] |
7. |
Denis, C. L.
(1984)
Genetics
108,
833-844 |
8. |
Denis, C. L.,
and Malvar, T.
(1990)
Genetics
124,
283-291 |
9. |
Liu, H. Y.,
Toyn, J. H.,
Chaing, Y. C.,
Draper, M. P.,
Johnston, L. H.,
and Denis, C. L
(1997)
EMBO J.
16,
5289-5298 |
10. | Draper, M. P., Liu, H. Y., Nelsbach, A. H., Mosley, S. P., and Denis, C. L. (1994) Mol. Cell. Biol. 14, 4522-4531[Abstract] |
11. | Draper, M. P., Salvadore, C., and Denis, C. L. (1995) Mol. Cell. Biol. 15, 3487-3495[Abstract] |
12. | Sakai, A., Chibazakura, T., Shimizu, Y., and Hishinuma, F. (1992) Nucleic Acids Res. 20, 6227-6233[Abstract] |
13. | Toyn, J. H., Araki, H., Sugino, A., and Johnston, L. H. (1991) Gene (Amst.) 104, 63-70[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Bai, Y.,
Salvadore, C.,
Chiang, Y. C.,
Collart, M. A.,
Liu, H. Y.,
and Denis, C. L.
(1999)
Mol. Cell. Biol.
19,
6642-6651 |
15. |
Lee, T. I.,
Wyrich, J. J.,
Koh, S. S.,
Jennings, E. G.,
Godbois, E. L.,
and Young, R. A.
(1998)
Mol. Cell. Biol.
18,
4455-4462 |
16. |
Albert, T. K.,
Lemaire, M.,
Berkum, N. L.,
Gentz, R.,
Collart, M. A.,
and Timmers, H. T. M.
(2000)
Nucleic Acids Res.
28,
809-817 |
17. | Nagai, K., Oubridge, C., Ito, N., Avis, J., and Evans, P. (1995) Trends Biochem. Sci. 20, 235-240[CrossRef][Medline] [Order article via Infotrieve] |
18. | Borden, K. L., and Freemont, P. S. (1996) Curr. Opin. Struct. Biol. 6, 395-401[CrossRef][Medline] [Order article via Infotrieve] |
19. | Saurin, A. J., Borden, K. L., Boddy, M. N., and Freemont, P. S. (1996) Trends Biochem. Sci. 21, 208-214[CrossRef][Medline] [Order article via Infotrieve] |
20. | Borden, K. L. (2000) J. Mol. Biol. 295, 1103-1112[CrossRef][Medline] [Order article via Infotrieve] |
21. | Freemont, P. S. (2000) Curr. Biol. 10, R84-R87[CrossRef][Medline] [Order article via Infotrieve] |
22. |
Joazeiro, C. A.,
Wing, S. S.,
Huang, H.,
Leverson, J. D.,
Hunter, T.,
and Liu, Y. C.
(1999)
Science
286,
309-312 |
23. |
Kamura, T.,
Koepp, D. M.,
Conrad, M. N.,
Skowyra, D.,
Moreland, R. J.,
Iliopoulos, O.,
Lane, W. S.,
Kaelin, W. G., Jr.,
Elledge, S. J.,
Conaway, R. C.,
Harper, J. W.,
and Conaway, J. W.
(1999)
Science
284,
657-661 |
24. |
Skowyra, D.,
Koepp, D. M.,
Kamura, T.,
Conrad, M. N.,
Conaway, R. C.,
Conaway, J. W.,
Elledge, S. J.,
and Harper, J. W.
(1999)
Science
284,
662-665 |
25. | Boddy, M. N., Freemont, P. S., and Borden, K. L. (1994) Trends Biochem. Sci. 19, 198-199[CrossRef][Medline] [Order article via Infotrieve] |
26. |
Regnier, C. H.,
Tomasetto, C.,
Moog-Lutz, C.,
Chenard, M. P.,
Wendling, C.,
Basset, P.,
and Rio, M. C.
(1995)
J. Biol. Chem.
270,
25715-25721 |
27. | Borden, K. L. (1998) Biochem. Cell Biol. 76, 351-358[CrossRef][Medline] [Order article via Infotrieve] |
28. | Barlow, P. N., Luisi, B., Milner, A., Elliott, M., and Everett, R. (1994) J. Mol. Biol. 237, 201-211[CrossRef][Medline] [Order article via Infotrieve] |
29. | Bordon, K. L., Boddy, M. N., Lally, J., O'Reilly, N. J., Martin, S., Howe, K., Solomon, E., and Freemont, P. S. (1995) EMBO J. 14, 1532-1541[Abstract] |
30. | Bellon, S. F., Rodgers, K. K., Schatz, D. G., Coleman, J. E., and Steitz, T. A. (1997) Nat. Struct. Biol. 4, 586-591[Medline] [Order article via Infotrieve] |
31. | Zheng, N., Wang, P., Jeffrey, P. D., and Pavletich, N. P. (2000) Cell 102, 533-539[Medline] [Order article via Infotrieve] |
32. | Sattler, M., Schleucher, J., and Griesinger, C. (1999) Prog. NMR Spec. 34, 93-158 |
33. | Bottomley, M. J., Macias, M. M., Liu, Z., and Sattler, M. (1999) J. Biomol. NMR 13, 381-385[CrossRef][Medline] [Order article via Infotrieve] |
34. | Düx, P., Whitehead, B., Boelens, R., Kaptein, R., and Vuister, G. W. (1997) J. Biomol. NMR 19, 301-306 |
35. | Neri, D., Szyperski, T., Otting, G., Senn, H., and Wüthrich, K. (1989) Biochemistry 28, 7510-7516[Medline] [Order article via Infotrieve] |
36. | Vuister, G. W., and Bax, A. (1993) J. Am. Chem. Soc. 115, 7772-7777 |
37. | Cordier, F., and Grzesiek, S. (1999) J. Am. Chem. Soc. 121, 1601-1602[CrossRef] |
38. | Delaglio, F., Grzesiek, S., Vuister, G. W., Zhu, G., Pfeifer, J., and Bax, A. (1995) J. Biomol. NMR 6, 277-293[Medline] [Order article via Infotrieve] |
39. | Kleywegt, G. J., Vuister, G. W., Padilla, A., Knegtel, R. M. A., Boelens, R., and Kaptein, R. (1993) J. Magn. Reson. Ser. B 102, 166-176[CrossRef] |
40. | Nilges, M., Clore, G. M., and Gronenborn, A. M. (1988) FEBS Lett. 229, 317-324[CrossRef][Medline] [Order article via Infotrieve] |
41. | Brünger, A. T. (1992) X-PLOR Version 3.1: A System for X-ray Crystallography and NMR , Yale University Press, New Haven, CT |
42. | Fletcher, C. M., Jones, D. N., Diamond, R., and Neuhaus, D. (1996) J. Biomol. NMR 8, 292-310 |
43. | Neuhaus, D., Nakaseko, Y., Schwabe, J. W. R., and Klug, A. (1992) J. Mol. Biol. 228, 637-651[Medline] [Order article via Infotrieve] |
44. | Laskowski, R. A., Rullman, J. A., MacArther, M. W., Kaptein, R., and Thornton, J. M. (1996) J. Biomol. NMR 8, 477-486[Medline] [Order article via Infotrieve] |
45. | Koradi, R., Billeter, M., and Wüthrich, K. (1996) J. Mol. Graph. 14, 51-55[CrossRef][Medline] [Order article via Infotrieve] |
46. | Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994) Nucleic Acids Res. 22, 4673-4680[Abstract] |
47. | Crowley, P., Ubbink, M., and Otting, G. (2000) J. Am. Chem. Soc. 122, 2968-2969[CrossRef] |
48. |
Goodfellow, B. J.,
Rusnak, F.,
Moura, I.,
Domke, T.,
and Moura, J. J. G.
(1998)
Protein Sci.
7,
928-937 |
49. | Ayhan, M., Xiao, Z., Lavery, M. J., Hamer, A. M., Nugent, K. W., Scrofani, S. D. B., Guss, M., and Wedd, A. G. (1996) Inorg. Chem. 35, 5902-5911[CrossRef] |