From the Institut de Génétique et de Biologie Moléculaire et Cellulaire, Université Louis Pasteur, 67400 Illkirch-Cedex, France
Received for publication, August 31, 2000, and in revised form, October 25, 2000
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ABSTRACT |
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The human MAT1 protein belongs to the
cyclin-dependent kinase-activating kinase complex,
which is functionally associated to the transcription/DNA repair factor
TFIIH. The N-terminal region of MAT1 consists of a C3HC4 RING finger,
which contributes to optimal TFIIH transcriptional activities. We
report here the solution structure of the human MAT1 RING finger domain
(Met1-Asp65) as determined by
1H NMR spectroscopy. The MAT1 RING finger domain presents
the expected MAT1 is one of the nine subunits of the human transcription/DNA
repair factor TFIIH, which is known to play a crucial role in the
transcription of class II genes as well as in DNA repair through the
nucleotide excision repair pathway (1). This factor may be resolved
in vivo and in vitro into two structural
subcomplexes: the TFIIH core and the cyclin-dependent
kinase (cdk)1-activating
kinase (CAK) complex (2, 3). The CAK complex is composed of the
catalytic subunit cdk7, the regulatory subunit cyclin H, and a third
partner, MAT1, originally defined as a stabilizing and activating
factor (4, 5). This complex is also found in its free form within the
cell and preferentially phosphorylates cdks known as key components of
the cell cycle progression (6). As part of the TFIIH factor, the CAK
complex phosphorylates different substrates of the transcription
apparatus including TATA box-binding protein, TFIIE, TFIIF, the
C-terminal domain of the largest subunit of RNA polymerase II, and
regulatory factors such as p53 and some nuclear receptors (7). The cdk7
kinase activity of the CAK complex is stimulated by a combined action
of cyclin H and MAT1 binding and cdk7 phosphorylation (8). Moreover,
the subunit MAT1 is involved in a substrate selection process choosing
either cdk or another transcription apparatus to be phosphorylated (9, 10). To further investigate the role of the CAK complex in
transcription when part of TFIIH and to elucidate the specific role of
MAT1, a structural study of all CAK components was undertaken. The
crystal structure of cyclin H was solved (11), and a structural model between cdk7 and cyclin H was built (12). Recently, a combination of
sequence analysis and biochemical data showed that MAT1 can be divided
into three functional domains: an N-terminal RING finger domain, a
central coiled coil domain, and a C-terminal domain rich in hydrophobic
residues. Functional analysis revealed that the C terminus strongly
interacts in vitro, as well as in vivo, with the
cdk7-cyclin H complex and stimulates cdk7 kinase activity (13).
The authors showed that the median domain of MAT1 is involved in CAK
anchoring to the core TFIIH through interactions with both XPD and
XPB helicases. It has also been shown that the deletion of the N
terminus, which presents the consensus sequence of a C3HC4 RING finger
domain, inhibits the basal transcription as well as the phosphorylation
of the C-terminal domain of RNA polymerase II when engaged in a
transcription complex (13). This enlightens the potential role
of the RING finger domain of MAT1 in the architecture of the
preinitiation complex of the transcription.
To complete the functional data available for the MAT1 N-terminal
domain and to provide a structural basis for further structure-function relationships, we determined its solution structure using proton NMR
spectroscopy. The comparison with previously reported RING finger
structures shows that the MAT1 RING finger domain presents a classical
Expression and Purification of the Recombinant RING Finger
Domain--
The nucleotide sequence encoding the fragment
corresponding to the RING finger domain of MAT1
(Met1-Asp65) was amplified by polymerase chain
reaction and inserted into the appropriate Escherichia coli
expression vector. The cDNA of the human MAT1 gene was amplified by
polymerase chain reaction using a forward primer, which introduces a
BamHI site at the 5' end, and a reverse primer containing a
stop codon and an EcoRI site at the 3' end. After digestion
by BamHI and EcoRI (New England Biolabs), the
polymerase chain reaction fragment was inserted into the pGEX-4T2
expression vector (Amersham Pharmacia Biotech). A starter
culture of 500 ml of LB containing 200 µg/ml ampicilin was inoculated
with the E. coli strain BL21
Cells were resuspended in 100 ml of buffer B (50 mM
Tris-HCl, pH 7.5, 500 mM NaCl) containing 2.5 mM NMR Spectroscopy--
40 µl of D2O was added to
the 400 µl of the protein solution for the lock, and
2,2-dimethyl-2-silapentane-5-sulfonate was used as the internal
chemical shift reference. Homonuclear TOCSY (14), NOESY (15),
and DQF-COSY (16) spectra were recorded at four temperatures (283, 290, 298, and 303 K) on either Bruker DRX600 or DMX750 spectrometers with
spectral widths of 7000 Hz (600 MHz) or 8333 Hz (750 MHz) in both
dimensions and a relaxation delay of 2 s. Water signal suppression
was achieved by presaturation or by using a WATERGATE sequence (17).
Slowly exchanging amide protons were identified by recording 70-ms
NOESY spectra at 283 K and at different delays after addition of
D2O to the lyophilized sample. Processing was performed on
an SGI Octane SE computer using the program FELIX 97 (Biosym
Technologies) and on an SGI INDY R5000 computer using XWIN-NMR
software (Bruker). Spectra were assigned with the FELIX 97 package
(Biosym Technologies) and the XEASY program (18). A single set
of resonances was assigned for 63 of the 65 residues of the MAT1
fragment; the two missing residues were the N-terminal Met and
Arg54. Stereospecific assignments of C Structure Calculations--
A first set of distance constraints
was obtained by classifying peak volumes measured on a 70-ms
NOESY spectrum recorded at 298 K as strong, medium, and weak,
corresponding to distances of 2.7, 3.7 and 5.0 Å, respectively. 60 structures were generated using the restrained simulated annealing
protocol implemented in the program X-PLOR 3.851 (20, 21). Eight
additional distances of 2.4 Å were added between the two zinc atoms
and the S
The final set of experimental constraints included 897 distances
derived from NOEs (75 intra-residue, 286 sequential, 210 medium
range, and 326 long range) and 22 distances derived from hydrogen
bonding patterns. 22 The MAT1 RING Domain Solution Structure--
To determine the
solution structure of the N-terminal RING finger domain of the human
MAT1 subunit, a polypeptide corresponding to residues
Met1-Asp65 was produced. The definition of the
domain boundaries was based on mild proteolysis experiments and on the
comparison of orthologous sequences. This domain, when expressed as a
GST fusion protein in E. coli, is soluble and can be easily
purified. After removal of the GST tag and subsequent gel filtration,
the MAT1 1-65 fragment led to a monodisperse solution and could be
concentrated up to 2 mM. As expected for a canonical C3HC4
RING finger domain (27), atomic absorption and mass spectrometry
experiments showed that the human MAT1 RING finger domain binds two
zinc atoms (data not shown).
A first analysis of NOESY spectra recorded on the MAT1 RING
finger domain indicated the presence of both
The distribution of the inter-residue NOE restraints used to calculate
the structure together with the C
Experimental restraints and structural statistics over the 20 lowest energy structures are summarized in Table
I. The C
The triple-stranded
The RING domain is stabilized by two mononuclear zinc sites separated
by 14 Å. Cys6, Cys9, Cys31, and
Cys34 form one zinc-binding site (C4), whereas
Cys26, Cys46, and Cys49 with
His28 form the second zinc-binding site (C3H). The first
cysteine pair (Cys6, Cys9) stabilizes the
N-terminal part of the peptide. The loop L1 containing the
Finally, the MAT1 RING finger core is stabilized by a network of highly
conserved hydrophobic residues among MAT1 orthologs (Fig.
2), namely Leu19 in the loop
L1; Met22, Leu30, and Val60 in the
internal face of the Structure Comparison with Known RING Finger Domains--
The
topology of the three
The comparison of the MAT1 RING structure with other available RING
structures confirms that the consensus C3HC4 zinc-binding sequence
defines a conserved structural motif, which constitutes a widely used
molecular scaffold. Sequence comparisons of various RING sequences
show, however, that this consensus sequence incorporates regions of
high sequence diversity with variable spacing between the conserved
zinc-binding residues. One of these regions is located between the
first two pairs of zinc-binding ligands and encompasses the loop L1. In
most RING sequences, this loop contains 10-12 residues, whereas the
MAT1 sequence incorporates 16-17 residues (Fig. 2B). The
observation of a well defined secondary structure element (helix
The use of stereospecific constraints on most of the H Functional Implications of MAT1 Structure--
In a recent paper,
Busso et al. (13) have established the role of the
N-terminal RING finger domain of MAT1 in the activation of
transcription in a TFIIH-dependent manner. They have also
observed that the presence of the RING finger domain allows an optimal phosphorylation of the RNA polymerase II C-terminal domain. In agreement with the general role of RING domains in mediating
protein-protein interactions (34), it has been suggested that the MAT1
RING domain interacts with other factors within the preinitiation
complex of transcription, although no partner has yet been found. When compared with other RING structures, the MAT1 RING domain presents specific features that could be involved in the MAT1 activities.
First, the occurrence of a stretch of conserved hydrophobic residues
located in the vicinity of the
A second specific structural feature of MAT1 concerns the presence of a
structured region including one turn of an
Analysis of the surface electrostatic potential shows that the MAT1
RING domain is highly positively charged because of the presence of
several basic side chains of arginines and lysines (Fig.
5). Whereas the extended distribution of
the nine positive charges observed at the domain surface is a specific
feature of higher eucaryote sequences of MAT1, it is worth noting that
the four positively charged residues that are conserved from human to
yeast (Lys20, Arg54, Lys55, and
Arg59) are located in the same area, forming a positive
patch. Two highly conserved acidic residues located on the helix
(Glu32 and Asp36) form a small negative patch
on the exposed side of the helix
The role of the MAT1 RING finger domain within the transcription
complex needs to be studied further by site-directed
mutagenesis. Two targets need to be identified from the
three-dimensional structure and probed: (i) the positively charged
residues that may be involved in the modulation of the CAK
phosphorylation activity through electrostatic interactions with either
the phosphorylated C-terminal domain, the DNA, or another component of
the preinitiation complex, and (ii) the solvent-exposed hydrophobic
residues in the strand
Since the first member of the Really Interesting New Gene protein
family was identified in 1991 (37), only a few structural data are yet
available, partly because of the natural propensity of these domains to
aggregate and precipitate when being expressed and concentrated. So far
no general structure-activity relationship for the RING finger family
has been established. The solution structure of the N-terminal part of
MAT1 is the fourth structure of a RING domain that is now available.
These new data provide interesting insights into the structure of the
loop region of variable length between the first two pairs of
zinc-binding residues that could be useful in other biological
contexts. Finally, the structural variability of the loop L1 and the
charge distribution at the surface of the RING domains could be
essential factors that modulate RING finger activities.
topology with two interleaved zinc-binding
sites conserved among the RING family. However, the presence of an
additional helical segment in the N-terminal part of the domain and a
conserved hydrophobic central
strand are the defining features of
this new structure and more generally of the MAT1 RING finger
subfamily. Comparison of electrostatic surfaces of RING finger
structures shows that the RING finger domain of MAT1 presents a
remarkable positively charged surface. The functional implications of
these MAT1 RING finger features are discussed.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
topology with a "cross-brace" arrangement of the eight
zinc-binding ligands. The MAT1 RING finger domain is characterized by
the presence of an additional short
-helix within the N-terminal
loop and by an extended basic surface. The functional implications of
these features, which are specific to all of the MAT1 RING finger
orthologous sequences, are discussed, as are the new insights brought
by this fourth high resolution structure of a RING finger.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
(DE3) transformed by the
pGEX-4T2 recombinant vector and grown overnight at 37 °C. Cells were
pelleted, resuspended in a fresh medium, and used to inoculate 6 liters
of LB medium containing 200 µg/ml ampicilin at an
A600 nm of 0.1. Cultures were grown at
37 °C to an A600 nm of 0.6-0.8, and the
expression of recombinant proteins was induced by addition of 0.6 mM
isopropyl-1-thio-
-D-galactopyranoside. After
4 h, cells were harvested, washed in buffer A (20 mM
Tris-HCl, pH 7.5, 150 mM NaCl, 20% glycerol), frozen
in liquid nitrogen, and stored at
80 °C.
-mercaptoethanol and disrupted by sonication for 10 min (pulse 2/8, T = 10 °C) using a 13-mm
probe with a Vibracell 72412 sonicator at 30% intensity. The cell
extract was then centrifuged for 2 h at 45,000 rpm at 4 °C in a
Beckman R60Ti rotor. The soluble extract containing the GST-RING finger
recombinant protein was incubated during 1 h at 4 °C with 4 ml
of GSH-Sepharose resin (Amersham Pharmacia Biotech)
preequilibrated in buffer B containing 2.5 mM
-mercaptoethanol. The resin was washed in a batch with 40 volumes of
buffer B. The adsorbed proteins were eluted with 2 × 4 ml of
buffer B containing 30 mM glutathione. The fractions
(4 ml) containing the GST-RING finger protein (as judged by
SDS-polyacrylamide gel electrophoresis) were pooled, and the GST fusion
protein was cleaved with bovine thrombin (Sigma) (3 units per mg of
recombinant fusion protein) at 4 °C during 12 h. The sample was
concentrated using a Centriprep device with a 3,000-Da cut-off
(Amicon). Digestion was stopped by addition of 5 mM
Pefabloc (Roche Molecular Biochemicals), and the fraction was then
subjected to gel filtration chromatography (Amersham Pharmacia Biotech;
2.6 × 60 cm at a flow rate of 2 ml/min) in buffer C (20 mM Tris-HCl, pH 7.5, 50 mM NaCl). Recombinant RING finger protein-containing fractions were pooled and concentrated on a Centriprep device with a 3,000-Da cut-off to a final concentration of approximately 20 mg/ml (2 mM). For NMR studies, the
sample was dialyzed against buffer C containing deuterated Tris.
methylene protons
were obtained for 30 residues on the basis of the patterns of HN-H
and H
-H
NOEs and JH
-H
scalar couplings (19).
of cysteine residues and between the side chain of
His28 and the second zinc atom according to the binding
pattern deduced from the primary sequence analysis. Several rounds of
structure calculations were then analyzed in an iterative manner with
successive incorporation of initially ambiguous distance restraints.
The structure was then refined by converting the NOE cross-peak volumes (V) into target distances (d) according to the
following relationship (22),
where dref was the
(dij
(Eq. 1)
6)
1/6 average
distance calculated for all distances ranging from 2.7 to 5 Å between amide, H
, and H
protons obtained from the first round of
calculations. Vref was calculated as the
arithmetic average over all corresponding volumes. Upper (d+) and lower
(d
) limit distance restraints were derived using the empirical
relationship proposed by M. Nilges.
(Eq. 2)
and 21
dihedral angles deduced from
JHN-H
couplings and from the secondary
structure analysis were used as weak constraints (
= ±50°
and
= ±40°). The topology of the peptide was modified to
include the coordination bounds between histidine and cysteine residues
and the two zinc atoms according to tetrahedral coordination values
(23). Structure calculations run using either N
2 or N
1 of the
His28 ring as the fourth ligand for the second zinc-binding
site allowed us to unambiguously assign N
1 as the zinc-bound atom. A
set of 51 structures was retained based on the following criteria: low total energy, no NOE violation greater than 0.5 Å, and no angle violation greater than 5°. Structures were visualized using MOLMOL (24), and their structural quality was analyzed with PROCHECK (25) and
WHATIF (26). The latter program has been used to define the
structurally equivalent positions and to superimpose MAT1 structure
onto the three available structures of RING finger domains, namely RAG1
(Protein Data Bank code 1rmd; resolution 2.1 Å), immediate
early equine herpes virus (IEEHV) (Protein Data Bank code 1chc;
NOE/angle dataset, 616/10; backbone/all atoms rmsd, 0.55/1.02
Å), and promyelocytic leukemia (PML) proto-oncoprotein (Protein
Data Bank code 1bor; NOE/angle dataset, 197/26; backbone/all atoms
rmsd, 0.88/1.40 Å). Figures showing three-dimensional structures and
electrostatic surface representations were prepared with MOLMOL (24).
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
and
secondary elements with a good dispersion of resonances. The sample behaves as a
monomer in solution because the average proton line widths are in a
good agreement with the expected values for a folded 7-kDa protein. NOE
connectivities observed between H
protons of Cys6,
Cys9 and Cys31, Cys34 clearly
indicate that these residues are forming one of the two zinc-binding
sites and therefore that the two zinc atoms are bound in a
cross-brace fashion, which is one of the defining features of
the RING family.
rmsd calculated along the peptide
chain are shown in Fig. 1A.
Except for a few regions where no long range NOE could be observed, the
experimental set of NOE-derived distances allows an accurate definition
of the three-dimensional structure of the human MAT1 RING finger domain, with a backbone rmsd of 0.67 Å.
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Fig. 1.
NMR data of the human MAT1 RING
finger domain. A, the number of NOE distances as a
function of residue number is shown in violet for
intra-residue and in blue, yellow, and
red for short, medium, and long range inter-residue,
respectively. The presence of intra-residue NOE distances indicates the
residues for which stereospecified H assignments were possible.
B, stereo view of the C
trace from the 20 lowest energy
superimposed NMR structures (rmsd 0.67 Å). The two zinc atoms and the
zinc-binding residues are shown in red. C,
schematic view of the
fold of the MAT1 RING finger
domain with the two zinc ligation sites (ZNI and ZNII).
-helices and
-strands are displayed with pink boxes and cyan
arrows, respectively. Secondary structure NOEs evidencing the
three-stranded
-sheet are shown as blue arrows. Hydrogen
bond constraints deduced from solvent exchange experiments are
indicated by red dashed lines.
backbone trace of the 20 lowest energy NMR structures is shown in Fig. 1B. The
N-terminal fragment of the human MAT1 subunit adopts the
fold typical of RING finger domains and presents an unusual one-turn
helix in its N terminus. The core of the domain consists of a
three-stranded antiparallel
-sheet, comprising residues
Leu21-Val23 (
1),
Thr29-Cys31 (
2), and
Arg59-Gln61 (
3) packed along a two-turn
-helix (helix
2, residues
Glu32-Val40).
Structural statistics of the MAT1 RING finger
sheet is clearly defined by an unambiguous
pattern of NOEs H
-HN, H
-H
, and HN-HN (Fig. 1C).
Slowly exchanging amide protons are observed for residues
Met22, Val23, Leu30, and
Gln61 in the
-strands, which indicate that they are
hydrogen-bonded. A regular pattern of H
-HN(i,i+3), H
-H
(i,i+3),
and H
-HN(i,i+4) NOEs (28) together with upfield-shifted H
resonances (29) and solvent-protected amide protons define two helical
regions (
1 and
2). Some regions of the peptide chain are less
well defined (local rmsd, ~1 Å) and correspond to loops that link
the secondary structure elements, namely loop L1, which encompasses
helix
1 (Thr12-Arg15), and loop L2 between
residues Val40 and Ser56. The Ramachandran plot
(data not shown) shows that 97% of the nonglycine and nonproline
residues are located in allowed regions; the few residues presenting
unusual
and
angles are systematically located in the loop regions.
1 helix
is connected to the central
1-strand, which is linked to
2 by a
short loop harboring the two zinc ligands Cys26 and
His28. A two-turn
-helix
(Glu32-Val40) is positioned between the
2-strand and the
3-strand and contains Cys34, which
is paired with Cys31 to form the third zinc-ligand pair. A
long loop (L2) comprising the fourth pair of zinc ligands (residues
Cys46, Cys49) connects the helix
2 to the
3-strand. The overall shape of the MAT1 RING finger domain is found
to be slightly elongated, with principal axis lengths of 13.5 × 10.0 × 19.5 Å.
sheet; Val35, Leu38,
and Phe39 in the helix
2; and Leu53 and
Phe58 in the loop L2.
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Fig. 2.
Three-dimensional structure of the
human MAT1 RING finger domain and structural sequence alignment of
selected RING finger domains. A, ribbon diagram of the
MAT1 RING finger domain structure from Gln4 to
Leu62. The right side shows the molecule after a
180° rotation around the vertical axis compared with the left
side. Secondary structure elements are shown in blue
and orange. The orange color highlights specific
features of the MAT1 structure that are discussed in the text. Highly
conserved hydrophobic residues in MAT1 orthologs are shown in
green. The two zinc ligation sites (ZNI and ZNII) are shown
in red. B, structural multiple sequence alignment
of RING finger domains from MAT1, RAG1, and IEEHV proteins. Plain
circles indicate structural equivalent positions (rmsd less than
2.5 Å) between MAT1 and RAG1 or IEEHV. The metal-coordinating residues
are shown in magenta. Conserved hydrophobic residues are
shown in green. Conserved positive and negative positions
are shown in blue and red, respectively. The
boxed positions indicate the tyrosine that is strictly
conserved within MAT1 sequences as discussed in the text. For the sake
of clarity, all currently known MAT1 RING finger sequences are
displayed, whereas only a subset of selected sequences are shown for
RAG1- and IEEHV-related proteins. The abbreviations used for the
alignment are as follows: hs, Homo
sapiens; mm, Mus musculus; xl,
Xenopus laevis; mg, Marthasterias
glacialis; dm, Drosophila melanogaster;
ce, Caenorhabditis elegans; sc,
Saccharomyces cerevisiae; sp,
Schizosaccharomyces pombe; om,
Oncorhynchus mykiss; en, Emericella
nidulans; hsveb, equine herpes virus; hsv,
herpes simplex virus; cl, Candida lypotica.
Accession numbers (referring to Swiss-Prot or TrEMBL protein data
bases) of proteins used in the alignment are as follows: MAT1_hs,
p51948; MAT1_mm, p51949; MAT1_xl, p51951; MAT1_mg, p51950; MAT1_dm,
o76487; MAT1_ce, o17245; MAT1_sc, p01365; MAT1_sp, o94684; rag1_mm,
p15919; unk_sp, O14212; trifd_mm, q9wts0; rag1_xl, q91829; rag1_om,
q91187; nuvA_en, q00178; icp0_hsveb, p28990; icp0_hsv4, o39303;
icp0_hsv2 h, p28284; rad18_sc, p10862; topoRS_hs, q9unr9; pex10_cl,
q9uvf7.
strands, together with the cross-brace
arrangement of the eight zinc-binding residues of MAT1 RING domain, is
similar to that observed in two RING atomic resolution structures that
have been reported: the structure from the IEEHV protein solved by NMR
(30) and the crystal structure of the human recombination-activating
protein RAG1 dimerization domain (31). A similar cross-brace
arrangement of the zinc-binding residues was also found in the
structure of human acute PML proto-oncoprotein (32). The ribbon
diagrams of the MAT1 RING structure, together with the three previously
reported structures of RING finger domains, are shown in Fig.
3. A data base search for superimposable
folds in the Protein Data Bank using the Dali program (33) finds
structural similarities between MAT1 and the RING motifs in the RAG1
dimerization domain (31) and in the IEEHV (30). The best structural
homology score is found for the superimposition of the MAT1 structure
onto the crystal structure of RAG1. Indeed, both structures can be superimposed for 43 C
equivalent atoms with an rmsd value of 1.7 Å,
whereas the comparison with the solution structure of the IEEHV RING
yields 28 equivalent C
atom positions that superimpose with an rmsd
value of 1.99 Å (the structurally equivalent positions are indicated
by plain circles in the alignment of Fig. 2B).
However, no significant superimposition could be obtained when
comparing the MAT1 RING structure with the solution structure of the
RING finger domain from the acute promyelocytic leukemia
proto-oncoprotein PML. It is worth mentioning that the weak structural
homology observed between the RING finger domains of the human MAT1
protein and PML was also observed when comparing those of RAG1 and PML (31).
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Fig. 3.
Ribbon diagrams of MAT1, RAG1,
IEEHV, and PML structures. Helices and -sheets are shown in
orange and blue, respectively. The two zinc
ligand sites are shown in red. The orientations of MAT1,
RAG1, IEEHV, and PML RING finger structures are identical to the
orientation used in Fig. 2. The side chains of conserved residues that
stabilize the hydrophobic core are shown in green. They
correspond to Met22, Leu30, and
Val35 in MAT1; Val37, Phe45, and
Ile50 in RAG1; and Met20, Phe28,
and Ile33 in IEEHV. The residues at equivalent positions in
PML are Leu21, Leu28, and
Leu33.
1)
in this region is noteworthy and constitutes a specific feature of the
MAT1 structure (Fig. 2A). In contrast, the loop L2
containing the fourth zinc ligand pair presents the same conformation
as in other RING structures despite the sequence divergence outside the
fourth pair of zinc ligands.
methylene
protons allows a precise determination of the side chain orientations
(angle
1), in particular for the zinc-binding residues. A detailed
analysis of the zinc ligation sites in the various RING structures
reveals that the second coordination site, ZNII, is well conserved
between the different structures. When comparing the ZNII binding sites
of MAT1 and RAG1 (Fig. 4), we found a
sharp superimposition, with an rmsd of 0.29 Å, of the four MAT1 ZNII ligand side chain heavy atoms (Cys26, His28,
Cys46, and Cys48) onto the corresponding atoms
of RAG1. In the same manner, the ZNII coordination sites of RAG1 and
IEEHV can be superimposed with an rmsd of 0.32 Å. The first
coordination site is less conserved between the three RING finger
structures. Indeed, the superimposition of the C
of the four ligands
(Cys6, Cys9, Cys31, and
Cys34) onto the equivalent C
of RAG1 is poor, yielding
an rmsd of 0.57 Å, a value similar to the one obtained when comparing
RAG1 and IEEHV (rmsd of 0.54 Å). In the RAG1 structure, the first
zinc-binding site is part of a binuclear cluster, with the
Cys29 (equivalent to Cys9 in MAT1) bridging two
zinc atoms. This feature of RAG1 may explain the observed local
structure differences around the first zinc-binding site.
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Fig. 4.
Comparison of zinc coordination
sites from MAT1 and RAG1 RING finger domains. The zinc ligand side
chains of MAT1 and RAG1 are represented in red and
yellow, respectively. Their corresponding backbones are
shown in light red and light yellow.
Superimposition was done on side chain heavy atoms of MAT1 and RAG1
residues in ZNI (Cys6, Cys9, Cys31,
Cys34; Cys26, Cys29,
Cys46, Cys49) with an rmsd of 0.57 Å and in
ZNII (Cys26, His28, Cys46,
Cys49; Cys41, His43,
Cys61, Cys64) with an rmsd of 0.29 Å. The
numbering corresponds to the MAT1 RING finger sequence.
1 strand (residues Leu19,
Leu21, Met22, and Val23)
constitutes an interesting feature. Among these conserved hydrophobic residues, two are involved in the hydrophobic packing of the RING structure, namely Leu19 and Met22, whereas the
two others (Leu21 and Val23) are exposed to the
solvent. Such a stretch of hydrophobic residues is unusual in RING
sequences (Pfam zf-C3HC4 (35)) and could be related to a regulation of
the MAT1 activity.
-helix (
1), which
corresponds to the sequence insertion between the first and second
pairs of zinc-binding ligands, only observed in MAT1 sequences.
Interestingly, this helix contains a solvent-exposed tyrosine residue
(Tyr14), which is strictly conserved among all MAT1
sequences, suggesting that the helix
1 might be involved in the
packing of the RING domain with another partner. This hypothesis is
supported by the comparison of the MAT1 RING structure with the crystal
structure of RAG1, which shows that the equivalent part in RAG1
interacts with its N-terminal C2H2 zinc finger (32). Moreover, the
solvent-exposed tyrosine could be a potential site for phosphorylation
and therefore be involved in activity regulation. Recently, the crystal
structure of a complex consisting of a portion of the c-Cbl
proto-oncogene protein bound to the ubiquitin-conjugating enzyme UbcH7
and a kinase peptide was reported (36). The structure reveals that the
loop L1 of the C-terminal C-cbl RING domain interacts closely with both
the N-terminal C-cbl tyrosine kinase binding domain and the
UbcH7 partner, thus emphasizing the functional role of this region in
RING domains.
2. It is worth noting that the two
C-terminal acidic residues (Glu64 and Asp65)
are also conserved but are disordered in the structure. The presence of
positively charged patches on protein surfaces seems to be a general
feature of the RING finger domains, but the basicity of the MAT1 RING
surface is remarkable, as shown in Fig. 5. It must be stressed that
most of the mutations that affect the function of PML and IEEHV RING
finger domains involve charged residues.
View larger version (53K):
[in a new window]
Fig. 5.
Electrostatic surface
representations of MAT1, RAG1, IEEHV, and PML structures. Positive
and negative charges are shown in blue and red,
respectively. The orientations of MAT1, RAG1, IEEHV, and PML RING
finger domain surfaces are identical to the one used in Fig. 2.
Underlined residues indicate that they are highly conserved
within MAT1, RAG1, IEEHV, and PML orthologous sequences.
1 that may directly affect the stability of
the preinitiation complex. It would be of particular interest to know
whether these residues are independently related to the two distinct
functions of the MAT1 RING finger domain: the phosphorylation of the
RNA polymerase II C-terminal domain and transcription activation. To
address these points, the building of specific mutants is currently under way.
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ACKNOWLEDGEMENTS |
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We express our gratitude to M. Suzuki and K. Yamasaki at the AIST-NIBHT CREST Center of Structural Biology, Tsukuba, Japan for providing access to the 800-MHz spectrometer and for help during data collection. We are grateful to D. Moras for fruitful discussions, T. Henry for protein expertise, C. Ling for technical assistance, and E. Kellenberger for help with structure calculations. We thank the Institut de Génétique et de Biologie Moléculaire et Cellulaire staff for oligonucleotides and DNA sequencing.
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FOOTNOTES |
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* This work was supported by CNRS, INSERM, the Hopital Universitaire de Strasbourg, and the Association de la Recherche contre le Cancer.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 1G25) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
Supported by grants from the ADRERUS/LILLY.
§ Both authors contributed equally to this work.
¶ Supported by grants from the Ministère de la Recherche et de l'Enseignement Supérieur.
To whom correspondence should be addressed. E-mail:
kieffer@esbs.u-strasbg.fr.
Published, JBC Papers in Press, October 30, 2000, DOI 10.1074/jbc.M007963200
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ABBREVIATIONS |
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The abbreviations used are: cdk, cyclin-dependent kinase; CAK, cdk-activating kinase; GST, glutathione S-transferase; NOE, nuclear Overhauser effect; rmsd, root mean square deviation; IEEHV, immediate early equine herpes virus; PML, promyelocytic leukemia.
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