Solution Structure of the N-terminal Domain of the Human TFIIH MAT1 Subunit

NEW INSIGHTS INTO THE RING FINGER FAMILY*

Virginie GervaisDagger§, Didier Busso§, Emeric Wasielewski, Arnaud Poterszman, Jean-Marc Egly, Jean-Claude Thierry, and Bruno Kieffer||

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



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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 beta alpha beta beta 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 beta  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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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 beta beta alpha beta 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 alpha -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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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 BL21lambda (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-beta -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.

Cells were resuspended in 100 ml of buffer B (50 mM Tris-HCl, pH 7.5, 500 mM NaCl) containing 2.5 mM beta -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 beta -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.

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 Cbeta methylene protons were obtained for 30 residues on the basis of the patterns of HN-Hbeta and Halpha -Hbeta NOEs and JHalpha -Hbeta scalar couplings (19).

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 Sgamma 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),


d=<FENCE><FR><NU>d<SUB><UP>ref</UP></SUB><SUP>−6</SUP></NU><DE>V<SUB><UP>ref</UP></SUB></DE></FR> · V</FENCE><SUP>−1/6</SUP> (Eq. 1)
where dref was the (dij-6)-1/6 average distance calculated for all distances ranging from 2.7 to 5 Å between amide, Halpha , and Hbeta 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.
d<UP>±</UP>=d±(0.125 · d<SUP>2</SUP>) (Eq. 2)

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 phi  and 21 psi  dihedral angles deduced from JHN-Halpha couplings and from the secondary structure analysis were used as weak constraints (Delta phi  = ±50° and Delta psi  = ±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 Nepsilon 2 or Ndelta 1 of the His28 ring as the fourth ligand for the second zinc-binding site allowed us to unambiguously assign Ndelta 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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 alpha  and beta  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 Hbeta 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.

The distribution of the inter-residue NOE restraints used to calculate the structure together with the Calpha 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 Hbeta assignments were possible. B, stereo view of the Calpha 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 alpha beta beta alpha beta fold of the MAT1 RING finger domain with the two zinc ligation sites (ZNI and ZNII). alpha -helices and beta -strands are displayed with pink boxes and cyan arrows, respectively. Secondary structure NOEs evidencing the three-stranded beta -sheet are shown as blue arrows. Hydrogen bond constraints deduced from solvent exchange experiments are indicated by red dashed lines.

Experimental restraints and structural statistics over the 20 lowest energy structures are summarized in Table I. The Calpha 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 beta beta alpha beta fold typical of RING finger domains and presents an unusual one-turn alpha  helix in its N terminus. The core of the domain consists of a three-stranded antiparallel beta -sheet, comprising residues Leu21-Val23 (beta 1), Thr29-Cys31 (beta 2), and Arg59-Gln61 (beta 3) packed along a two-turn alpha -helix (helix alpha 2, residues Glu32-Val40).


                              
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Table I
Structural statistics of the MAT1 RING finger

The triple-stranded beta -sheet is clearly defined by an unambiguous pattern of NOEs Halpha -HN, Halpha -Halpha , and HN-HN (Fig. 1C). Slowly exchanging amide protons are observed for residues Met22, Val23, Leu30, and Gln61 in the beta -strands, which indicate that they are hydrogen-bonded. A regular pattern of Halpha -HN(i,i+3), Halpha -Hbeta (i,i+3), and Halpha -HN(i,i+4) NOEs (28) together with upfield-shifted Halpha resonances (29) and solvent-protected amide protons define two helical regions (alpha 1 and alpha 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 alpha 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 phi  and psi  angles are systematically located in the loop regions.

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 alpha 1 helix is connected to the central beta 1-strand, which is linked to beta 2 by a short loop harboring the two zinc ligands Cys26 and His28. A two-turn alpha -helix (Glu32-Val40) is positioned between the beta 2-strand and the beta 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 alpha 2 to the beta 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.

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 beta  sheet; Val35, Leu38, and Phe39 in the helix alpha 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.

Structure Comparison with Known RING Finger Domains-- The topology of the three beta  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 Calpha equivalent atoms with an rmsd value of 1.7 Å, whereas the comparison with the solution structure of the IEEHV RING yields 28 equivalent Calpha 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 beta -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.

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 alpha 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.

The use of stereospecific constraints on most of the Hbeta methylene protons allows a precise determination of the side chain orientations (angle kappa 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 Calpha of the four ligands (Cys6, Cys9, Cys31, and Cys34) onto the equivalent Calpha 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.

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 beta 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.

A second specific structural feature of MAT1 concerns the presence of a structured region including one turn of an alpha -helix (alpha 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 alpha 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.

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 alpha 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.



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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.

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 beta 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.

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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

* 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/).

Dagger 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


    ABBREVIATIONS

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES


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