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

Abstract

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 by1H NMR spectroscopy. The MAT1 RING finger domain presents the expected βαββ 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 β 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 discussed1G25. 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 by1H NMR spectroscopy. The MAT1 RING finger domain presents the expected βαββ 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 β 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 discussed1G25. cyclin-dependent kinase cdk-activating kinase glutathione S-transferase nuclear Overhauser effect root mean square deviation immediate early equine herpes virus promyelocytic leukemia 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 (1Frit P. Bergmann E. Egly J.M. Biochimie ( Paris ). 1999; 81: 27-38Crossref PubMed Scopus (55) Google Scholar). This factor may be resolvedin vivo and in vitro into two structural subcomplexes: the TFIIH core and the cyclin-dependent kinase (cdk)1-activating kinase (CAK) complex (2Drapkin R. Roy G.L. Cho H. Akoulitchev S. Reinberg D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6488-6493Crossref PubMed Scopus (140) Google Scholar, 3Reardon J.T. Ge H. Gibbs E. Sancar A. Hurwitz J. Pan Z. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6482-6487Crossref PubMed Scopus (99) Google Scholar). 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 (4Tassan J.P. Jaquenoud M. Fry A.M. Frutiger S. Hughes G.J. Nigg E.A. EMBO J. 1995; 14: 5608-5617Crossref PubMed Scopus (172) Google Scholar, 5Devault A. Martinez A.M. Fesquet D. Labbe J.C. Morin N. Tassan J.P. Nigg E.A. Cavadore J.C. Doree M. EMBO J. 1995; 14: 5027-5036Crossref PubMed Scopus (201) Google Scholar). 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 (6Kaldis P. Cell. Mol. Life Sci. 1999; 55: 284-296Crossref PubMed Scopus (185) Google Scholar). 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 (7Riedl T. Egly J.M. Gene Expr. 2000; 9: 3-13Crossref PubMed Scopus (47) Google Scholar). The cdk7 kinase activity of the CAK complex is stimulated by a combined action of cyclin H and MAT1 binding and cdk7 phosphorylation (8Nigg E.A. Curr. Opin. Cell Biol. 1996; 8: 312-317Crossref PubMed Scopus (127) Google Scholar). Moreover, the subunit MAT1 is involved in a substrate selection process choosing either cdk or another transcription apparatus to be phosphorylated (9Rossignol M. Kolb-Cheynel I. Egly J.M. EMBO J. 1997; 16: 1628-1637Crossref PubMed Scopus (168) Google Scholar,10Yankulov K.Y. Bentley D.L. EMBO J. 1997; 16: 1638-1646Crossref PubMed Scopus (153) Google Scholar). 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 (11Andersen G. Poterszman A. Egly J.M. Moras D. Thierry J.C. FEBS Lett. 1996; 397: 65-69Crossref PubMed Scopus (28) Google Scholar), and a structural model between cdk7 and cyclin H was built (12Andersen G. Busso D. Poterszman A. Hwang J.R. Wurtz J.M. Ripp R. Thierry J.C. Egly J.M. Moras D. EMBO J. 1997; 16: 958-967Crossref PubMed Scopus (76) Google Scholar). 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 (13Busso D. Keriel A. Sandrock B. Poterszman A. Gileadi O. Egly J.M. J. Biol. Chem. 2000; 275: 22815-22823Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). 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 (13Busso D. Keriel A. Sandrock B. Poterszman A. Gileadi O. Egly J.M. J. Biol. Chem. 2000; 275: 22815-22823Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). 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 ββαβ 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 α-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. 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 coliexpression vector. The cDNA of the human MAT1 gene was amplified by polymerase chain reaction using a forward primer, which introduces aBamHI 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 BL21λ(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 anA 600 nm of 0.1. Cultures were grown at 37 °C to an A 600 nm of 0.6–0.8, and the expression of recombinant proteins was induced by addition of 0.6 mmisopropyl-1-thio-β-d-galactopyranoside. After 4 h, cells were harvested, washed in buffer A (20 mmTris-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 mmTris-HCl, pH 7.5, 500 mm NaCl) containing 2.5 mm β-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 mmβ-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 mmPefabloc (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. 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 (14Bax A. Davis D.G. J. Magn. Reson. 1985; 61: 306-320Google Scholar), NOESY (15Jeener J. Meier B.H. Bachmann P. Ernst R.R. J. Chem. Phys. 1979; 71: 4546-4553Crossref Scopus (4845) Google Scholar), and DQF-COSY (16Rance M. Sorensen O.W. Bodenhausen G. Wagner G. Ernst R.R. Wüthrich K. Biochem. Biophys. Res. Commun. 1983; 117: 479-485Crossref PubMed Scopus (2597) Google Scholar) 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 (17Piotto M. Saudek V. Sklenar V. J. Biomol. NMR. 1992; 2: 661-665Crossref PubMed Scopus (3539) Google Scholar). 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 (18Bartels C. Xia T.-H. Billeter M. Güntert M. Wüthrich K. J. Biomol. NMR. 1995; 5: 1-10Crossref PubMed Scopus (1607) Google Scholar). 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 Cβ methylene protons were obtained for 30 residues on the basis of the patterns of HN-Hβ and Hα-Hβ NOEs and J Hα-Hβscalar couplings (19Wagner G. Braun W. Havel T.F. Schaumann T. Go N. Wüthrich K. J. Mol. Biol. 1987; 196: 611-639Crossref PubMed Scopus (635) Google Scholar). 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 60 structures were using the in the program M. A.M. FEBS Lett. PubMed Scopus Google Scholar, Scholar). additional distances of were added between the two and the of residues and between the chain of and the to the binding the sequence of structure were then in an with of distance The structure was then by the volumes into distances to the M. NMR Full Text PDF Scopus Google Scholar), 1 was the distance for all distances to 5 between and protons obtained the first of was as the all corresponding and distance were using the by M. 2 The final set of constraints distances NOEs medium and and distances and couplings and the structure analysis were used as constraints = and = The topology of the was to the between and residues and the two to A. PubMed Scopus Google Scholar). using either or of the as the fourth for the zinc-binding site to as the A set of structures was on the and were using R. Billeter M. Wüthrich K. J. Mol. 1996; 14: Scopus Google Scholar), and structural was with R. J.M. J. Biomol. NMR. 1996; 8: PubMed Scopus Google Scholar) and G. J. Mol. 8: PubMed Scopus Google Scholar). The program has been used to the and to MAT1 structure the three available structures of RING finger resolution immediate early equine herpes virus and promyelocytic leukemia structures and electrostatic were with R. Billeter M. Wüthrich K. J. Mol. 1996; 14: Scopus Google Scholar). To the solution structure of the N-terminal RING finger domain of the human MAT1 a corresponding to residues was The of the domain was on and on the comparison of orthologous This domain, when as a GST fusion protein in E. is soluble and can be After of the GST and gel the MAT1 fragment to a solution and be concentrated to 2 As expected for a C3HC4 RING finger domain Curr. Opin. Biol. 1996; PubMed Scopus Google Scholar), and showed that the human MAT1 RING finger domain two A first analysis of NOESY spectra recorded on the MAT1 RING finger domain the presence of both and β with a of The sample as a in solution the proton widths are in a with the expected for a between protons of and that these residues are one of the two zinc-binding sites and that the two are in a which is one of the defining features of the RING family. The of the used to the structure with the the chain are shown in 1 A. for a be the set of distances an of the structure of the human MAT1 RING finger domain, with a of and structural the 20 structures are in The of the 20 NMR structures is shown in 1 B. The N-terminal fragment of the human MAT1 subunit the ββαβ of RING finger and presents an in its N The core of the domain consists of a residues and a α-helix residues of the MAT1 RING for = to an of 20 structures with the 60 in in additional in in between the of the 20 of of to an of 20 structures with the 60 between the of the 20 in a new The is defined by an of NOEs and 1 Slowly exchanging amide protons are for residues and in the which that are A of and NOEs K. NMR of and Google Scholar) with resonances D. A. J. Biomol. NMR. 1995; 5: PubMed Scopus Google Scholar) and amide protons two helical and of the chain are well defined and to that the structure loop which and loop between residues and The shows that of the and residues are in the residues and are in the loop The RING domain is by two sites by and form one zinc-binding site and with form the zinc-binding site The first the N-terminal part of the The loop containing the is to the central which is to by a short loop the two and A α-helix is between the and the and which is with to form the third A loop the fourth of the to the The of the MAT1 RING finger domain is found to be with of × × the MAT1 RING finger core is by a of conserved hydrophobic residues among MAT1 in the loop and in the internal of the β and in the and and in the loop The topology of the three β with the arrangement of the eight zinc-binding residues of MAT1 RING domain, is to that in two RING resolution structures that been the structure the protein solved by NMR B. A. M. R. J. Mol. Biol. PubMed Scopus Google Scholar) and the crystal structure of the human protein domain D.G. Biol. 1997; PubMed Scopus Google Scholar). A arrangement of the zinc-binding residues was also found in the structure of human J. S. K. E. EMBO J. 1995; 14: PubMed Scopus Google Scholar). The of the MAT1 RING with the three previously reported structures of RING finger are shown in A data for in the using the program C. J. Mol. Biol. PubMed Scopus Google Scholar) structural between MAT1 and the RING in the domain D.G. Biol. 1997; PubMed Scopus Google Scholar) and in the B. A. M. R. J. Mol. Biol. PubMed Scopus Google Scholar). The structural is found for the of the MAT1 structure the crystal structure of both structures can be for with an of the comparison with the solution structure of the RING that with an of are by in the of 2 However, be obtained when the MAT1 RING structure with the solution structure of the RING finger domain the promyelocytic leukemia It is that the structural between the RING finger of the human MAT1 protein and was also when of and D.G. Biol. 1997; PubMed Scopus Google Scholar). The comparison of the MAT1 RING structure with available RING structures that the consensus C3HC4 zinc-binding sequence a conserved structural which a used of RING that this consensus sequence of high sequence with between the conserved zinc-binding residues. of these is between the first two of zinc-binding and the loop RING sequences, this loop the MAT1 sequence residues 2 The of a well defined structure in this region is and a specific of the MAT1 structure 2 the loop containing the fourth presents the as in RING structures the sequence the fourth of ligands. The of constraints on of the methylene protons a of the chain in for the zinc-binding residues. A analysis of the sites in the RING structures that the is well conserved between the different the binding sites of MAT1 and we found a with an of of the four MAT1 chain and the corresponding of the the sites of and can be with an of The first site is conserved between the three RING finger the of the of the four and the of is an of a to the one obtained when and of the the first zinc-binding site is part of a with the to in two This of may the structure the first zinc-binding a Busso (13Busso D. Keriel A. Sandrock B. Poterszman A. Gileadi O. Egly J.M. J. Biol. Chem. 2000; 275: 22815-22823Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) the role of the N-terminal RING finger domain of MAT1 in the of transcription in a also that the presence of the RING finger domain an optimal phosphorylation of the RNA polymerase II C-terminal with the role of RING in interactions J. Mol. Biol. 2000; PubMed Scopus Google Scholar), has been that the MAT1 RING domain interacts with factors within the preinitiation complex of has been with RING the MAT1 RING domain presents specific features that be involved in the MAT1 activities. the of a of conserved hydrophobic residues in the of the strand and an these conserved hydrophobic two are involved in the hydrophobic of the RING and the two and are to the a of hydrophobic residues is in RING A. E. R. Res. 1999; PubMed Scopus Google and be to a of the MAT1 A specific structural of MAT1 the presence of a region including one of an α-helix which to the sequence between the first and of zinc-binding in MAT1 this a which is conserved among all MAT1 sequences, that the be involved in the of the RING domain with another This is by the comparison of the MAT1 RING structure with the crystal structure of which shows that the part in interacts with its N-terminal finger J. S. K. E. EMBO J. 1995; 14: PubMed Scopus Google Scholar). Moreover, the be a potential site for phosphorylation and be involved in activity Recently, the crystal structure of a complex of a of the protein to the and a kinase was reported N. P. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The structure that the loop of the C-terminal RING domain interacts with both the N-terminal kinase binding domain and the partner, the functional role of this region in RING of the electrostatic potential shows that the MAT1 RING domain is positively charged of the presence of basic of and the extended of the nine at the domain is a specific of of MAT1, is that the four positively charged residues that are conserved human to and are in the a conserved residues on the and form a on the of the It is that the two C-terminal residues and are also conserved are in the The presence of positively charged on protein surfaces to be a of the RING finger the of the MAT1 RING is as shown in It be that of the that the of and RING finger charged residues. The role of the MAT1 RING finger domain within the transcription complex to be further by to be identified the structure and the positively charged residues that may be involved in the of the CAK phosphorylation activity through electrostatic interactions with either the phosphorylated C-terminal domain, the or another of the preinitiation complex, and the hydrophobic residues in the strand that may the of the preinitiation It be of to these residues are to the two of the MAT1 RING finger the phosphorylation of the RNA polymerase II C-terminal domain and transcription To these the of specific is the first of the Gene protein was identified in J. Cell. Full Text PDF PubMed Scopus Google Scholar), a structural data are of the of these to and when and for the RING finger has been The solution structure of the N-terminal part of MAT1 is the fourth structure of a RING domain that is new data provide insights into the structure of the loop region of between the first two of zinc-binding residues that be in the structural of the loop and the at the of the RING be factors that RING finger activities. We to M. and K. at the of for to the and for during data We are to D. Moras for T. for protein C. for and E. for with structure We the for and DNA