Solution NMR structure of MED25(391–543) comprising the activator-interacting domain (ACID) of human mediator subunit 25

  • Alexander Eletsky
  • William T. Ruyechan
  • Rong Xiao
  • Thomas B. Acton
  • Gaetano T. Montelione
  • Thomas Szyperski
Article

Abstract

The solution NMR structure of protein MED25(391–543), comprising the activator interacting domain (ACID) of subunit 25 of the human mediator, is presented along with the measurement of polypeptide backbone heteronuclear 15N-{1H} NOEs to identify fast internal motional modes. This domain interacts with the acidic transactivation domains of Herpes simplex type 1 (HSV-1) protein VP16 and the Varicella-zoster virus (VZV) major transactivator protein IE62, which initiate transcription of viral genes. The structure is similar to the β-barrel domains of the human protein Ku and the SPOC domain of human protein SHARP, and provides a starting point to understand the structural biology of initiation of HSV-1 and VZV gene activation. Homology models built for the two ACID domains of the prostate tumor overexpressed (PTOV1) protein using the structure of MED25(391–543) as a template suggest that differential biological activities of the ACID domains in MED25 and PTOV1 arise from modulation of quite similar protein–protein interactions by variable residues grouped around highly conserved charged surface areas.

Keywords

ACID MED25 Mediator complex PTOV Structural genomics 

Abbreviations

ACID

Activator-interacting domain

CBP

CREB-binding protein

HDAC

Histone deacetylase complex

HSV-1

Herpes simplex virus type 1

NESG

Northeast Structural Genomics Consortium

MED25

Subunit 25 of the human mediator complex

NOE

Nuclear Overhauser effect

PDB

Protein Data Bank

PTOV

Prostate tumor overexpressed

RMSD

Root mean square deviation

SHARP

SMRT/HDAC1-associated repressor protein

SPOC

Spen paralog and ortholog C-terminal domain

TAD

Transactivation domain

VBD

VP16-binding domain

VZV

Varicella-zoster virus

Introduction

The Mediator complex [1] has been identified in all eukaryotes thus far investigated for its presence [2], and appears to be an essential part of the RNA polymerase type II (RNAP II) machinery for gene transcription [3]. Mediator is composed of up to thirty protein subunits [3, 4], some of which interact with cellular transcriptional activators (such as Sp1, p53, the vitamin D receptor, the adenovirus E1A protein) [4, 5], as well as herpes simplex type 1 virus (HSV-1) and varicella-zoster virus (VZV) transcriptional activators [6, 7, 8, 9]. These interactions result in a structural change of Mediator and subsequent RNAP II binding resulting in assembly of the transcription pre-initiation complex [10].

Subunit 25 of the human mediator (MED25) comprises 747 residues and was identified as the target of the acidic transactivation domains (TADs) of the HSV-1 protein VP16 [6, 7], and the VZV major transactivator protein IE62 [8, 9]. Although these two TADs do not exhibit any significant sequence similarity, they are both largely unstructured in solution [9, 11] and contain a high content of acidic and aliphatic amino acid residues [9, 11, 12]. Furthermore, both TADs bind to the polypeptide segment of MED25 comprising approximately residues 390 to 540, which was named activator-interacting domain (ACID), VP16-binding domain (VBD) or prostate tumor overexpressed (PTOV) protein domain [6, 7, 13]. Given the high content of basic amino residues in ACID and the fact that mutation of certain Phe residues in the TADs significantly decreases transactivation [9, 12, 14], one would expect that the formation of a TAD-ACID complex involves both electrostatic as well as hydrophobic interactions [15, 16]. Since the VP16 TAD folds upon binding to other cofactors [12, 17], it might very well be that the TADs fold upon binding to the ACID domain. Considering that ACID domain also interacts with the histone transacetylase CBP, which is involved in chromatin remodeling [13, 18], the structure of MED25 ACID domain is of key importance for understanding the structural biology of MED25 and its role in transcriptional activation.

MED25(391–543) (UniProt accession number Q71SY5) comprises the MED25 ACID domain and belongs to the Pfam [19] domain family PF11232 currently containing 50 members. These include the two ACID domains of protein PTOV1 which is overexpressed in prostate cancer tumors [20]. MED25(391–543) was selected as a community nominated target by the Protein Structure Initiative and assigned to the Northeast Structural Genomics Consortium (NESG; http://www.nesg.org; target ID HR6188A) for structure determination.

Materials and methods

MED25(391–543) was cloned, expressed, and purified following standard protocols developed by the NESG for production of uniformly (U) 13C,15N-labeled protein samples [21, 22]. Briefly, the 391–543 fragment of Q71SY5 from Homo sapiens was cloned into pET15_NESG, a derivative of pET15 (Novagen), yielding the plasmid HR6188A-391–543-14.15. The resulting construct contains ten nonnative residues at the N-terminus (MGHHHHHHSH) to facilitate protein purification. Escherichia coli BL21 (DE3) pMGK cells, a rare codon enhanced strain, were transformed with HR6188A-391–543-14.15, and cultured in MJ9 minimal medium containing (15NH4)2SO4 and U-13C-glucose as sole nitrogen and carbon sources. U-13C,15N MED25(391–543) was purified using an AKTAxpress (GE Healthcare) based two step protocol consisting of IMAC (HisTrap HP) and gel filtration (HiLoad 26/60 Superdex 75) chromatography. The final yield of purified U-13C,15N-MED25(391–543) (>98% homogenous by SDS-PAGE; 19.7 kDa by MALDI-TOF mass spectrometry) was ~23 mg/L. In addition, a uniformly 15N- and 5% biosynthetically directed fractionally 13C-labeled sample 5% 13C;U-15N-MED25(391–543) was produced using a mixture of 95% unlabeled and 5% U-13C-glucose as the sole carbon source [23]. The NMR samples of U-13C,15N-MED25(391–543) and 5% 13C,U-15N-MED25(391–543) were prepared at respective concentrations of ~0.7 and ~0.4 mM in 90% H2O/10% D2O solution containing 20 mM MES (pH 6.5), 100 mM NaCl, 10 mM DTT, 5 mM CaCl2, and 0.02% NaN3. An isotropic overall rotational correlation time of about 9 ns was inferred from 15N spin relaxation times, indicating that MED25(391–543) is monomeric in solution. This was confirmed by analytical gel-filtration (Agilent Technologies) and static light scattering (Wyatt Technology Co.), using protocols described previously [22].

The following spectra were recorded for U-13C;15N-MED25(391–543) at 25 °C on (i) a Varian INOVA 750 spectrometer (total measurement time 4 days) equipped with a conventional 1H{13C,15N} probe: [15N,1H]-HSQC, HNCO, CBCA(CO)NH, HBHA(CO)NH, HN(CA)CO, aliphatic and aromatic (H)CCH, (H)CCH-TOCSY [24], and (ii) a Bruker AVANCE 900 spectrometer (total measurement time 6 days) equipped with a cryogenic 1H{13C,15N} probe: 2D [15N,1H]-HSQC, aliphatic and aromatic 2D constant-time [13C,1H]-HSQC, 3D HNCACB, 3D HNCA, simultaneous 3D 15N/13Caliphatic/13Caromatic-resolved [1H,1H]-NOESY [25] (mixing time 70 ms), 2D long-range [15N,1H]-HSQC for determining the tautomeric states of His residues, and 2D (HB)CB(CGCDCE)HE [26]. To identify fast internal motional modes, a 2D NMR experiment to measure 15N-{1H} heteronuclear NOEs [27] was performed with 4 s pre-saturation of the amide protons using a Varian INOVA 750 spectrometer (measurement time 4 days). 2D constant-time [13C,1H]-HSQC spectra were recorded as described [28] for 5% 13C;U-15N-MED25(391–543) on the Varian INOVA 750 spectrometer to obtain stereo-specific assignments for Val and Leu isopropyl groups [23]. All NMR spectra were processed using the programs PROSA v6.4 [29] and TopSpin v1.4 (Bruker Biospin), and analyzed using programs CARA [30] and XEASY [31]. Sequence-specific backbone (HN, Hα, N, C′, Cα) and Hβ/Cβ resonance assignments were obtained by using the program AutoAssign [32, 33]. Resonance assignment of side-chains was accomplished using aliphatic and aromatic 3D (H)CCH, 3D (H)CCH-TOCSY, 2D (HB)CB(CGCDCE)HE and 3D 15N/13Caliphatic/13Caromatic-resolved NOESY. Side chains assignments and ε-protonated neutral tautomeric state of His 435 and His 502 were obtained using 2D long-range [15N,1H]-HSQC [34]. Overall, for residues 391–543 sequence-specific resonance assignments were obtained for 99.3% of backbone (Fig. S1) and 100.0% of side chain resonances assignable with the NMR experiments listed above (i.e., excluding Pro 15N, Lys and Arg side chain amino groups, hydroxyl protons of Ser, Thr and Tyr, thiol protons of Cys, carboxyl atoms of Asp and Glu, Cε1, Hε1 and imino groups of His, and non-protonated aromatic carbons). Chemical shifts were deposited in the BioMagResBank on 11/24/2010 (BMRB ID: 17323). 1H-1H upper distance limit constraints for structure calculation were obtained from 3D 15N/13Caliphatic/13Caromatic-resolved [1H,1H]-NOESY, and backbone dihedral angle constraints for residues located in well-defined regular secondary structure elements were derived from chemical shifts using the program TALOS+ [35].

Automated NOE assignment was performed iteratively with CYANA v3.0 [36, 37], and the results were verified by interactive spectral analysis. Stereospecific assignments of methylene protons were performed with the GLOMSA module and the final structure calculation was performed with CYANA followed by refinement of selected conformers in an ‘explicit water bath’ [38] using the program CNS v1.2 [39]. Validation of the 20 refined conformers was performed with the Protein Structure Validation Software (PSVS) server [40]. Homology models of the PTOV1 ACID domains were generated using the SWISS-MODEL server [41], starting with the lowest-energy MED25(391–543) conformer as a template.

Results and discussion

A high-quality structure of the 152-residue domain construct MED25(391–543) was obtained (Table 1; Fig. 1a, b) and the coordinates were deposited in the Protein Data Bank [42] (http://www.rcsb.org) on 11/24/2010 (accession code 2L6U). MED25(391–543) exhibits a mixed α/β fold resembling the shape of a stein: seven β-strands form a barrel of topology A(↑)B(↓)D(↑)G(↓)F(↑)E(↓)C(↑) with strands C, E, F and G flanked by α-helices I and III, and helix II connecting strands D and E at one end of the barrel. The locations of the regular secondary structure elements are: β-strands A (residues 399–409), B (424–433), C (447–454), D (468–473), E (494–499), F (510–516) and G (521–527), and α-helices I (455–466; with a kink between Gly 462 and Pro 463), II (481–491) and III (531–540). β-strands A and B are connected by a long flexibly disordered loop comprising residues 410–423, and residues Ser 517, Lys 518, Lys 519 and Lys 520 form a type I β-turn connecting β-strands F and G.
Table 1

Statistics for MED25(391–543) structure

Completeness of resonance assignments (%)a

 Backbone/side-chain

99.3/100.0

Completeness of stereospecific assignments (%)

 Val and Leu isopropyl/βCH2/αCH2 of Gly

97/26/20

Conformation-restricting distance constraints

 Intraresidue [i = j]

596

 Sequential [|i − j| = 1]

622

 Medium range [1 < |i − j| < 5]

428

 Long range [|i − j| ≥ 5]

1,016

 Total

2,662

Dihedral angle constraints (φ/ψ)

60/60

Average number of constraints per residue

18.3

Average number of long-range distance constraints per residue

6.7

CYANA target function (Å2)

0.19 ± 0.04

Average number of distance constraint violations per conformer

 0.2–0.5 Å

8.2

 >0.5 Å

0.0

Average number of dihedral angle constraint violations per conformer

 >10°

0.0

Average RMSD from mean coordinates (Å)

 Regular secondary structure elementsb, backbone heavy atoms

0.6

 Regular secondary structure elementsb, all heavy atoms

1.1

 Ordered residuesc, backbone heavy atoms

0.7

 Ordered residuesc, all heavy atoms

1.2

Global quality scoresc (raw/Z-scored)

 PROCHECK G-factor (φ and ψ)

−0.38/−1.18

 PROCHECK G-factor (all dihedral angles)

−0.24/−1.42

 MOLPROBITY clash score

12.25/−0.58

 Verify3D

0.39/−1.12

 ProsaII

0.47/−0.74

RPF scorese

 Recall/precision/F-measure

0.993/0.876/0.931

 DP-score

0.841

Ramachandran plot summary (%)c

 Most favored regions

94.9

 Additionally allowed regions

5.3

 Generously allowed regions

0.2

 Disallowed regions

0.0

aResidues 391–543, excluding Pro 15N, Lys and Arg side chain amino groups, hydroxyl protons of Ser, Thr and Tyr, thiol protons of Cys, carboxyl atoms of Asp and Glu, Cε1, Hε1 and imino groups of His, and non-protonated aromatic carbons

bResidues in regular secondary structure elements: 399–409, 424–433, 468–473, 521–527, 510–516, 494–499, 447–454, 455–461, 463–465, 481–491, 531–540

cOrdered residues: 399–409, 424–432, 443–465, 468–473, 480–500, 509–515, 518–528, 531–541

dCalibrated relative to a set of high-resolution X-ray crystal structures for which the corresponding mean structure-quality score corresponds to Z score = 0.0 [40]

eAs described in [52]

Fig. 1

a Stereoview of the ensemble of 20 conformers representing the solution structure of MED25(391–543) obtained after superposition of the Cα atoms of the regular secondary structure elements superimposed for minimal RMSD. Residues 391–395 and the disordered N-terminal tag are omitted for clarity, and the N- and C-termini are labeled. b Ribbon diagram of the lowest-energy conformer of MED25(391–543): α-helices I, II and III are shown in red and yellow, β-strands A to G are shown in cyan, and other polypeptide segments are shown in gray. ce Superposition of NMR structure ensembles of MED25(391–543) in blue with other MED25 ACID domain structures in orange: c MED25(391–548), PDB code 2L23 [43]; d MED25(391–553), PDB code 2KY6 [44]; e MED25(394–543), PDB code 2XNF [45]. The Cα atoms of consensus regular secondary structure elements being superimposed for minimal RMSD and the side-chain heavy atoms of Trp 402, Trp 408, Trp 444, His 499 and Phe 500 are shown. f Representation of the electrostatic surface potential of MED25(391–543) calculated with MOLMOL [51]

In parallel with the structure determination of MED25(391–543) presented here, the NMR structures of MED25(391–548) (PDB accession code: 2L23) [43], MED25(391–553) (PDB accession code: 2KY6) [44], and MED25(394–543) (PDB accession code: 2XNF) [45] were solved. These four structures are the first structural representatives for protein family PF11232. Comparison with our MED25(391–543) structure (Fig. 1c–e) reveals that the spatial arrangement of the regular structure elements is very similar: the root mean square deviation (RMSD) calculated between the mean coordinates of the backbone heavy atoms N, Cα and C′ is only 0.97, 1.03 and 1.00 Å, respectively. Moreover, the long loop connecting β-strands A and B is highly disordered in all structures. Differences are observed, however, for the conformation of the N- and C-terminal segments which is likely due to variations in construct lengths. Notably, the structure presented here, as well as the structures of MED25(391–553) [44] and MED25(394–543) [45], exhibit better defined conformations of Trp 408, His 499 and Phe 500 side chains when compared with MED25(391–548) [43]. However, in MED25(394–543) [45] the indole moiety of Trp 408 is flipped around χ2 when compared with the other structures. Furthermore, the side-chain conformations of Trp 402 and Trp 444 are distinctly different in the MED25(391–548) [43] when compared with the other structures. Since the chemical shifts of MED25(391–548) [43] and MED25(394-543) [45] are not available in the BMRB, it remains to be seen if differences in resonance assignments and/or the completeness of resonance assignment account for the observed structural differences.

Inspection of heteronuclear 15N-{1H} NOEs (Fig. S2a) shows that polypeptide backbone segments exhibiting increased global displacements (Figs. 1, S2b), that is, a reduced precision of the structural description, are affected by the presence of fast internal motional modes. Most prominently, the long loop connecting β-strands A and B is highly flexibly disordered and exhibits the largest displacements. Increased disorder manifested in lower 15N-{1H} NOEs and increased displacements is likewise observed for the loops connecting β-strands B and C, E and F, the loop connecting β-strand D and helix II, and the β-turn between strands F and G. In contrast, the 15N-{1H} NOE measured for Gly 493, which is located between α-helix II and β-strand E, is comparably low while the displacements of Gly 493 and its neighboring residues are hardly increased. This finding suggests that the internal motion registered by this NOE primarily affects the N-H moiety of Gly 493. Furthermore, reduced 15N-{1H} NOEs are also observed for the kinked α-helix I (average: 0.73 ± 0.06) while the corresponding global displacements are only slightly higher than in the other regular structure elements. In fact, except for the terminal α-helix III (average 0.76 ± 0.07), the average 15N-{1H} NOEs are significantly higher for the other regular secondary structure elements: 0.81 ± 0.03 (β-strand A), 0.79 ± 0.09 (B), 0.82 ± 0.04 (C), 0.82 ± 0.05 (D), 0.81 ± 0.04 (α-helix II), 0.80 ± 0.04 (E), 0.84 ± 0.01 (F), and 0.81 ± 0.06 (G). Since the binding site for VP16 C-terminal domain, as judged from chemical shift perturbations [43], includes residues Gln 456, Leu 458, Thr 459, Phe 465 and Arg 466 located in helix I, as well as Asn 438 in the loop connecting β-strands B and C, and Lys519 in the β-turn connecting β-strands F and G, it may very well be that the increased flexibility of these polypeptide segments are important for MED25-TAD complex formation.

The electrostatic potential of MED25(391–543) (Fig. 1f) exhibits two distinct positively charged regions on opposite sides of the protein molecule which are likely important for TAD binding. Furthermore, there is an extended groove on the surface which is ‘wrapped around’ the protein and passes through the two positively charged regions. A total of 42 residues, most of which are positively charged or hydrophobic (Ser 396, Lys 398, Trp 408, Gln 409, Tyr 432, Glu 437, Asn 438, Leu 439, Gln 455, Arg 469, Met 470, Gln 472, Phe 473, His 474, Phe 475, Lys 478, Lys 484, Tyr 487, Arg 488, Met 490, Gly 491, Gly 493, Phe 494, Pro 500, His 502, Thr 503, Ala 504, Cys 506, Glu 507, Val 508, Arg 509, Leu 514, Tyr 515, Ser 517, Lys 519, Lys 520, Ile 521, Phe 522, Met 523, Tyr 528, Gln 530) participate in forming the groove. Consistent with the hypothesis that the charged patches and the groove are important for TAD binding, substitution of amino acid residues 447–450 or 484–488 to Ala resulted in a decreased interaction with the IE62 TAD [9] and VP16 TAD (Yamamoto and Ruyechan, unpublished results).

A search of the PDB for structurally similar proteins using the DALI server [46] resulted in only two significant hits (Fig. 2a, b) other than MED25(391–548): (i) the β-barrel domains of the Ku70/Ku80 heterodimer (PDB accesion codes 1JEY, 1JEQ; highest Z-score 8.5; 108 residues with 10% sequence identity aligned with an RMSD value of 3.1 Å for Cα atoms) and (ii) the Spen paralog and orthlog C-terminal (SPOC) domain of SMRT/HDAC1-associated repressor protein (SHARP) (PDB accession code 1OW1; Z-score 7.2; 107 residues; 19%; 2.9 Å). Ku70/Ku80 is implicated in a wide range of functions, including DNA helicase activity, transcriptional activation, but mainly in double-stranded DNA break repair. In these crystal structures the β-barrel domains of Ku make extensive contacts with each other, as well as with other regions of the opposite subunit and the phosphate backbone of the DNA. SHARP, on the other hand, binds to nuclear receptors and represses transcription by interacting with histone deacetylase complexes (HDAC) [47, 48], and the acidic co-repressor SMRT/NCoR [47, 49] via the C-terminal SPOC domain.
Fig. 2

a Ribbon diagrams of MED25(391–543) in blue and the SPOC domain of SHARP (1OW1) in orange after superposition of Cα atoms identified by DALI. b Same for the β-barrel domain of Ku70 subunit (1JEY) with the extended loop of residues 276–340 interacting with DNA being omitted for clarity. Regions of Ku80 subunit and DNA backbone interacting with the β-barrel domain are shown in grey. c Sequence alignment of human MED25 and PTOV1 fragments comprising the ACID domains. The regular secondary structure elements of MED25(391–543) are indicated above the alignment. The figure was generated with the program Chroma v1.0 (http://www.llew.org.uk/chroma/) using its default coloring scheme: negative (red), positive (dark blue), charged (magenta), polar (light blue), Ser/Thr (cyan), tiny (light green), small (dark green), aliphatic (grey on yellow), aromatic (blue on yellow), big (violet on pale yellow), hydrophobic (black on yellow). d Ribbon diagram of the homology model of the PTOV1N-terminal domain. Residues which are conserved in both ACID domains as well as MED25(391–543) are depicted in grey. Residues which are conserved in the two PTOV1 ACID domains are shown in red, while residues which are not conserved in the two ACID domains are shown in blue. Substitutions among the following groups of residues are considered as being conserved: (i) Leu, Val, Ile, Ala, Phe, Met; (ii) Glu, Asp; (iii) Arg, Lys; (iv) Ser, Thr, Cys. e Same as d but for the homology model of the PTOV1 C-terminal domain

MED25(391–543) exhibits 81 and 73% sequence identity with the N- and C-terminal ACID domains of human PTOV1 protein, respectively (Fig. 2c). The high sequence identity allowed us to generate high-quality homology models of the PTOV1 ACID domains (Fig. 2d, e). Inspection of the models shows that residues which are different in MED25(391–543) and the PTOV1 ACID domains are grouped peripherally around the highly conserved charged surface regions. This finding indicates that the differential biological activity of ACID domains in MED25 and PTOV1 [50] arises from a modulation of quite similar protein-protein interaction patterns by these peripheral residues.

Notes

Acknowledgments

We thank R. Shastry, C. Ciccosanti, H. Janjua, and G.V.T. Swapna for contributions in sample preparation, and J. K. Everett and S. Bhattacharya for helpful discussions. This work was supported by the National Institutes of Health, grant numbers: U54 GM094597 (T.S. and G.T.M.) and R01 AI18449 (W.T.R.). Prof. T. Szyperski is a member of the New York Structural Biology Center. The Center is a STAR center supported by the New York State Office of Science, Technology, and Academic Research. 900 MHz spectrometer was purchased with funds from NIH, USA, the Keck Foundation, New York State, and the NYC Economic Development Corporation. Support was also obtained from the University at Buffalo’s Center for Computational Research.

Supplementary material

10969_2011_9115_MOESM1_ESM.doc (1.1 mb)
Supplementary material 1 (DOC 1170 kb)

References

  1. 1.
    Kim YJ, Bjorklund S, Li Y, Sayre MH, Kornberg RD (1994) A multiprotein mediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA-polymerase-II. Cell 77:599–608PubMedCrossRefGoogle Scholar
  2. 2.
    Malik S, Roeder RG (2005) Dynamic regulation of pol II transcription by the mammalian Mediator complex. Trends Biochem Sci 30:256–263PubMedCrossRefGoogle Scholar
  3. 3.
    Casamassimi A, Napoli C (2007) Mediator complexes and eukaryotic transcription regulation: an overview. Biochimie 89:1439–1446PubMedCrossRefGoogle Scholar
  4. 4.
    Conaway RC, Sato S, Tomomori-Sato C, Yao TT, Conaway JW (2005) The mammalian Mediator complex and its role in transcriptional regulation. Trends Biochem Sci 30:250–255PubMedCrossRefGoogle Scholar
  5. 5.
    Bourbon HM, Aguilera A, Ansari AZ, Asturias FJ, Berk AJ, Bjorklund S, Blackwell TK, Borggrefe T, Carey M, Carlson M, Conaway JW, Conaway RC, Emmons SW, Fondell JD, Freedman LP, Fukasawa T, Gustafsson CM, Han M, He X, Herman PK, Hinnebusch AG, Holmberg S, Holstege FC, Jaehning JA, Kim YJ, Kuras L, Leutz A, Lis JT, Meisterernest M, Naar AM, Nasmyth K, Parvin JD, Ptashne M, Reinberg D, Ronne H, Sadowski I, Sakurai H, Sipiczki M, Sternberg PW, Stillman DJ, Strich R, Struhl K, Svejstrup JQ, Tuck S, Winston F, Roeder RG, Kornberg RD (2004) A unified nomenclature for protein subunits of Mediator complexes linking transcriptional regulators to RNA polymerase II. Mol Cell 14:553–557PubMedCrossRefGoogle Scholar
  6. 6.
    Mittler G, Stuhler T, Santolin L, Uhlmann T, Kremmer E, Lottspeich F, Berti L, Meisterernst M (2003) A novel docking site on Mediator is critical for activation by VP16 in mammalian cells. EMBO J 22:6494–6504PubMedCrossRefGoogle Scholar
  7. 7.
    Yang FJ, DeBeaumont R, Zhou S, Naar AM (2004) The activator-recruited cofactor/Mediator coactivator subunit ARC92 is a functionally important target of the VP16 transcriptional activator. Proc Natl Acad Sci USA 101:2339–2344PubMedCrossRefGoogle Scholar
  8. 8.
    Yang M, Hay J, Ruyechan WT (2008) Varicella-zoster virus IE62 protein utilizes the human mediator complex in promoter activation. J Virol 82:12154–12163PubMedCrossRefGoogle Scholar
  9. 9.
    Yamamoto S, Eletsky A, Szyperski T, Hay J, Ruyechan WT (2009) Analysis of the varicella-zoster virus IE62N-terminal acidic transactivating domain and its interaction with the human mediator complex. J Virol 83:6300–6305PubMedCrossRefGoogle Scholar
  10. 10.
    Taatjes DJ (2010) The human mediator complex: a versatile, genome-wide regulator of transcription. Trends Biochem Sci 35:315–322PubMedCrossRefGoogle Scholar
  11. 11.
    Perera LP, Mosca JD, Ruyechan WT, Hayward GS, Straus SE, Hay J (1993) A major transactivator of varicella-zoster virus, the immediate-early protein IE62, contains a potent N-terminal activation domain. J Virol 67:4474–4483PubMedGoogle Scholar
  12. 12.
    Jonker HRA, Wechselberger RW, Boelens R, Folkers GE, Kaptein R (2005) Structural properties of the promiscuous VP16 activation domain. Biochemistry 44:827–839PubMedCrossRefGoogle Scholar
  13. 13.
    Lee HK, Park UH, Kim EJ, Um SJ (2007) MED25 is distinct from TRAP220/MED1 in cooperating with CBP for retinoid receptor activation. EMBO J 26:3545–3557PubMedCrossRefGoogle Scholar
  14. 14.
    Cress WD, Triezenberg SJ (1991) Critical structural elements of the VP16 transcriptional activation domain. Science 251:87–90PubMedCrossRefGoogle Scholar
  15. 15.
    Hermann S, Berndt KD, Wright AP (2001) How transcriptional activators bind target proteins. J Biol Chem 276:40127–40132PubMedCrossRefGoogle Scholar
  16. 16.
    Ferreira ME, Hermann S, Prochasson P, Workman JL, Berndt KD, Wright APH (2005) Mechanism of transcription factor recruitment by acidic activators. J Biol Chem 280:21779–21784PubMedCrossRefGoogle Scholar
  17. 17.
    Langlois C, Mas C, Di Lello P, Jenkins LMM, Legault P, Omichinski JG (2008) NMR structure of the complex between the Tfb1 subunit of TFIIH and the activation domain of VP16: structural similarities between VP16 and p53. J Am Chem Soc 130:10596–10604PubMedCrossRefGoogle Scholar
  18. 18.
    Rana R, Surapureddi S, Kam W, Ferguson S, Goldstein JA (2011) Med25 is required for RNA Pol II recruitment to specific promoters thus regulating xenobiotic and lipid metabolism in human liver. Mol Cell Biol 31:466–481PubMedCrossRefGoogle Scholar
  19. 19.
    Finn RD, Mistry J, Tate J, Coggill P, Heger A, Pollington JE, Gavin OL, Gunasekaran P, Ceric G, Forslund K, Holm L, Sonnhammer ELL, Eddy SR, Bateman A (2010) The Pfam protein families database. Nucleic Acids Res 38:D211–D222PubMedCrossRefGoogle Scholar
  20. 20.
    Benedit P, Paciucci R, Thomson TM, Valeri M, Nadal M, Caceres C, de Torres I, Estivill X, Lozano JJ, Morote J, Reventos J (2001) PTOV1, a novel protein overexpressed in prostate cancer containing a new class of protein homology blocks. Oncogene 20:1455–1464PubMedCrossRefGoogle Scholar
  21. 21.
    Acton TB, Gunsalus KC, Xiao R, Ma LC, Aramini J, Baran MC, Chiang YW, Climent T, Cooper B, Denissova NG, Douglas SM, Everett JK, Ho CK, Macapagal D, Rajan PK, Shastry R, Shih LY, Swapna GVT, Wilson M, Wu M, Gerstein M, Inouye M, Hunt JF, Montelione GT (2005) Robotic cloning and protein production platform of the Northeast Structural Genomics Consortium. In: Nuclear magnetic resonance of biological macromolecules, Part C. Elsevier, San Diego, pp 210–243Google Scholar
  22. 22.
    Xiao R, Anderson S, Aramini J, Belote R, Buchwald WA, Ciccosanti C, Conover K, Everett JK, Hamilton K, Huang YJ, Janjua H, Jiang M, Kornhaber GJ, Lee DY, Locke JY, Ma LC, Maglaqui M, Mao L, Mitra S, Patel D, Rossi P, Sahdev S, Sharma S, Shastry R, Swapna GVT, Tong SN, Wang DY, Wang HA, Zhao L, Montelione GT, Acton TB (2010) The high-throughput protein sample production platform of the Northeast Structural Genomics Consortium. J Struct Biol 172:21–33PubMedCrossRefGoogle Scholar
  23. 23.
    Neri D, Szyperski T, Otting G, Senn H, Wuthrich K (1989) Stereospecific nuclear magnetic resonance assignments of the methyl groups of valine and leucine in the DNA-binding domain of the 434 repressor by biosynthetically directed fractional 13C labeling. Biochemistry 28:7510–7516PubMedCrossRefGoogle Scholar
  24. 24.
    Cavanagh J, Fairbrother WJ, Palmer AG III, Rance M, Skelton NJ (2007) Protein NMR spectroscopy: principles and practice. Academic Press, AmsterdamGoogle Scholar
  25. 25.
    Shen Y, Atreya HS, Liu GH, Szyperski T (2005) G-matrix Fourier transform NOESY-based protocol for high-quality protein structure determination. J Am Chem Soc 127:9085–9099PubMedCrossRefGoogle Scholar
  26. 26.
    Yamazaki T, Formankay JD, Kay LE (1993) 2-Dimensional NMR experiments for correlating C-13-beta and H-1-delta/epsilon chemical-shifts of aromatic residues in C-13-labeled proteins via scalar couplings. J Am Chem Soc 115:11054–11055CrossRefGoogle Scholar
  27. 27.
    Renner C, Schleicher M, Moroder L, Holak TA (2002) Practical aspects of the 2D N-15-{H-1}-NOE experiment. J Biomol NMR 23:23–33PubMedCrossRefGoogle Scholar
  28. 28.
    du Penhoat CH, Li Z, Atreya HS, Kim S, Yee A, Xiao R, Murray D, Arrowsmith CH, Szyperski T (2005) NMR solution structure of Thermotoga maritima protein TM1509 reveals a Zn-metalloprotease-like tertiary structure. J Struct Funct Genomics 6:51–62CrossRefGoogle Scholar
  29. 29.
    Guntert P, Dotsch V, Wider G, Wuthrich K (1992) Processing of multidimensional NMR data with the new software PROSA. J Biomol NMR 2:619–629CrossRefGoogle Scholar
  30. 30.
    Keller R (2004) The computer aided resonance assignment tutorial. CANTINA Verlag, GoldauGoogle Scholar
  31. 31.
    Bartels C, Xia TH, Billeter M, Guntert P, Wuthrich K (1995) The program Xeasy for computer-supported NMR spectral-analysis of biological macromolecules. J Biomol NMR 6:1–10CrossRefGoogle Scholar
  32. 32.
    Zimmerman DE, Kulikowski CA, Huang YP, Feng WQ, Tashiro M, Shimotakahara S, Chien CY, Powers R, Montelione GT (1997) Automated analysis of protein NMR assignments using methods from artificial intelligence. J Mol Biol 269:592–610PubMedCrossRefGoogle Scholar
  33. 33.
    Moseley HNB, Monleon D, Montelione GT (2001) Automatic determination of protein backbone resonance assignments from triple resonance nuclear magnetic resonance data. Method Enzymol 339:91–108CrossRefGoogle Scholar
  34. 34.
    Pelton JG, Torchia DA, Meadow ND, Roseman S (1993) Tautomeric states of the active-site histidines of phosphorylated and unphosphorylated III(Glc), a signal-transducing protein from Escherichia coli, using 2-dimensional heteronuclear NMR techniques. Protein Sci 2:543–558PubMedCrossRefGoogle Scholar
  35. 35.
    Cornilescu G, Delaglio F, Bax A (1999) Protein backbone angle restraints from searching a database for chemical shift and sequence homology. J Biomol NMR 13:289–302PubMedCrossRefGoogle Scholar
  36. 36.
    Guntert P, Mumenthaler C, Wuthrich K (1997) Torsion angle dynamics for NMR structure calculation with the new program DYANA. J Mol Biol 273:283–298PubMedCrossRefGoogle Scholar
  37. 37.
    Herrmann T, Guntert P, Wuthrich K (2002) Protein NMR structure determination with automated NOE assignment using the new software CANDID and the torsion angle dynamics algorithm DYANA. J Mol Biol 319:209–227PubMedCrossRefGoogle Scholar
  38. 38.
    Linge JP, Williams MA, Spronk C, Bonvin A, Nilges M (2003) Refinement of protein structures in explicit solvent. Proteins Struct Funct Genet 50:496–506PubMedCrossRefGoogle Scholar
  39. 39.
    Brunger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse-Kunstleve RW, Jiang JS, Kuszewski J, Nilges M, Pannu NS, Read RJ, Rice LM, Simonson T, Warren GL (1998) Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr 54:905–921PubMedCrossRefGoogle Scholar
  40. 40.
    Bhattacharya A, Tejero R, Montelione GT (2007) Evaluating protein structures determined by structural genomics consortia. Proteins Struct Funct Bioinf 66:778–795CrossRefGoogle Scholar
  41. 41.
    Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22:195–201PubMedCrossRefGoogle Scholar
  42. 42.
    Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, Bourne PE (2000) The Protein Data Bank. Nucleic Acids Res 28:235–242PubMedCrossRefGoogle Scholar
  43. 43.
    Bontems F, Verger A, Dewitte F, Lens Z, Baert J-L, Ferreira E, Launoit Yd, Sizun C, Guittet E, Villeret V, Monté D (2011) NMR structure of the human mediator MED25 ACID domain. J Struct Biol 174:245–251PubMedCrossRefGoogle Scholar
  44. 44.
    Milbradt AG, Kulkarni M, Yi TF, Takeuchi K, Sun ZYJ, Luna RE, Selenko P, Naar AM, Wagner G (2011) Structure of the VP16 transactivator target in the mediator. Nat Struct Mol Biol 18:410–415PubMedCrossRefGoogle Scholar
  45. 45.
    Vojnic E, Mourao A, Seizl M, Simon B, Wenzeck L, Lariviere L, Baumli S, Baumgart K, Meisterernst M, Sattler M, Cramer P (2011) Structure and VP16 binding of the Mediator Med25 activator interaction domain. Nat Struct Mol Biol 18:404–409PubMedCrossRefGoogle Scholar
  46. 46.
    Holm L, Sander C (1995) Dali—a network tool for protein structure comparison. Trends Biochem Sci 20:478–480PubMedCrossRefGoogle Scholar
  47. 47.
    Shi YH, Downes M, Xie W, Kao HY, Ordentlich P, Tsai CC, Hon M, Evans RM (2001) Sharp, an inducible cofactor that integrates nuclear receptor repression and activation. Genes Dev 15:1140–1151PubMedCrossRefGoogle Scholar
  48. 48.
    Oswald F, Kostezka U, Astrahantseff K, Bourteele S, Dillinger K, Zechner U, Ludwig L, Wilda M, Hameister H, Knochel W, Liptay S, Schmid RM (2002) SHARP is a novel component of the Notch/RBP-J kappa signalling pathway. EMBO J 21:5417–5426PubMedCrossRefGoogle Scholar
  49. 49.
    Ariyoshi M, Schwabe JWR (2003) A conserved structural motif reveals the essential transcriptional repression function of Spen proteins and their role in developmental signaling. Genes Dev 17:1909–1920PubMedCrossRefGoogle Scholar
  50. 50.
    Youn H-S, Park U-H, Kim E-J, Um S-J (2011) PTOV1 antagonizes MED25 in RAR transcriptional activation. Biochem Biophys Res Commun 404:239–244PubMedCrossRefGoogle Scholar
  51. 51.
    Koradi R, Billeter M, Wuthrich K (1996) MOLMOL: a program for display and analysis of macromolecular structures. J Mol Graph 14:51–55PubMedCrossRefGoogle Scholar
  52. 52.
    Huang YJ, Powers R, Montelione GT (2005) Protein NMR recall, precision, and F-measure scores (RPF scores): structure quality assessment measures based on information retrieval statistics. J Am Chem Soc 127:1665–1674PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Alexander Eletsky
    • 1
    • 2
  • William T. Ruyechan
    • 3
    • 4
  • Rong Xiao
    • 5
    • 6
    • 7
  • Thomas B. Acton
    • 5
    • 6
    • 7
  • Gaetano T. Montelione
    • 5
    • 6
    • 7
  • Thomas Szyperski
    • 1
    • 2
  1. 1.Department of ChemistryThe State University of New York at BuffaloBuffaloUSA
  2. 2.Northeast Structural Genomics ConsortiumBuffaloUSA
  3. 3.Department of Microbiology and ImmunologyThe State University of New York at BuffaloBuffaloUSA
  4. 4.Northeast Structural Genomics ConsortiumBuffaloUSA
  5. 5.Center of Advanced Biotechnology and Medicine, Department of Molecular Biology and BiochemistryRutgers, The State University of New JerseyPiscatawayUSA
  6. 6.Department of Biochemistry, Robert Wood Johnson Medical SchoolUniversity of Medicine and Dentistry of New JerseyPiscatawayUSA
  7. 7.Northeast Structural Genomics ConsortiumPiscatawayUSA

Personalised recommendations