Model of the Mediator middle module based on protein cross-linking

Laurent Larivie` re 1 Clemens Plaschka 1 Martin Seizl 1 Evgeniy V. Petrotchenko 0 Larissa Wenzeck 1 Christoph H. Borchers 0 Patrick Cramer 1 0 Department of Biochemistry and Microbiology, Genome British Columbia Protein Centre, University of Victoria , No. 3101-4464 Markham Street, Vancouver Island Technology Park , Victoria, BC V8Z7X8, Canada 1 Gene Center Munich and Department of Biochemistry, Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians-Universita t M unchen , Feodor-Lynen-Str. 25, 81377 Munich, Germany The essential core of the transcription coactivator Mediator consists of two conserved multiprotein modules, the head and middle modules. Whereas the structure of the head module is known, the structure of the middle module is lacking. Here we report a 3D model of a 6-subunit Mediator middle module. The model was obtained by arranging crystal structures and homology models of parts of the module based on lysine-lysine cross-links obtained by mass spectrometric analysis. The model contains a central tetramer formed by the heterodimers Med4/Med9 and Med7/Med21. The Med7/Med21 heterodimer is flanked by subunits Med10 and Med31. The model is highly extended, suggests that the middle module is flexible and contributes to a molecular basis for detailed structure-function studies of RNA polymerase II regulation. - INTRODUCTION Mediator is a central and conserved coactivator complex required for gene transcription by RNA polymerase (Pol) II (16). Mediator connects gene-specific transcription factors and the general Pol II machinery. Mediator from the yeast Saccharomyces cerevisiae has a molecular mass of 1.4 MDa and consists of 25 subunits that were assigned to four modules called head, middle, tail and kinase modules. The head and middle modules constitute the functional core of Mediator (7). The Mediator core subunits are conserved throughout eukaryotes (8). The crystal structure of the 7-subunit Mediator head module has been solved at 4.3- A resolution for S. cerevisiae (9,10) and at 3.4-A resolution for Schizosaccharomyces pombe (11). The structure of the middle module remains unknown. The S. cerevisiae middle module comprises four essential subunits, Med4, Med7, Med10 (Nut2) and Med21 (Srb7), and three nonessential subunits, Med1, Med9 (Cse2) and Med31 (Soh1). Detailed structural information on parts of the middle module is limited to two subcomplexes, the heterodimers Med7N/Med31 (12) and Med7C/Med21 (13), where Med7N and Med7C correspond to the N- and C-terminal regions of Med7, respectively. We previously reported the expression and purification of a recombinant 7-subunit Mediator middle module (14), and found that the high intrinsic flexibility of the module prevents its crystallization. To investigate the 3D subunit architecture of the middle module, we report here a new protocol for the heterologous expression and purification of a 6-subunit middle module lacking subunit Med1. We subjected the purified middle module to chemical lysinelysine cross-linking and identified pairs of cross-linked sites by mass spectrometry (CX-MS). CX-MS is a novel and powerful method for obtaining the subunit architecture of large protein complexes (15). We previously applied CX-MS to multiprotein complexes involved in transcription (1618). By combining the cross-linking information with known structures and structure-based homology modeling, we derived an architectural model of the Mediator middle module that provides the relative orientation of subunits and guides future structural and mechanistic studies of Mediator function. MATERIALS AND METHODS Preparation of a 6-subunit Mediator middle module Bacterial co-expression of the S. cerevisiae Mediator middle module was performed using a single plasmid based on a pCDFDuet-1 vector (Novagen), shown schematically in Figure 1A. Open reading frames were cloned sequentially and additional ribosomal binding sites were introduced as described (13). Med31 harbors a deca-histidine tag at its N-terminus. The exact sequence of the construct is available on request. The middle module was expressed in Escherichia coli BL21 CodonPlus(DE3)RIL cells (Stratagene). Cells were grown in Luria broth medium at 37 C to an optical density of 0.5 at 600 nm. Expression was induced with 0.5 mM isopropyl-b-D-1thiogalactopyranoside for 16 h at 18 C. Cells were lysed by sonication in buffer A [50 mM Tris pH 8.0, 150 mM sodium chloride, 5 mM dithiothreitol (DTT)] containing protease inhibitors (19). After centrifugation, the supernatant was loaded onto a 2-ml Ni-NTA agarose bead column (QIAGEN) equilibrated in buffer A. The column was washed with buffer A containing increasing concentrations of imidazole (0, 20, 50 mM). The complex was eluted with buffer A containing 300 mM imidazole. The middle module was further purified by anion exchange chromatography with a 1-ml HiTrap Q HP column (GE Healthcare). The column was equilibrated in buffer B (50 mM Tris pH 8.0, 50 mM sodium chloride, 2 mM DTT), and proteins were eluted with a linear gradient from 50 mM to 1 M sodium chloride in buffer B. Fractions containing middle module were applied to a HiLoad 16/600 Superdex 200-pg (GE Healthcare) exclusion column equilibrated in buffer C (20 mM HEPESKOH pH 7.5, 150 mM potassium acetate, 10% (v/v) glycerol, 2 mM DTT). The protein complex was concentrated to 3 mg/ml, flash-frozen and stored at 80 C. Chemical protein cross-linking The pure middle module was cross-linked using isotopically coded cyanurbiotindipropionyl succinimide (CBDPS, Creative Molecules Inc.) (20). The middle module was diluted to 0.5 mg/ml with buffer D (1 phosphate buffered saline, 2 mM DTT). CBDPS was dissolved in DMSO to 10 mM. To determine the optimal ratio of CBDPS to middle module, we mixed 3 mg of middle module with CBDPS at a concentration of 0.051.5 mM, and incubated for 30 min at 30 C. The reaction was stopped by addition of 0.5 M NH4HCO3 to a final concentration of 40 mM and incubation for 10 min at room temperature, and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) (Figure 1B). The optimum concentration of CBDPS was considered to result in a higher molecular weight band. We used a final concentration of 0.7 mM CBDPS. The cross-linked sample was dialyzed twice in dialysis buttons (Hampton Research) against 20 ml of buffer D. Trypsin and/or GluC were, respectively, added in a 1:10 or 1:1 ratio of protease to middle module and incubated overnight at 37 C. Proteases were then inhibited by addition of 10 mM 4-(2-aminoethyl)benzenesulfonylfluoride and 20 mM phenylmethanesulfonylfluoride, and incubation for 10 min at room temperature. Affinity enrichment was performed with monomeric avidin beads (ThermoFisher) equilibrated with 0.1 M ammonium acetate. The amount of bead slurry was adjusted to a ratio of 1:10 of total CBDPS to bead capacity (1.2 mg/ml). The sample was loaded five times. The column was washed with 300 ml of ammonium acet (...truncated)