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

9266–9273 Nucleic Acids Research, 2013, Vol. 41, No. 20 doi:10.1093/nar/gkt704 Published online 11 August 2013 Model of the Mediator middle module based on protein cross-linking Laurent Larivière1, Clemens Plaschka1, Martin Seizl1, Evgeniy V. Petrotchenko2, Larissa Wenzeck1, Christoph H. Borchers2 and Patrick Cramer1,* 1 Gene Center Munich and Department of Biochemistry, Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität München, Feodor-Lynen-Str. 25, 81377 Munich, Germany and 2Department 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 Received June 1, 2013; Revised July 17, 2013; Accepted July 18, 2013 ABSTRACT 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 (1–6). 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-Å resolution for S. cerevisiae (9,10) and at 3.4-Å 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 lysine–lysine 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 (16–18). 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 *To whom correspondence should be addressed. Tel: +49 89 2180 76951; Fax: +49 89 2180 76998; Email: ß The Author(s) 2013. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Research, 2013, Vol. 41, No. 20 9267 Figure 1. Preparation and CX-MS analysis of the Mediator middle module. (A) Schematic representation of the plasmid used for Mediator middle module recombinant expression. Coding sequences are colored according to a code used throughout (Med4, cyan; Med7, orange; Med9, brown; Med10, slate; Med21, magenta; Med31, green). Co-expression was driven from a single plasmid with two T7 promoters, one for bicistronic expression of Med9 and Med4, and one for tetracistronic expression of Med31, Med10, Med7 and Med21. His tag, deca-histidine tag; ori, origin of replication; lacI, gene encoding Lac repressor; RBS, ribosome binding site; Sm, streptomycin resistance gene. (B) SDS-PAGE analysis of the middle module cross-linked with different concentrations of CBDPS. (C) Fragmentation spectrum of a cross-linked peptide. 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. Chemic (...truncated)