Hexagonal crystal form reveals highly flexible dimer of dimers of the C-terminal domain of transcription cofactor PC4

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Regulation of gene transcription is fundamental for the living cell. Human positive cofactor 4 (PC4) is a well-studied transcription cofactor involved in the regulation of class II activator-dependent gene transcription, where it can act both as a
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  Hexagonal crystal form reveals highly flexible dimer of dimersof the C-terminal domain of transcription cofactor PC4 S.H.W. Scheres, J.M.H. van den Elsen, M. Meisterernst  1 , J. Kroon, P. Gros  Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands  1 Laboratory for Molecular Biology-Genzentrum, Ludwig-Maximilians-University, Munich, Germany  INTRODUCTIONRegulation of gene transcription is fundamental for the living cell. Human positive cofactor 4 (PC4)is a well-studied transcription cofactor involved in the regulation of class II activator-dependentgene transcription, where it can act both as a coactivator and as a repressor. The C-terminal domainof PC4 (residues 63-127) binds single stranded (ss) DNA and heteroduplex DNA. This bindingmode is involved in the repressive function of the protein [1,2]. The N-terminal domain (residues 1-62) plays a role in transcription coactivation and contains two serine-rich stretches which providethe phosphorylation sites in PC4. It has been suggested that upon phosphorylation PC4 switchesfrom the coactivating mode to the repressive one [3]. Both domains are required for binding todouble stranded (ds) DNA. Until now the mechanism of coactivation as well as the biologicalfunction of binding to dsDNA has remained unclear. The crystal structure of the C-terminal domainof PC4 has been solved in our lab in 1997 [4]. Numerous attempts to crystallize the entire proteinhave failed so far. In order to gain better insights of the coactivating mode of PC4, the first serine-rich stretch of the protein has been removed and this new construct (comprising residues 22-127)has been over-expressed in  E.coli and subjected to crystallographic studies.RESULTSHexagonal crystals appeared after two months of incubation using the hanging drop method. Thesecrystals grew to a suitable size during an additional 2-3 months. Mass spectrometric and SDS-PAGE analyses revealed that a degradation product had been crystallized, containing only residues 55-127. Diffraction data up to 2.6 Å resolution were collected from these crystals at the EMBLbeamline BW7B. The structure was solved by molecular replacement (using the C-terminal domainas a search model), and refined to an R-factor of 21.2% (Rfree=24.6%).The P6 1 crystals contain a dimer of dimers in the asymmetric unit (see figure 1). No model could bebuilt for the N-terminal 55 SSSS 58 sequence, indicating that this serine-rich stretch is disordered. Thetemperature factors of the model are generally very high (the average B-factor of all protein atomsis 70.1 Å 2 ) and especially the N-termini and the ssDNA binding sites are highly flexible. The dimer-dimer interface is similar to the ones observed in the ‘wide octamer’ in the triclinic crystal form of the C-terminal domain, suggesting that this dimerization of dimers could be relevant for thebiological function of PC4.  Figure 1: The dimer of dimers in the asymmetric unit References [1]S. Werten et al. J. Mol. Biol. 276, 367 (1998)[2]S. Werten et al. EMBO J. 17, 5103 (1998)[3]S. Malik  et al. Proc. Natl. Acad. Sci. USA 95, 2192 (1998)[4]J. Brandsen et al. , Nature Struc. Biol. 4, 900 (1997)   \ R X U I L J X U H 
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