MED25 (ARC92/ACID1) is a 747 residues subunit specific to higher eukaryote Mediator complex, an essential component of the RNA polymerase II general transcriptional machinery. MED25 is a target of the Herpes simplex virus transactivator protein VP16. MED25 interacts with VP16 through a central MED25 PTOV (Prostate tumour overexpressed)/ACID (Activator interacting domain) domain of unknown structure. As a first step towards understanding the mechanism of recruitment of transactivation domains by MED25, we report here the NMR structure of the MED25 ACID domain. The domain architecture consists of a closed B-barrel with seven strands (B1-B7) and three a-helices (H1-H3), an architecture showing similarities to that of the SPOC (Spen paralog and ortholog C-terminal domain) domain-like superfamily. Preliminary NMR chemical shift mapping showed that VP16 H2 (VP16C) interacts with MED25 ACID through one face of the beta-barrel, defined by strands B4-B7-B6. (C) 2010 Elsevier Inc. All rights reserved.