Expression of peripheral antigens in the thymus has been implicated in T cell tolerance and autoimmunity. Here we identified medullary thymic epithelial cells as being a unique cell type that expresses a diverse range of tissue-specific antigens. We found that this promiscuous gene expression was a cell-autonomous property of medullary epithelial cells and was maintained during the entire period of thymic T cell output. It may facilitate tolerance induction to self-antigens that would otherwise be temporally or spatially secluded from the immune system. However, the array of promiscuously expressed self-antigens appeared random rather than selected and was not confined to secluded self-antigens.

No abstract

The replication of many retroviruses is mediated by a transcriptional activator protein, Tat, which activates RNA polymerase II at the level of transcription elongation. Tat interacts with Cyclin T1 of the positive transcription-elongation factor P-TEFb to recruit the transactivation-response TAR RNA, which acts as a promoter element in the transcribed 5' end of the viral long terminal repeat. Here we present the structure of the cyclin box domain of Cyclin T1 in complex with the Tat protein from the equine infectious anemia virus and its corresponding TAR RNA. The basic RNA-recognition motif of Tat adopts a helical structure whose flanking regions interact with a cyclin T-specific loop in the first cyclin box repeat. Together, both proteins coordinate the stem-loop structure of TAR. Our findings show that Tat binds to a surface on Cyclin T1 similar to where recognition motifs from substrate and inhibitor peptides were previously found to interact within Cdk-cyclin pairs.

Formins induce the nucleation and polymerization of unbranched actin filaments. They share three homology domains required for profilin binding, actin polymerization, and regulation. Diaphanous-related formins (DRFs) are activated by GTPases of the Rho/Rac family, whose interaction with the N-terminal formin domain is thought to displace a C-terminal Diaphanous-autoregulatory domain (DAD). We have determined the structure of the N-terminal domains of FHOD1 consisting of a GTPase-binding domain (GBD) and the DAD-recognition domain FH3. In contrast to the formin mDia1, the FHOD1-GBD reveals a ubiquitin superfold as found similarly in c-Raf1 or PI3 kinase. This GBD is recruited by Rac and Ras GTPases in cells and plays an essential role for FHOD1-mediated actin remodeling. The FHOD1-FH3 domain is composed of five armadillo repeats, similarly to other formins. Mutation of one residue in the predicted DAD-interaction surface efficiently activates FHOD1 in cells. These results demonstrate that DRFs have evolved different molecular solutions to govern their autoregulation and GTPase specificity.

Rhodobacter capsulatus xanthine dehydrogenase (XDH) is an (␣␤) 2 heterotetrameric cytoplasmic enzyme that resembles eukaryotic xanthine oxidoreductases in respect to both amino acid sequence and structural fold. To obtain a detailed understanding of the mechanism of substrate and inhibitor binding at the active site, we solved crystal structures of R. capsulatus XDH in the presence of its substrates hypoxanthine, xanthine, and the inhibitor pterin-6-aldehyde using either the inactive desulfo form of the enzyme or an active site mutant (E B 232Q) to prevent substrate turnover. The hypoxanthine-and xanthine-bound structures reveal the orientation of both substrates at the active site and show the importance of residue Glu B -232 for substrate positioning. The oxygen atom at the C-6 position of both substrates is oriented toward Arg B -310 in the active site. Thus the substrates bind in an orientation opposite to the one seen in the structure of the reduced enzyme with the inhibitor oxypurinol. The tightness of the substrates in the active site suggests that the intermediate products must exit the binding pocket to allow first the attack of the C-2, followed by oxidation of the C-8 atom to form the final product uric acid. Structural studies of pterin-6-aldehyde, a potent inhibitor of R. capsulatus XDH, contribute further to the understanding of the relative positioning of inhibitors and substrates in the binding pocket. Steady state kinetics reveal a competitive inhibition pattern with a K i of 103.57 ؎ 18.96 nM for pterin-6-aldehyde.

Hexim1 is a cellular protein that associates with the positive transcription elongation factor b (P-TEFb) to regulate RNA polymerase II elongation of nascent mRNA transcripts. It directly binds to Cyclin T1 of P-TEFb and inhibits the kinase activity of Cdk9, leading to an arrest of transcription elongation. Here, we report the solution structure of the Cyclin T binding domain (TBD) of Hexim1 that forms a parallel coiled-coil homodimer composed of two segments and a preceding alpha helix that folds back onto the first coiled-coil unit. NMR titration, fluorescence, and immunoprecipitation experiments revealed the binding interface to Cyclin T1, which covers a large surface on the first coiled-coil segment. Electrostatic interactions between an acidic patch on Hexim1 and positively charged residues of Cyclin T1 drive the complex formation that is confirmed by mutagenesis data on Hexim1 mediated transcription regulation in cells. Thus, our studies provide structural insights how Hexim1 recognizes the Cyclin T1 subunit of P-TEFb, which is a key step toward the regulation of transcription elongation.Cyclin T1 ͉ NMR spectroscopy ͉ transcription elongation T ranscriptional elongation by RNA polymerase II (pol II) is a highly regulated process that generates full-length RNA transcripts and coordinates transcription with pre-mRNA processing (1). The positive transcription elongation factor b (P-TEFb) is a key regulator for the transition from abortive to productive elongation of mature mRNA molecules (2). The core form of P-TEFb is a heterodimer between the cyclin-dependent kinase 9 (Cdk9) and its regulatory subunit Cyclin T1 (CycT1) or the minor forms T2 or K. Transcription activation by P-TEFb depends on the kinase activitiy of Cdk9, which phosphorylates the C-terminal domain (CTD) of the largest subunit of RNA pol II to stimulate the processivity of elongation (3). Initially, P-TEFb has been found to play a critical role in stimulating the transcription of HIV-1 genes, where the viral transactivator protein Tat recruits P-TEFb to stalled RNA pol II molecules (2). At present, P-TEFb is considered a global transcriptional elongation factor important for the expression of most RNA pol II-regulated genes (4).Recently, it has been found that P-TEFb itself is subject to a tight regulation, because approximately half of nuclear P-TEFb in HeLa cells is sequestered in an inactive state. This inactive complex is composed of the Cdk9/CycT1 heterodimer bound to the abundant small nuclear RNA 7SK and the regulatory protein Hexim1 (5-10). Whereas 7SK is supposed to serve as a molecular scaffold to mediate the interaction of Hexim1 with P-TEFb, Hexim1 binds directly to the cyclin box repeats of CycT1 and inhibits the kinase activity of Cdk9. The other half of P-TEFb heterodimers is catalytically active and binds the bromodomain protein, Brd4 (11, 12), which has high affinity for the acetylated tails of core histones H3 and H4 and can bind the Mediator complex.Hexim1 was initially identified as a protein whose expression is induced in...

The human immunodeficiency virus 1 (HIV-1) Nef protein is a pathogenicity factor required for effective progression to AIDS, which modulates host cell signaling pathways and T-cell receptor internalization. We have determined the crystal structure of Nef, allele SF2, in complex with an engineered SH3 domain of human Hck showing unnaturally tight binding and inhibitory potential toward Nef. This complex provides the most complete Nef structure described today, and explains the structural basis of the high affinity of this interaction. Intriguingly, the 33-residue C-terminal flexible loop is resolved in the structure by its interactions with a highly conserved hydrophobic groove on the core domain of an adjacent Nef molecule. The loop mediates the interaction of Nef with the cellular adaptor protein machinery for the stimulated internalization of surface receptors. The endocytic dileucine-based sorting motif is exposed at the tip of the acidic loop, giving the myristoylated Nef protein a distinctly dipolar character. The intermolecular domain assembly of Nef provides insights into a possible regulation mechanism for cargo trafficking.

SummaryThe positive transcription elongation factor b (P-TEFb) is an essential regulator of viral gene expression during the life cycle of HIV-1. Its Cyclin T1 subunit forms a ternary complex with the viral Tat protein and the TAR RNA element thereby activating cyclin dependent kinase 9 (Cdk9), which stimulates transcription at the level of chain elongation. We report the structure of the cyclin box domain of human Cyclin T1 at a resolution of 2.67 Å. The structure was obtained by crystallographic analysis of a fusion protein composed of Cyclin T1 linked to the transactivator protein Tat from equine infectious anemia virus (EIAV), which is functionally and structurally related to HIV-1 Tat. The conserved cyclin box domain of Cyclin T1 exhibits structural features for interaction with physiological binding partners such as Cdk9. A recognition site for Cdk/Cyclin substrates is partly covered by a Cyclin T-specific insert, suggesting specific interactions with regulatory factors. The previously identified Tat/TAR recognition motif (TRM) forms a C-terminal helix that is partly occluded in the cyclin box repeat interface, while cysteine 261 is accessible to form an intermolecular zinc finger with Tat. Residues of the TRM contribute to a positively charged groove that may directly attract RNA molecules. The EIAV Tat protein instead appeared undefined from the electron density map suggesting that it is highly disordered. Functional experiments confirmed the TAR binding properties of the fusion protein and suggested residues on the second cyclin box repeat to contribute to Tat stimulated transcription.