The TRAP coactivator complex is a large, multisubunit complex of nuclear proteins which associates with nuclear hormone receptors (NRs) in the presence of cognate ligand and stimulates NR-mediated transcription. A single subunit, TRAP220, is thought to target the entire complex to a liganded receptor through a domain containing two of the signature LXXLL motifs shown previously in other types of coactivator proteins to be essential for mediating NR binding. In this work, we demonstrate that each of the two LXXLL-containing regions, termed receptor binding domains 1 and 2 (RBD-1 and RBD-2), is differentially preferred by specific NRs. The retinoid X receptor (RXR) displays a weak yet specific activation function 2 (AF2)-dependent preference for RBD-1, while the thyroid hormone receptor (TR), vitamin D 3 receptor (VDR), and peroxisome proliferator-activated receptor all exhibit a strong AF2-dependent preference for RBD-2. Using site-directed mutagenesis, we show that preference for RBD-2 is due to the presence of basic-polar residues on the amino-terminal end of the core LXXLL motif. Furthermore, we show that the presence and proper spacing of both RBD-1 and RBD-2 are required for an optimal association of TRAP220 with RXR-TR or RXR-VDR heterodimers bound to DNA and for TRAP220 coactivator function. On the basis of these results, we suggest that a single molecule of TRAP220 can interact with both subunits of a DNA-bound NR heterodimer.Nuclear hormone receptors (NRs) make up a family of ligand-activated transcription factors that regulate the expression of target genes involved in development, differentiation, and homeostasis (39,47,60). The ligands for NRs, small hydrophobic molecules including steroids, retinoids, thyroid hormone, and vitamin D 3 , bind to the C-terminal ligand binding domain (LBD) of their cognate NR and induce conformational changes which modulate receptor activity (44). Transcriptional activation by NRs can be mediated by two separable activation functions (AFs): AF1, located at the N terminus (30,45,56), and AF2, located in the LBD (3,11,15,52). While AF1 is poorly conserved among NR family members, the AF2 domain is highly conserved and essential for ligand-dependent activation (11,47). Recent structural studies suggest that ligand binding regulates AF2 activity by changing the stereospecific position of the most C-terminal LBD ␣ helix (helix 12), a motif previously shown to be indispensable for AF2 function (44, 47). The ligand-induced repositioning of helix 12 places it in close proximity to ␣ helices 3, 4, and 5 and is thought to generate a hydrophobic binding surface for transcriptional coactivator proteins (12,25,46,53).The best-characterized NR coactivators identified thus far are members of the SRC/p160 family of proteins, which include SRC-1/NCoA-1, TIF2/GRIP1, and pCIP/RAC3/AIB-1/ ACTR/TRAM-1 (reviewed in references 42 and 58). While the exact mechanism of action of the SRC/p160 proteins is unclear, their ability to associate with histone acetyltransferases (HATs) such as CBP/p300 (...

Peptides fused to the coat proteins of filamentous phages have found widespread applications in antigen display, the construction of antibody libraries, and biopanning. However, such systems are limited in terms of the size and number of the peptides that may be incorporated without compromising the fusion proteins' capacity to self-assemble. We describe here a system in which the molecules to be displayed are bound to pre-assembled polymers. The polymers are T4 capsids and polyheads (tubular capsid variants) and the display molecules are derivatives of the dispensable capsid protein SOC. In one implementation, SOC and its fusion derivatives are expressed at high levels in Escherichia coli, purified in high yield, and then bound in vitro to separately isolated polyheads. In the other, a positive selection vector forces integration of the modified soc gene into a soc-deleted T4 genome, leading to in vivo binding of the display protein to progeny virions. The system is demonstrated as applied to C-terminal fusions to SOC of (1) a tetrapeptide; (2) the 43-residue V3 loop domain of gp120, the human immunodeficiency virus type-I (HIV-I) envelope glycoprotein; and (3) poliovirus VPl capsid protein (312 residues). SOC-V3 displaying phage were highly antigenic in mice and produced antibodies reactive with native gp120. That the fusion protein binds correctly to the surface lattice was attested in averaged electron micrographs of polyheads. The SOC display system is capable of presenting up to -I O 3 copies per capsid and >IO4 copies per polyhead of V3-sized domains. Phage displaying SOC-VPI were isolated from a 1:106 mixture by two cycles of a simple biopanning procedure, indicating that proteins of at least 35 kDa may be accommodated.Keywords: bacteriophage T4; capsid assembly; human immunodeficiency virus antigens; phage display; protein engineering Filamentous phage-based display systems (Smith, 1985) have found widespread use in molecular biology, including many immunologic applications such as antigen presentation and the immunoisolation of desired recombinants (biopanning) (Marks et al., 1992;Smith et al ., 1993;Williamson et al., 1993). However, with filamentous phages, peptides that may be displayed from the major coat protein are limited in size to 6-10 amino acid residues (Kishchenko et al., 1994;Iannolo et al., 1995), although somewhat

No abstract

No abstract

No abstract