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Ligand-dependent activation of gene transcription by nuclear receptors is dependent on the recruitment of coactivators, including a family of related NCoA/SRC factors, via a region containing three helical domains sharing an LXXLL core consensus sequence, referred to as LXDs. In this manuscript, we report receptor-specific differential utilization of LXXLL-containing motifs of the NCoA-1/SRC-1 coactivator. Whereas a single LXD is sufficient for activation by the estrogen receptor, different combinations of two, appropriately spaced, LXDs are required for actions of the thyroid hormone, retinoic acid, peroxisome proliferator-activated, or progesterone receptors. The specificity of LXD usage in the cell appears to be dictated, at least in part, by specific amino acids carboxy-terminal to the core LXXLL motif that may make differential contacts with helices 1 and 3 (or 3') in receptor ligand-binding domains. Intriguingly, distinct carboxy-terminal amino acids are required for PPARgamma activation in response to different ligands. Related LXXLL-containing motifs in NCoA-1/SRC-1 are also required for a functional interaction with CBP, potentially interacting with a hydrophobic binding pocket. Together, these data suggest that the LXXLL-containing motifs have evolved to serve overlapping roles that are likely to permit both receptor-specific and ligand-specific assembly of a coactivator complex, and that these recognition motifs underlie the recruitment of coactivator complexes required for nuclear receptor function.

The inhibitor of apoptosis proteins (IAPs) constitute an evolutionarily conserved family of homologous proteins that suppress apoptosis induced by multiple stimuli. Some IAP family proteins, including XIAP, cIAP-1, and cIAP-2, can bind and directly inhibit selected caspases, a group of intracellular cell death proteases. These caspase-inhibiting IAP family proteins all contain three tandem BIR domains followed by a RING zinc finger domain. To determine the structural basis for caspase inhibition by XIAP, we analyzed the effects of various fragments of this IAP family protein on caspase activity in vitro and on apoptosis suppression in intact cells. The RING domain of XIAP failed to inhibit the activity of recombinant caspases-3 or -7, whereas a fragment of XIAP encompassing the three tandem BIR domains potently inhibited these caspases in vitro and blocked Fas (CD95)-induced apoptosis when expressed in cells. Further dissection of the XIAP protein demonstrated that only the second of the three BIR domains (BIR2) was capable of binding and inhibiting these caspases. The apparent inhibition constants (Ki) for BIR2-mediated inhibition of caspases-3 and -7 were 2-5 nM, indicating that this single BIR domain possesses potent anti-caspase activity. Expression of the BIR2 domain in cells also partially suppressed Fas-induced apoptosis and blocked cytochrome c-induced processing of caspase-9 in cytosolic extracts, whereas BIR1 and BIR3 did not. These findings identify BIR2 as the minimal caspase-inhibitory domain of XIAP and indicate that a single BIR domain can be sufficient for binding and inhibiting caspases.

Thirty-six monoclonal antibodies from mice and three from rats were raised against a peptide corresponding to the immunodominant domain of the transmembrane gp41 protein of human immunodeficiency virus (HIV) type 1 (LGLWGCSGKLIC; amino acid residues 598 to 609). Of these, three monoclonal antibodies from the mice and one from a rat also reacted with the corresponding peptide derived from the HIV type 2 transmembrane gp41 protein (amino acid residues 593 to 603; NSWGCAFRQVC). Immunochemical studies using a variety of synthetic peptides indicated that the cross-reactivity was due to antibody binding to CSGKLIC of HIV type 1 or CAFRQVC of HIV type 2. Single amino acid substitutions for a cysteine at either the amino or the carboxy end of the peptide interrupted antibody binding, indicating that the site recognized was the Cys-XXXXX-Cys loop. Similar results were obtained when the 11-mer HIV type 2 gp41 peptide (amino acids 593 to 603) was inoculated into mice to raise monoclonal antibodies. In this instance, of 30 monoclonal antibodies developed, 4 reacted with both HIV type 1 and HIV type 2 peptides. The conformation of a seven-residue peptide, CSGKLIC, corresponding to residues 603 to 609 of the gp41 immunodominant epitope of HIV-1 was investigated by proton nuclear magnetic resonance spectroscopy. The immunologically active form of CSGKLIC contains an intramolecular disulfide bond and maintains a preference for a folded conformation, apparently including a type I reverse turn about the residues SGKL. No such preference is observed for the reduced form of the peptide, which contains two thiol groups. The presence of the disulfide bond is thus integral to the formation of the structure of the loop in solution. In agreement with this finding, elimination of the possibility of loop formation by substitution of S for C at the amino or carboxy termini of the 7-mer is accompanied by the failure of antibody binding to this peptide.