Bacteriophage SPP1 tail tube protein self-assembles into ß-structure-rich tubes.

The majority of known bacteriophages have long tails that serve for bacterial target recognition and viral DNA delivery into the host. These structures form a tube from the viral capsid to the bacterial cell. The tube is formed primarily by a helical array of tail tube protein (TTP) subunits. In phages with a contractile tail, the TTP tube is surrounded by a sheath structure. Here, we report the first evidence that a phage TTP, gp17.1 of siphophage SPP1, self-assembles into long tubes in the absence of other viral proteins. gp17.1 does not exhibit a stable globular structure when monomeric in solution, even if it was confidently predicted to adopt the ß-sandwich fold of phage λ TTP. However, Fourier transform infrared and nuclear magnetic resonance spectroscopy analyses showed that its ß-sheet content increases significantly during tube assembly, suggesting that gp17.1 acquires a stable ß-sandwich fold only after self-assembly. EM analyses revealed that the tube is formed by hexameric rings stacked helicoidally with the same organization and helical parameters found for the tail of SPP1 virions. These parameters were used to build a pseudo-atomic model of the TTP tube. The large loop spanning residues 40-56 is located on the inner surface of the tube, at the interface between adjacent monomers and hexamers. In line with our structural predictions, deletion of this loop hinders gp17.1 tube assembly in vitro and interferes with SPP1 tail assembly during phage particle morphogenesis in bacteria.

Structure-based design of a potent artificial transactivation domain based on p53.

Malfunctions in transcriptional regulation are associated with a number of critical human diseases. As a result, there is considerable interest in designing artificial transcription activators (ATAs) that specifically control genes linked to human diseases. Like native transcriptional activator proteins, an ATA must minimally contain a DNA-binding domain (DBD) and a transactivation domain (TAD) and, although there are several reliable methods for designing artificial DBDs, designing artificial TADs has proven difficult. In this manuscript, we present a structure-based strategy for designing short peptides containing natural amino acids that function as artificial TADs. Using a segment of the TAD of p53 as the scaffolding, modifications are introduced to increase the helical propensity of the peptides. The most active artificial TAD, termed E-Cap-(LL), is a 13-mer peptide that contains four key residues from p53, an N-capping motif and a dileucine hydrophobic bridge. In vitro analysis demonstrates that E-Cap-(LL) interacts with several known p53 target proteins, while in vivo studies in a yeast model system show that it is a 20-fold more potent transcriptional activator than the native p53-13 peptide. These results demonstrate that structure-based design represents a promising approach for developing artificial TADs that can be combined with artificial DBDs to create potent and specific ATAs.

NMR structure of the complex between the Tfb1 subunit of TFIIH and the activation domain of VP16: structural similarities between VP16 and p53.

The Herpes Simplex Virion Protein 16 (VP16) activates transcription through a series of protein/protein interactions involving its highly acidic transactivation domain (TAD). The acidic TAD of VP16 (VP16TAD) has been shown to interact with several partner proteins both in vitro and in vivo, and many of these VP16 partners also bind the acidic TAD of the mammalian tumor suppressor protein p53. For example, the TADs of VP16 and p53 (p53TAD) both interact directly with the p62/Tfb1 (human/yeast) subunit of TFIIH, and this interaction correlates with their ability to activate both the initiation and elongation phase of transcription. In this manuscript, we use NMR spectroscopy, isothermal titration calorimetery (ITC) and site-directed mutagenesis studies to characterize the interaction between the VP16TAD and Tfb1. We identify a region within the carboxyl-terminal subdomain of the VP16TAD (VP16C) that has sequence similarity with p53TAD2 and binds Tfb1 with nanomolar affinity. We determine an NMR structure of a Tfb1/VP16C complex, which represents the first high-resolution structure of the VP16TAD in complex with a target protein. The structure demonstrates that like p53TAD2, VP16C forms a 9-residue alpha-helix in complex with Tfb1. Comparison of the VP16/Tfb1and p53/Tfb1 structures clearly demonstrates how the viral activator VP16C and p53TAD2 shares numerous aspects of binding to Tfb1. Despite the similarities, important differences are observed between the p53TAD2/Tfb1 and VP16C/Tfb1 complexes, and these differences demonstrate how selected activators such as p53 depend on phosphorylation events to selectively regulate transcription.

p53 and TFIIEalpha share a common binding site on the Tfb1/p62 subunit of TFIIH.

The general transcription factor IIH is recruited to the transcription preinitiation complex through an interaction between its p62/Tfb1 subunit and the alpha-subunit of the general transcription factor IIE (TFIIEalpha). We have determined that the acidic carboxyl terminus of TFIIEalpha (TFIIEalpha(336-439)) directly binds the amino-terminal PH domain of p62/Tfb1 with nanomolar affinity. NMR mapping and mutagenesis studies demonstrate that the TFIIEalpha binding site on p62/Tfb1 is identical to the binding site for the second transactivation domain of p53 (p53 TAD2). In addition, we demonstrate that TFIIEalpha(336-439) is capable of competing with p53 for a common binding site on p62/Tfb1 and that TFIIEalpha(336-439) and the diphosphorylated form (pS46/pT55) of p53 TAD2 have similar binding constants. NMR structural studies reveal that TFIIEalpha(336-439) contains a small domain (residues 395-433) folded in a novel betabetaalphaalphaalpha topology. NMR mapping studies demonstrate that two unstructured regions (residues 377-393 and residues 433-439) located on either side of the folded domain appear to be required for TFIIEalpha(336-439) binding to p62/Tfb1 and that these two unstructured regions are held close to each other in three-dimensional space by the novel structured domain. We also demonstrate that, like p53, TFIIEalpha(336-439) can activate transcription in vivo. These results point to an important interplay between the general transcription factor TFIIEalpha and the tumor suppressor protein p53 in regulating transcriptional activation that may be modulated by the phosphorylation status of p53.

PTHrP fragments 1-16 and 1-23 do not bind to either the ETA or the ETB endothelin receptors.

Because of some isofunctional similarities with endothelin-1 (ET-1), it has been suggested that PTHrP(1-16) and PTHrP(1-23) could interact with osteoblast cells via ETA receptors. To document this interaction, we used the thoracic rat aorta and the guinea-pig lung parenchyma paradigms as ETA and ETB models, respectively. In addition, we also performed a series of competition experiments against [125I]ET-1, using transfected cells expressing the ETA or ETB receptor. So far, no agonistic nor antagonistic activities were observed in the ETA and ETB bioassays with the PTHrP fragments. Furthermore, both fragments were unable to displace [125I]ET-1 bound to cells expressing the ETA or ETB receptor.

Development of agonists of endothelin-1 exhibiting selectivity towards ETA receptors.

(1) Endothelin-1 (ET-1) is a bicyclic 21-amino-acid peptide causing a potent and sustained vasoconstriction, mainly through the ET(A) receptor subtype. So far, no selective ET(A) agonists are described in the literature. (2) A series of truncated and chemically modified ET-1 analogues were obtained through solid-phase peptide synthesis and their biological activity was assessed on rat thoracic aorta rings (ET(A) receptors) and guinea-pig lung parenchyma strips (ET(B) receptors). (3) Structure-activity studies led to the identification of ET-1 fragments exhibiting an ET(A) selective agonistic activity. (4) In particular, [D-Lys(9)]cyclo(11-15) ET-1(9-21) was the most potent peptide. It appeared as a full agonist of ET(A) receptors, being under two orders of magnitude less potent than ET-1 (EC(50): 2.3 x 10(-7) vs 6.8 x 10(-9) M). Interestingly, even a linear formylated analogue, [Ala(11,15), Trp(For)(21)]ET-1(9-21), showed a selective ET(A) activity (EC(50): 3.0 x 10(-6) M). None of the numerous analogues of the series exhibited substantial effects in the guinea-pig lung parenchyma bioassay. (5) Thus, this study describes the first compounds showing a significant bioactivity in an ET(A) pharmacological preparation while being inactive in an ET(B) paradigm. They show that the ET-1 pharmacophores, responsible for the ET(A)-mediated actions, are located within the 9-21 segment of the molecule. Moreover, the bicyclic structure of ET-1 does not appear as essential for the ET(A)-related vasoconstriction. Results also suggest that the positive charge of the Lys(9) side chain participates in an intramolecular ionic bond with the carboxylate function of Asp(18).