Showing papers by "Stephen W. Fesik published in 1996"

Discovering High-Affinity Ligands for Proteins: SAR by NMR

Abstract: A nuclear magnetic resonance (NMR)-based method is described in which small organic molecules that bind to proximal subsites of a protein are identified, optimized, and linked together to produce high-affinity ligands. The approach is called "SAR by NMR" because structure-activity relationships (SAR) are obtained from NMR. With this technique, compounds with nanomolar affinities for the FK506 binding protein were rapidly discovered by tethering two ligands with micromolar affinities. The method reduces the amount of chemical synthesis and time required for the discovery of high-affinity ligands and appears particularly useful in target-directed drug research.

X-ray and NMR structure of human Bcl-xL, an inhibitor of programmed cell death.

Abstract: THE Bcl-2 family of proteins regulate programmed cell death by an unknown mechanism. Here we describe the crystal and solution structures of a Bcl-2 family member, Bcl-xL (ref. 2). The structures consist of two central, primarily hydrophobic alpha-helices, which are surrounded by amphipathic helices. A 60-residue loop connecting helices alpha1 and alpha2 was found to be flexible and non-essential for anti-apoptotic activity. The three functionally important Bcl-2 homology regions (BH1, BH2 and BH3) are in close spatial proximity and form an elongated hydrophobic cleft that may represent the binding site for other Bcl-2 family members. The arrangement of the alpha-helices in Bcl-xL is reminiscent of the membrane translocation domain of bacterial toxins, in particular diphtheria toxin and the colicins. The structural similarity may provide a clue to the mechanism of action of the Bcl-2 family of proteins.

NMR structure and mutagenesis of the Fas (APO-1/CD95) death domain.

Abstract: PROGRAMMEDcell death (apoptosis) mediated by the cytokine receptor Fas is critical for the removal of autoreactive T cells1, the mechanism of immune privilege2,3, and for maintenance of immune-system homeostasis4. Signalling of programmed cell death involves the self-association of a conserved cytoplasmic region of Fas called the death domain5–7 and interaction with another death-domain-containing protein, FADD8 (also known as MORT1)9. Although death domains are found in several proteins10, their three-dimensional structure and the manner in which they interact is unknown. Here we describe the solution structure of the Fas death domain, as determined by NMR spectroscopy. The structure consists of six antiparallel, amphi-pathic α-helices arranged in a novel fold. From the structure and from site-directed mutagenesis, we have identified the region of the death domain involved in self-association and binding to the downstream signalling partner FADD.

Structural basis for IL-4 receptor phosphopeptide recognition by the IRS-1 PTB domain.

Abstract: We present the NMR structure of the PTB domain of insulin receptor substrate-1 (IRS-1) complexed to a tyrosine-phosphorylated peptide derived from the IL-4 receptor. Despite the lack of sequence homology and different binding specificity, the overall fold of the protein is similar to that of the Shc PTB domain and closely resembles that of PH domains. However, the PTB domain of IRS-1 is smaller than that of She (110 versus 170 residues) and binds to phosphopeptides in a distinct manner. We explain the phosphopeptide binding specificity based on the structure of the complex and results of site-directed mutagenesis experiments.

Evidence for a role for the phosphotyrosine-binding domain of Shc in interleukin 2 signaling

Abstract: Stimulation via the T-cell growth factor interleukin 2 (IL-2) leads to tyrosine phosphorylation of Shc, the interaction of Shc with Grb2, and the Ras GTP/GDP exchange factor, mSOS. Shc also coprecipitates with the IL-2 receptor (IL-2R), and therefore, may link IL-2R to Ras activation. We have further characterized the Shc-IL-2R interaction and have made the following observations. (i) Among the two phosphotyrosine-interaction domains present in Shc, the phosphotyrosine-binding (PTB) domain, rather than its SH2 domain, interacts with the tyrosine-phosphorylated IL-2R beta chain. Moreover, the Shc-PTB domain binds a phosphopeptide derived from the IL-2R beta chain (corresponding to residues surrounding Y338, SCFTNQGpYFF) with high affinity. (ii) In vivo, mutant IL-2R beta chains lacking the acidic region of IL-2Rbeta (which contains Y338) fail to phosphorylate Shc. Furthermore, when wild type or mutant Shc proteins that lack the PTB domain were expressed in the IL-2-dependent CTLL-20 cell line, an intact Shc-PTB domain was required for Shc phosphorylation by the IL-2R, which provides further support for a Shc-PTB-IL-2R interaction in vivo. (iii) PTB and SH2 domains of Shc associate with different proteins in IL-2- and T-cell-receptor-stimulated lysates, suggesting that Shc, through the concurrent use of its two different phosphotyrosine-binding domains, could assemble multiple protein complexes. Taken together, our in vivo and in vitro observations suggest that the PTB domain of Shc interacts with Y338 of the IL-2R and provide evidence for a functional role for the Shc-PTB domain in IL-2 signaling.

Backbone dynamics of the C-terminal domain of Escherichia coli topoisomerase I in the absence and presence of single-stranded DNA.

Abstract: The backbone dynamics of the C-terminal DNA-binding domain of Escherichia coli topoisomerase I has been characterized in the absence and presence of single-stranded DNA by NMR spectroscopy. 15N spin-lattice relaxation times (T1), spin-spin relaxation times (T2), and heteronuclear NOEs were determined for the uniformly 15N-labeled protein. These data were analyzed by using the model-free formalism to derive the model-free parameters (S2, tau e, and R(ex)) for each backbone N-H bond vector and the overall molecular rotational correlation time (tau m)., The molecular rotational correlation time tau m was determined to be 7.49 +/- 0.36 ns for the free and 12.7 +/- 1.07 ns for the complexed protein. Several residues were found to be much more mobile than the average, including 11 residues at the N-terminus, 2 residues at the C-terminus, and residues 25 and 31-35 which are located in a region of the protein that binds to DNA. The binding of ssDNA to the free protein causes a slight increase in the order parameters (S2) for a small number of residues and a slight decrease in the order parameters (S2) for the majority of the residues. In particular, upon binding to ssDNA, the mobility of the first alpha-helix and the two beta-sheets was slightly increased, and the mobility of a few specific residues in the loops/turns was restricted. These results differ from the previous studies on the backbone dynamics of molecular complexes in which reduced mobilities were typically observed upon ligand binding.

Solution structure of the DNA-binding domain of a human papillomavirus E2 protein: evidence for flexible DNA-binding regions.

Abstract: The three-dimensional structure of the DNA-binding domain of the E2 protein from human papillomavirus-31 was determined by using multidimensional heteronuclear nuclear magnetic resonance (NMR) spectroscopy. A total of 1429 NMR-derived distance and dihedral angle restraints were obtained for each of the 83-residue subunits of this symmetric dimer. The average root mean square deviations of 20 structures calculated using a distance geometry-simulated annealing protocol are 0.59 and 0.90 A for the backbone and all heavy atoms, respectively, for residues 2−83. The structure of the human virus protein free in solution consists of an eight-stranded β-barrel and two pairs of α-helices. Although the overall fold of the protein is similar to the crystal structure of the bovine papillomavirus-1 E2 protein when complexed to DNA, several small but interesting differences were observed between these two structures at the subunit interface. In addition, a β-hairpin that contacts DNA in the crystal structure of the prot...

Biphenyl hydroxamate inhibitors of matrix metalloproteinases

Abstract: Compounds of formula ##STR1## or a pharmaceutically acceptable salt thereof inhibit matrix metalloproteinases and TNFα secretion and are useful in the treatment of inflammatory disease states. Also disclosed are matrix metalloproteinases and TNFα secretion inhibiting compositions and a method for inhibiting matrix metalloproteinases and TNFα secretion.

NMR Structures of Proteins Involved in Signal Transduction

Abstract: Signal transduction is a complicated process that may involve several steps mediated by specific intermolecular interactions. Recently, three-dimensional structures of proteins involved in signal transduction have been obtained and have greatly aided in our understanding of these processes at the molecular level.1,2 In this paper, three-dimensional structures of three proteins that are involved in signal transduction will be described, including (1) a protein tyrosine phosphatase, (2) a pleckstrin homology (PH) domain, and (3) the DNA-binding domain of a member of the ets family of transcription factors, Fli-1, in the DNA-bound form.

Inhibiteurs de metalloproteinases matricielles, a l'hydroxamate biphenylique

Abstract: L'invention concerne des composes de formule (I) ou un sel pharmaceutiquement acceptable de ceux-ci, qui inhibent la secretion des metalloproteinases matricielles et de TNFα et sont utiles dans le traitement de pathologies inflammatoires. Elle porte aussi sur des compositions inhibant la secretion de metalloproteinases matricielles et de TNFα et sur une methode d'inhibition de la secretion de metalloproteinases matricielles et de TNFα.