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

Huang, Baohua; Eberstadt, Matthias; Olejniczak, Edward T.; Meadows, Robert P.; Fesik, Stephen W.

doi:10.1038/384638a0

Cited by 366 publications

(334 citation statements)

References 28 publications

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“…In the case of Fas, either solubility conditions would need to be explored for an already folded domain or the boundaries of the domain would have to be extended slightly to enhance solubility. The recent publication of the solution structure of the Fas death domain (Huang et al, 1996) confirms our results presented above, namely that residues 210-305 do indeed delineate the folded domain. However, the construct employed by Huang et al (1996) was slightly longer, comprising residues 202-3 19, which presumably increased its solubility.…”

Section: Jr Huth Et Alsupporting

confidence: 87%

“…The recent publication of the solution structure of the Fas death domain (Huang et al, 1996) confirms our results presented above, namely that residues 210-305 do indeed delineate the folded domain. However, the construct employed by Huang et al (1996) was slightly longer, comprising residues 202-3 19, which presumably increased its solubility. In addition, the structure of the Fas death domain was determined at pH 4.0 in 50 mM sodium acetate and 50 mM NaCl, which may also have enhanced the solubility, whereas our spectrum was recorded in the absence of any salt at pH 4.3.…”

Section: Jr Huth Et Alsupporting

confidence: 87%

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Design of an expression system for detecting folded protein domains and mapping macromolecular interactions by NMR

et al. 1997

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Two protein expression vectors have been designed for the preparation of NMR samples. The vectors encode the immunoglobulin-binding domain of streptococcal protein G (GB1 domain) linked to the N-terminus of the desired proteins. This fusion strategy takes advantage of the small size, stable fold, and high bacterial expression capability of the GB 1 domain to allow direct NMR spectroscopic analysis of the fusion protein by ' H-l5N correlation spectroscopy.Using this system accelerates the initial assessment of protein NMR projects such that, in a matter of days, the solubility and stability of a protein can be determined. In addition, "N-labeling of peptides and their testing for DNA binding are facilitated. Several examples are presented that demonstrate the usefulness of this technique for screening protein/DNA complexes, as well as for probing ligand-receptor interactions, using iSN-labeled GBI-peptide fusions and unlabeled target.Keywords: domain structure; GBI-fusion protein; intermolecular interactions; NMR When investigating whether a protein or protein-DNA complex is suitable for NMR structural analysis, several technical goals must be met. First, the size, solubility, and stability of the protein domain must be optimized. Second, the protein should be expressed in a host (usually bacteria), allowing for easy and inexpensive labeling with "C and "N. A third challenge pertains to the preparation of protein-DNA complexes where both the protein and DNA sequences must be optimized to achieve a complex that is most amenable to structural investigation; that is, the system should not exhibit intermediate exchange on the NMR time scale. The practical size limit for structure determination by current solution NMR methodology is approximately 40 kDa, so that, for many proteins, subcloning of a smaller domain that retains functional qualities of the full-length protein is required. All too frequently, however, engineering of protein domains results in limited solubility and/or low stability, often to the point where many engineered proteins are partially or predominately unfolded. In practice, many variations of a protein sequence may be evaluated by NMR

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Section: Jr Huth Et Alsupporting

confidence: 87%

Section: Jr Huth Et Alsupporting

confidence: 87%

Design of an expression system for detecting folded protein domains and mapping macromolecular interactions by NMR

et al. 1997

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“…58 (A backbone overlay of the 10 lowest energy NMR structures is shown.) Subsequently, this motif has been identified in a wide variety of proteins, including other dimeric helical bundles (FIS DNA-binding protein 116 and Rop mutant 68 ), intramolecularly folded helical bundles (FAS death domain 117 and N-terminal domain of the δ subunit of the ATPsynthase 118 ), and β-sheet proteins (the IgG fold 119 ), and multimeric proteins with both α/β structures (p53 tetramerization domain 120 ). Glu7 is not involved in stabilizing the native state of α 2 D. Superposition of the 10 lowest energy NMR-derived structures (left panel) shows that Glu7 adopts multiple conformations typical of solvent-exposed residues (hydrophobic core residues in gray).…”

Section: Resultsmentioning

confidence: 99%

De NovoDesign of Helical Bundles as Models for Understanding Protein Folding and Function

et al. 2000

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De novo protein design has proven to be a powerful tool for understanding protein folding, structure, and function. In this Account, we highlight aspects of our research on the design of dimeric, four-helix bundles. Dimeric, four-helix bundles are found throughout nature, and the history of their design in our laboratory illustrates our hierarchic approach to protein design. This approach has been successfully applied to create a completely native-like protein. Structural and mutational analysis allowed us to explore the determinants of native protein structure. These determinants were then applied to the design of a dinuclear metal-binding protein that can now serve as a model for this important class of proteins.Protein folding is an important and multifaceted scientific problem. 1-8 One facet of this problem involves the prediction of three-dimensional structure from amino acid sequence, an endeavor the importance of which has been highlighted by the recent release of gene sequences of many whole organisms. As the size of the protein structural database expands, it will be increasingly possible to assign amino acid sequences to a given structural family on the basis of the homology of their sequences with proteins of known structure. Another facet of the folding problem is the delineation of the kinetic mechanism by which an unfolded protein chain undergoes the transition from a random coil with an astronomical number of configurations to a uniquely folded structure. A final facet of the protein folding problem is to understand, at the atomic level, the detailed physicochemical features that kinetically and thermodynamically direct the formation of an unique, cooperatively folded, native structure. This latter endeavor is particularly important as it lays the groundwork for the design of novel inhibitors of natural proteins as well as the construction of novel proteins and related biopolymers not found in nature.De novo protein design is an approach to the protein folding problem that critically tests our understanding of all these facets of this problem. 9,10 This method involves the design of a sequence that is intended to fold into a predetermined three-dimensional structure without the sequence being patterned after any natural protein. Through an iterative process of design and rigorous characterization, the principles governing protein folding and function can be evaluated. Thus, "failures" highlight gaps in our understanding, whereas "successes" confirm the principles used in the design and often provide simple model systems to further

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“…Fas expression is present in many tumours and tumour cell lines including in SCLC Muller et al, 1997;Niehans et al, 1997;Fulda et al, 1998;Kawasaki et al, 2000). After trimerisation of Fas on the cell membrane by extracellular FasL (Huang et al, 1996), Fas-associated Death Domain (FADD) and caspase-8 bind to the intracellular death domains of Fas and induce a death signal in the cell . This leads to the activation of a cascade of caspases and eventually to cell death.…”

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confidence: 99%

Indomethacin-induced activation of the death receptor-mediated apoptosis pathway circumvents acquired doxorubicin resistance in SCLC cells

et al. 2005

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Small-cell lung cancers (SCLCs) initially respond to chemotherapy but are often resistant at recurrence. A potentially new method to overcome resistance is to combine classical chemotherapeutic drugs with apoptosis induction via tumour necrosis factor (TNF) death receptor family members such as Fas. The doxorubicin-resistant human SCLC cell line GLC 4 -Adr and its parental doxorubicinsensitive line GLC 4 were used to analyse the potential of the Fas-mediated apoptotic pathway and the mitochondrial apoptotic pathway to modulate doxorubicin resistance in SCLC. Western blotting showed that all proteins necessary for death-inducing signalling complex formation and several inhibitors of apoptosis were expressed in both lines. The proapototic proteins Bid and caspase-8, however, were higher expressed in GLC 4 -Adr. In addition, GLC 4 -Adr expressed more Fas (3.1x) at the cell membrane. Both lines were resistant to anti-Fas antibody, but plus the protein synthesis inhibitor cycloheximide anti-Fas antibody induced 40% apoptosis in GLC 4 -Adr. Indomethacin, which targets the mitochondrial apoptotic pathway, induced apoptosis in GLC 4 -Adr but not in GLC 4 cells. Surprisingly, in GLC 4 -Adr indomethacin induced caspase-8 and caspase-9 activation as well as Bid cleavage, while both caspase-8 and caspase-9 specific inhibitors blocked indomethacin-induced apoptosis. In GLC 4 -Adr, doxorubicin plus indomethacin resulted in elevated caspase activity and a 2.7-fold enhanced sensitivity to doxorubicin. In contrast, no effect of indomethacin on doxorubicin sensitivity was observed in GLC 4 . Our findings show that indomethacin increases the cytotoxic activity of doxorubicin in a doxorubicin-resistant SCLC cell line partly via the death receptor apoptosis pathway, independent of Fas.

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

Cited by 366 publications

References 28 publications

Design of an expression system for detecting folded protein domains and mapping macromolecular interactions by NMR

Design of an expression system for detecting folded protein domains and mapping macromolecular interactions by NMR

De NovoDesign of Helical Bundles as Models for Understanding Protein Folding and Function

Indomethacin-induced activation of the death receptor-mediated apoptosis pathway circumvents acquired doxorubicin resistance in SCLC cells

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