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Showing papers on "Conformational change published in 2002"


Journal ArticleDOI
06 Sep 2002-Cell
TL;DR: It is shown that a highly bent integrin conformation is physiological and has low affinity for biological ligands.

1,134 citations


Journal ArticleDOI
12 Dec 2002-Nature
TL;DR: It is shown that recognition by receptor-binding-site antibodies induces conformational change, and conformational masking enables HIV-1 to maintain receptor binding and simultaneously to resist neutralization.
Abstract: The ability of human immunodeficiency virus (HIV-1) to persist and cause AIDS is dependent on its avoidance of antibody-mediated neutralization. The virus elicits abundant, envelope-directed antibodies that have little neutralization capacity. This lack of neutralization is paradoxical, given the functional conservation and exposure of receptor-binding sites on the gp120 envelope glycoprotein, which are larger than the typical antibody footprint and should therefore be accessible for antibody binding. Because gp120-receptor interactions involve conformational reorganization, we measured the entropies of binding for 20 gp120-reactive antibodies. Here we show that recognition by receptor-binding-site antibodies induces conformational change. Correlation with neutralization potency and analysis of receptor-antibody thermodynamic cycles suggested a receptor-binding-site 'conformational masking' mechanism of neutralization escape. To understand how such an escape mechanism would be compatible with virus-receptor interactions, we tested a soluble dodecameric receptor molecule and found that it neutralized primary HIV-1 isolates with great potency, showing that simultaneous binding of viral envelope glycoproteins by multiple receptors creates sufficient avidity to compensate for such masking. Because this solution is available for cell-surface receptors but not for most antibodies, conformational masking enables HIV-1 to maintain receptor binding and simultaneously to resist neutralization.

904 citations


Journal ArticleDOI
TL;DR: These long-range structural rearrangements of the entire integrin molecule involving multiple interdomain contacts appear closely linked to conformational changes in the I domain, which result in increased affinity and competence for ligand binding.
Abstract: Integrins are a structurally elaborate family of heterodimers that mediate divalent cation-dependent cell adhesion in a wide range of biological contexts. The inserted (I) domain binds ligand in the subset of integrins in which it is present. Its structure has been determined in two alternative conformations, termed open and closed. In striking similarity to signaling G proteins, rearrangement of a Mg(2+)-binding site is linked to large conformational movements in distant backbone regions. Mutations have been used to stabilize either the closed or open structures. These show that the snapshots of the open conformation seen only in the presence of a ligand or a ligand mimetic represent a high-affinity, ligand-binding conformation, whereas those of the closed conformation correspond to a low-affinity conformation. The C-terminal alpha-helix moves 10 A down the side of the domain in the open conformation. Locking in the conformation of the preceding loop is sufficient to increase affinity for ligand 9000-fold. This C-terminal "bell-rope" provides a mechanism for linkage to conformational movements in other domains. The transition from the closed to open conformation has been implicated in fast (<1 s) regulation of integrin affinity in response to activation signals from inside the cell. Recent integrin structures and functional studies reveal interactions between beta-propeller, I, and I-like domains in the headpiece, and a critical role for integrin EGF domains in the stalk region. These studies suggest that the headpiece of the integrin faces down toward the membrane in the inactive conformation and extends upward in a "switchblade"-like opening motion upon activation. These long-range structural rearrangements of the entire integrin molecule involving multiple interdomain contacts appear closely linked to conformational changes in the I domain, which result in increased affinity and competence for ligand binding.

537 citations


Journal ArticleDOI
28 Jun 2002-Cell
TL;DR: It is shown that ligand engagement of TCR-CD3 induces a conformational change that exposes a proline-rich sequence in CD3 epsilon and results in recruitment of the adaptor protein Nck, which is critical for maturation of the immune synapse and for T cell activation.

448 citations


Journal ArticleDOI
TL;DR: This work proposes a new signaling mechanism, whereby prolyl isomerization catalytically induces conformational changes in proteins following phosphorylation to regulate protein function, and suggests this post-phosphorylation mechanism might play an important role in cell growth control and diseases such as cancer and Alzheimer's.

339 citations


Journal ArticleDOI
TL;DR: The thermodynamics and kinetics of the interaction of dihydrofolate reductase with methotrexate have been studied by using fluorescence, stopped-flow, and single-molecule methods, and the association and dissociation rate constants were determined.
Abstract: The thermodynamics and kinetics of the interaction of dihydrofolate reductase (DHFR) with methotrexate have been studied by using fluorescence, stopped-flow, and single-molecule methods. DHFR was modified to permit the covalent addition of a fluorescent molecule, Alexa 488, and a biotin at the N terminus of the molecule. The fluorescent molecule was placed on a protein loop that closes over methotrexate when binding occurs, thus causing a quenching of the fluorescence. The biotin was used to attach the enzyme in an active form to a glass surface for single-molecule studies. The equilibrium dissociation constant for the binding of methotrexate to the enzyme is 9.5 nM. The stopped-flow studies revealed that methotrexate binds to two different conformations of the enzyme, and the association and dissociation rate constants were determined. The single-molecule investigation revealed a conformational change in the enzyme-methotrexate complex that was not observed in the stopped-flow studies. The ensemble averaged rate constants for this conformation change in both directions is about 2-4 s(-1) and is attributed to the opening and closing of the enzyme loop over the bound methotrexate. Thus the mechanism of methotrexate binding to DHFR involves multiple steps and protein conformational changes.

293 citations


Journal ArticleDOI
TL;DR: This version of FCS/FRET on immobilized single molecules is demonstrated to be a powerful technique in the study of conformational dynamics of biomolecules over time scales ranging from microseconds to seconds.
Abstract: Fluorescence correlation spectroscopy (FCS) of fluorescence resonant energy transfer (FRET) on immobilized individual fluorophores was used to study the Mg2+-facilitated conformational change of an RNA three-helix junction, a structural element that initiates the folding of the 30S ribosomal subunit. Transitions of the RNA junction between open and folded conformations resulted in fluctuations in fluorescence by FRET. Fluorescence fluctuations occurring between two FRET states on the millisecond time scale were found to be dependent on Mg2+ and Na+ concentrations. Correlation functions of the fluctuations were used to determine transition rates between the two conformations as a function of Mg2+ or Na+ concentration. Both the opening and folding rates were found to vary with changing salt conditions. Assuming specific binding of divalent ions to RNA, the Mg2+ dependence of the observed rates cannot be explained by conformational change induced by Mg2+ binding/unbinding, but is consistent with a model in which the intrinsic conformational change of the RNA junction is altered by uptake of Mg2+ ion(s). This version of FCS/FRET on immobilized single molecules is demonstrated to be a powerful technique in the study of conformational dynamics of biomolecules over time scales ranging from microseconds to seconds.

257 citations


Journal ArticleDOI
TL;DR: The structure of an archaeal Sir2 enzyme bound to an acetylated p53 peptide reveals that the substrate binds in a cleft in the enzyme, forming an enzyme-substrate beta sheet with two flanking strands in Sir2-Af2.

239 citations


Journal ArticleDOI
TL;DR: Results suggest that acid conditions increase the susceptibility of the molecule to conformational change and dissociation, and enhance the rate of fibrillation by providing a charged environment in which the attractive forces between the protein molecules is increased.

234 citations


Journal ArticleDOI
TL;DR: Using atomic force microscopy (AFM), conformational changes of the cytoplasmic and extracellular surfaces of native connexin 26 gap junction plaques suggest that calcium ions induce conformationalChanges affecting the structure of both the hemichannels and the intact channels forming cell–cell contacts.
Abstract: Gap junction channels mediate communication between adjacent cells. Using atomic force microscopy (AFM), we have imaged conformational changes of the cytoplasmic and extracellular surfaces of native connexin 26 gap junction plaques. The cytoplasmic domains of the gap junction surface, imaged at submolecular resolution, form a hexameric pore protruding from the membrane bilayer. Exhibiting an intrinsic flexibility, these cytoplasmic domains, comprising the C-terminal connexin end, reversibly collapse by increasing the forces applied to the AFM stylus. The extracellular connexon surface was imaged after dissection of the gap junction with the AFM stylus. Upon injection of Ca(2+) into the buffer solution, the extracellular channel entrance reduced its diameter from 1.5 to 0.6 nm, a conformational change that is fully reversible and specific among the divalent cations tested. Ca(2+) had a profound effect on the cytoplasmic surface also, inducing the formation of microdomains. Consequently, the plaque height increased by 0.6 nm to 18 nm. This suggests that calcium ions induce conformational changes affecting the structure of both the hemichannels and the intact channels forming cell-cell contacts.

233 citations


Journal ArticleDOI
TL;DR: It is shown that studies of conformational changes in proteins at a single molecule level and in the native in vivo context of a living cell are now possible.

Journal ArticleDOI
TL;DR: Hsp90 inactivation impedes interaction of androgen-bound GFP-NLS-AR with nuclear components and inhibits transcriptional activity, and it is concluded that hsp90s are required for the acquisition of active conformation in agonist-bound AR to regulate nuclear transfer, nuclear matrix binding, and transcriptionalactivity.
Abstract: Activation of the androgen receptor (AR) is induced by ligand binding through conformational changes leading to control of gene expression. Antiandrogens compete with androgens for AR occupancy and subsequently block at least one step in AR action. Analysis of nuclear transfer kinetics using the GFP-AR fusion protein and partial proteolysis analysis provided evidence that the ligand-bound receptor was in equilibrium between at least two distinct conformations, leading to the production of 35 and 29 kDa trypsin-resistant fragments. It also indicated that this equilibrium may regulate the rate of nuclear transfer. The slowing of nuclear transfer by antiandrogens was correlated with the amount of receptor in conformation leading to the 35 kDa trypsin-resistant fragment. To establish the role of heat shock protein (hsp) 90 activity in antiandrogenic action, the effect of geldanamycin (GA) was evaluated in both in vitro assays and live cells. We demonstrated that in vitro hsp90s are required to stabilize the receptor in the inactive conformation and that hsp90 activity is involved in the integrity and nuclear transfer of agonist- and antagonist-bound AR. Furthermore, nuclear transfer is not the only step affected by GA since this compound was also active on a constitutively nuclear AR (GFP-NLS-AR). Hsp90 inactivation impedes interaction of androgen-bound GFP-NLS-AR with nuclear components and inhibits transcriptional activity. We conclude that hsp90s are required for the acquisition of active conformation in agonist-bound AR to regulate nuclear transfer, nuclear matrix binding, and transcriptional activity. Pure antiandrogens block the transconformational change of AR in an intermediary complex unable to acquire the active conformation and to dissociate the hsp90.

Journal ArticleDOI
TL;DR: It is proposed that an intramolecular interaction between the spacer region (Sp) and the C‐terminus in RPTPα prohibited intermolecular interactions, and stress factors such as H2O2, UV and heat shock induced reversible, free radical‐dependent, intermolescular interactions between RPTP α and RPTPβ, suggesting an inducible switch in conformation and binding.
Abstract: The presence of two protein-tyrosine phosphatase (PTP) domains is a striking feature in most transmembrane receptor PTPs (RPTPs). The function of the generally inactive membrane-distal PTP domain (RPTP-D2) is unknown. Here we report that an intramolecular interaction between the spacer region (Sp) and the C-terminus in RPTPα prohibited intermolecular interactions. Interestingly, stress factors such as H2O2, UV and heat shock induced reversible, free radical-dependent, intermolecular interactions between RPTPα and RPTPα-SpD2, suggesting an inducible switch in conformation and binding. The catalytic site cysteine of RPTPα-SpD2, Cys723, was required for the H2O2 effect on RPTPα. H2O2 induced a rapid, reversible, Cys723-dependent conformational change in vivo, as detected by fluorescence resonance energy transfer, with cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) flanking RPTPα-SpD2 in a single chimeric protein. Importantly, H2O2 treatment stabilized RPTPα dimers, resulting in inactivation. We propose a model in which oxidative stress induces a conformational change in RPTPα-D2, leading to stabilization of RPTPα dimers, and thus to inhibition of RPTPα activity.

Journal ArticleDOI
TL;DR: The results suggest that the binding of HN protein to the receptor induces the conformational change of residues near the hydrophobic surface of Hn protein and that this change triggers the activation of the F protein, which initiates membrane fusion.
Abstract: Paramyxovirus infects cells by initially attaching to a sialic acid-containing cellular receptor and subsequently fusing with the plasma membrane of the cells. Hemagglutinin-neuraminidase (HN) protein, which is responsible for virus attachment, interacts with the fusion protein in a virus type-specific manner to induce efficient membrane fusion. To elucidate the mechanism of HN-promoted membrane fusion, we characterized a series of Newcastle disease virus HN proteins whose surface residues were mutated. Fusion promotion activity was substantially altered in only the HN proteins with a mutation in the first or sixth β sheet. These regions overlap the large hydrophobic surface of HN; thus, the hydrophobic surface may contain the fusion promotion domain. Furthermore, a comparison of the HN structure crystallized alone or in complex with 2-deoxy-2,3-dehydro-N-acetylneuraminic acid revealed substantial conformational changes in several loops within or near the hydrophobic surface. Our results suggest that the binding of HN protein to the receptor induces the conformational change of residues near the hydrophobic surface of HN protein and that this change triggers the activation of the F protein, which initiates membrane fusion.

Journal ArticleDOI
TL;DR: This work reports that when one AAA protein, Hsp104, engages polypeptide at the C-terminal peptide-binding region, the ATPase cycle of the C/terminal nucleotide-binding domain (NBD2) drives a conformational change in the middle region, demonstrating the crucial role this region plays in transducing signals from one end of the molecule to the other.

Journal ArticleDOI
TL;DR: The results demonstrate that the α1 helix undergoes conformational alterations during integrin activation and suggest that Mn2+ acts as a potent activator of β1integrins because it can promote a shift in the position of this helix.

Journal ArticleDOI
TL;DR: Evidence is provided here that BimEL induces Bax conformational change and apoptosis through a Bcl-XL-suppressible but heterodimerization-independent mechanism, and that native Bax is attached loosely on the surface of isolated mitochondria, which undergoes conformational changes and insertion into mitochondrial membrane upon stimulation by Bim EL, Bak-BH3 peptide, or freeze/thaw damage.

Journal ArticleDOI
25 May 2002-Virology
TL;DR: There is a thermodynamic equilibrium between the different states of G between pH 6 and pH 7.5 and studies on the mechanism leading to low pH induced fusion inactivation indicated that a large number of G molecules is required for stable hydrophobic interaction of the virus with the target membrane.

Journal ArticleDOI
TL;DR: The increased structure introduced into N-IC74 upon light chain binding suggests a mechanism by which LC8 and Tctex-1 may regulate the assembly of the dynein complex.
Abstract: The interactions of three subunits of cytoplasmic dynein from Drosophila melanogaster, LC8, Tctex-1, and the N-terminal domain of IC74 (N-IC74, residues 1-289), were characterized in vitro by affinity methods, limited proteolysis, and circular dichroism spectroscopy. These subunits were chosen for study because they are presumed to promote the assembly of the complex and to be engaged in the controlled binding and release of cargo. Limited proteolysis and mass spectrometry of N-IC74 in the presence of LC8 and Tctex-1 localized binding of Tctex-1 to the vicinity of K104 and K105, and localized binding of LC8 to the region downstream of K130. Circular dichroism, fluorescence, sedimentation velocity, and proteolysis studies indicate that N-IC74 has limited secondary and tertiary structure at near physiological solution conditions. Upon addition of LC8, N-IC74 undergoes a significant conformational change from largely unfolded to a more ordered structure. This conformational change is reflected in increased global protection of N-IC74 from proteolytic digestion following the interaction, and in a significant change in the CD signal. A smaller but reproducible change in the CD spectra was observed upon Tctex-1 binding as well. The increased structure introduced into N-IC74 upon light chain binding suggests a mechanism by which LC8 and Tctex-1 may regulate the assembly of the dynein complex.

Journal ArticleDOI
TL;DR: The structure of Escherichia coli core RNA polymerase was determined by cryo-electron microscopy and image processing of helical crystals to a nominal resolution and a very large conformational change of the T. aquaticus RNAP x-ray structure was required to fit the E. coli map.
Abstract: The structure of Escherichia coli core RNA polymerase (RNAP) was determined by cryo-electron microscopy and image processing of helical crystals to a nominal resolution of 15 A. Because of the high sequence conservation between the core RNAP subunits, we were able to interpret the E. coli structure in relation to the high-resolution x-ray structure of Thermus aquaticus core RNAP. A very large conformational change of the T. aquaticus RNAP x-ray structure, corresponding to opening of the main DNA/RNA channel by nearly 25 A, was required to fit the E. coli map. This finding reveals, at least partially, the range of conformational flexibility of the RNAP, which is likely to have functional implications for the initiation of transcription, where the DNA template must be loaded into the channel.

Journal ArticleDOI
TL;DR: The putative roles of spin-state and steric hindrance were evaluated by the structure determination of ferrous CO-bound BjFixLH and correlating its features with other ligand-bound structures, suggesting a more complicated mechanism than solely spin state or ligand sterics.
Abstract: Structures of the Bradyrhizobium japonicum FixL heme domain have been determined in the absence and presence of specific ligands to elucidate the detailed features of its O2 sensing mechanism. The putative roles of spin-state and steric hindrance were evaluated by the structure determination of ferrous CO-bound BjFixLH and correlating its features with other ligand-bound structures. As found for NO-BjFixLH, no protein conformational change was observed in CO-BjFixLH, suggesting a more complicated mechanism than solely spin state or ligand sterics. To evaluate the role of oxidation state, the structure of the ferrous deoxy-BjFixLH was determined. The structure of deoxy-BjFixLH was found to be virtually identical to the structure of the ferric met-BjFixLH. The role of hydrogen bonding of substrates to a heme-pocket water was evaluated by determining the structure of BjFixLH bound to 1-methyl-imidazole that cannot form a hydrogen bond with this water. In this case, the heme-mediated conformational change was observed, limiting the potential importance of this interaction. Finally, the structure of cyanomet-BjFixLH was revisited to rule out concerns regarding the partial occupancy of the cyanide ligand in a previous structure. In the revised structure, Arg 220 was found to move into the heme pocket to form a hydrogen bond to the bound cyanide ligand. The implications of these results on FixL's sensing mechanism are discussed.

Journal ArticleDOI
TL;DR: The results show a startling interplay between the two ligands, which both exert control over the conformation of the domain to influence its electron transfer properties, and NADPH binding will probably close the conformational lock in vivo, preventing electron transfer to the oxygenase domain and the resultant stimulation of nitric oxide synthesis.

Journal Article
TL;DR: It is demonstrated that EpoB induces apoptosis through a Bcl-2-suppressible pathway that controls a conformational change of the proapoptotic Bax protein.
Abstract: Epothilone B is a novel nontaxane antimicrotubule agent that is active even against paclitaxel (Taxol)-resistant cancer cells. The present study further explores the mechanisms underlying epothilone B-mediated cytotoxicity in human breast cancer cells. We show that BMS-247550 (EpoB), a novel epothilone B analogue, induces cell cycle arrest at the G(2)-M phase transition and subsequent apoptotic cell death of MDA-MB-468 (468) cells. Treating cells with EpoB triggers a conformational change in the Bax protein and its translocation from the cytosol to the mitochondria, which is accompanied by cytochrome c release from the inter-membrane space of mitochondria into the cytosol. Overexpression of Bcl-2 delays Bax conformational change, cytochrome c release, and apoptosis induced by EpoB. Conversely, the Bcl-2 antagonist Bak-BH3 peptide or HA14-1 compound abrogates the antiapoptotic effects of Bcl-2 and enhances apoptosis of 468 cells pretreated with EpoB (to induce mitotic arrest). In synchronized 468 cells, EpoB is more potent in inducing Bax conformational change and apoptosis at G(2)-M phase compared with G(1)-S phase of the cell cycle. Taken together, these findings demonstrate that EpoB induces apoptosis through a Bcl-2-suppressible pathway that controls a conformational change of the proapoptotic Bax protein. The enhanced cytotoxicity of EpoB by blocking Bcl-2 at mitochondria implies a potential application of the combination of EpoB and Bcl-2 antagonists in the treatment of human breast cancer.

Journal ArticleDOI
TL;DR: Using the beta2-adrenergic receptor as a model system, evidence is obtained for an evolutionary conserved activation mechanism where disruption of intramolecular interactions between TM3 and TM6 leads to a major conformational change of TM6 relative to the rest of the receptor.
Abstract: Our understanding of how G-protein-coupled receptors (GPCRs) operate at the molecular level has been considerably improved over the last few years. The application of advanced biophysical techniques as well as the availability of high-resolution structural information has allowed insight both into conformational changes accompanying GPCR activation and the underlying molecular mechanism governing transition of the receptor between its active and inactive states. Using the beta2-adrenergic receptor as a model system we have obtained evidence for an evolutionary conserved activation mechanism where disruption of intramolecular interactions between TM3 and TM6 leads to a major conformational change of TM6 relative to the rest of the receptor. This conclusion was based on experiments in which environmentally sensitive, sulfhydryl-reactive fluorophores were site-selectively incorporated into wild-type and mutant beta2-adrenergic receptors purified from Sf-9 insect cells. Our studies have also raised important questions regarding kinetics of receptors activation. These questions should be addressed in the future by application of techniques that will allow for simultaneous measurement of conformational changes and receptor activation. At the current stage we are exploring the possibility of reaching this goal by direct in situ labeling of the beta2-adrenergic receptor in Xenopus laevis oocytes with conformationally sensitive fluorescent probes and parallel detection of receptor activation by co-expression with the cAMP sensitive Cl- channel CFTR (cystic fibrosis transmembrane conductance regulator) and electrophysiological measurements.

Journal ArticleDOI
TL;DR: These studies bridge static microscopic crystal structures with macroscopic kinetic data by delineating a specific sequence and suggest new lines of experimentation in the study of polymerase mechanisms, which should provide further insights into mechanisms of error discrimination and DNA synthesis fidelity.

Journal ArticleDOI
TL;DR: Conformational change(s) elicited by phosphorylation of the R domain within the PDE5 holoenzyme may also cause or participate in stimulating catalysis, and Migration in native polyacrylamide gels suggests that either cGMP binding orosphorylation produces distinct conformers of theR domain.

Journal ArticleDOI
TL;DR: Protein film voltammetry of chicken liver sulfite oxidase (SO) bound at the pyrolytic graphite "edge" or modified gold electrodes shows that catalytic electron transport is controlled by the inherent electrochemical characteristics of the heme b domain and conformational changes that allow intramolecular electron transfer with the molybdenum active site.
Abstract: Protein film voltammetry of chicken liver sulfite oxidase (SO) bound at the pyrolytic graphite “edge” or modified gold electrodes shows that catalytic electron transport is controlled by the inherent electrochemical characteristics of the heme b domain and conformational changes that allow intramolecular electron transfer with the molybdenum active site. In the absence of sulfite, a single nonturnover electrochemical signal is observed at +90 mV (vs SHE) that is assigned to heme b. In the presence of sulfite, this signal transforms into a catalytic wave at similar potential. The shape and negligible pH dependence of this wave indicate that catalytic turnover is controlled by the one-electron transfers through heme b. The smaller turnover numbers obtained in this experiment (kcat ≈ 2−4 s-1, as compared to 100 s-1 in solution) suggest that only a small fraction of SO is bound at the electrode in a manner that permits the conformational change necessary for fast interdomain electron transfer.

Journal ArticleDOI
TL;DR: The solution NMR structure is reported for Ca(2+)-loaded S100B bound to a 12-residue peptide, TRTK-12, from the actin capping protein CapZ (alpha1 or alpha2 subunit, residues 265-276:TRTKIDWNKILS).

Journal ArticleDOI
TL;DR: Measurements of the relaxation rates of protonated base carbon and nitrogen nuclei reveal a broad range of dynamic heterogeneity exhibited by hexanucleotide loop, the dinucleotide bulge, and the A-form helical regions of the HIV-2 TAR-argininamide complex.

Journal ArticleDOI
TL;DR: The kinetics and conformational changes of alpha(1)-antitrypsin polymerization at pH 4 are characterized using tryptophan fluorescence, circular dichroism, turbidity changes and thioflavin T binding to describe a novel kinetic mechanism of serine proteinase inhibitor polymerization.