Showing papers on "Conformational change published in 1999"
TL;DR: A large conformational change of a ∼15-amino-acid region (the neck linker) in kinesin is detected and visualized using electron paramagnetic resonance, fluorescence resonance energy transfer, pre-steady state kinetics and cryo-electron microscopy.
Abstract: Kinesin motors power many motile processes by converting ATP energy into unidirectional motion along microtubules. The force-generating and enzymatic properties of conventional kinesin have been extensively studied; however, the structural basis of movement is unknown. Here we have detected and visualized a large conformational change of an approximately 15-amino-acid region (the neck linker) in kinesin using electron paramagnetic resonance, fluorescence resonance energy transfer, pre-steady state kinetics and cryo-electron microscopy. This region becomes immobilized and extended towards the microtubule 'plus' end when kinesin binds microtubules and ATP, and reverts to a more mobile conformation when gamma-phosphate is released after nucleotide hydrolysis. This conformational change explains both the direction of kinesin motion and processive movement by the kinesin dimer.
771 citations
TL;DR: Using a quantitative 3D approach termed vector geometry mapping (VGM), this tertiary structural diversity of EF‐hand proteins and its correlation with target recognition are discussed.
Abstract: The EF-hand motif, which assumes a helix-loop-helix structure normally responsible for Ca2+ binding, is found in a large number of functionally diverse Ca2+ binding proteins collectively known as the EF-hand protein superfamily. In many superfamily members, Ca2+ binding induces a conformational change in the EF-hand motif, leading to the activation or inactivation of target proteins. In calmodulin and troponin C, this is described as a change from the closed conformational state in the absence of Ca2+ to the open conformational state in its presence. It is now clear from structures of other EF-hand proteins that this "closed-to-open" conformational transition is not the sole model for EF-hand protein structural response to Ca2+. More complex modes of conformational change are observed in EF-hand proteins that interact with a covalently attached acyl group (e.g., recoverin) and in those that dimerize (e.g., S100B, calpain). In fact, EF-hand proteins display a multitude of unique conformational states, together constituting a conformational continuum. Using a quantitative 3D approach termed vector geometry mapping (VGM), we discuss this tertiary structural diversity of EF-hand proteins and its correlation with target recognition.
265 citations
TL;DR: It is proposed that in wild-type bacteriorhodopsin and in most mutants, this conformational change between the M1 and M2 states is likely to make an important contribution towards efficiently switching proton accessibility of the Schiff base from the extracellular side to the cytoplasmic side of the membrane.
246 citations
TL;DR: Analysis of the pheromone-binding protein from B. mori PBP in Escherichia coliperiplasm demonstrated that its tertiary structure was sensitive to pH changes and that a dramatic conformational transition occurred between pH 6.0 and 5.0.
243 citations
TL;DR: Fluorescence measurements indicate conformational changes in the α subunit after calmodulin binding and Ca2+ binding to theα subunit–calmodulin complex that result in channel gating, and a state model forCa2+ gating was developed that simulates alterations in SK channel Ca2-sensitivity and cooperativity associated with mutations in CaM.
Abstract: Small conductance Ca2+-activated potassium channels (SK channels) are coassembled complexes of pore-forming SK α subunits and calmodulin. We proposed a model for channel activation in which Ca2+ binding to calmodulin induces conformational rearrangements in calmodulin and the α subunits that result in channel gating. We now report fluorescence measurements that indicate conformational changes in the α subunit after calmodulin binding and Ca2+ binding to the α subunit–calmodulin complex. Two-hybrid experiments showed that the Ca2+-independent interaction of calmodulin with the α subunits requires only the C-terminal domain of calmodulin and is mediated by two noncontiguous subregions; the ability of the E-F hands to bind Ca2+ is not required. Although SK α subunits lack a consensus calmodulin-binding motif, mutagenesis experiments identified two positively charged residues required for Ca2+-independent interactions with calmodulin. Electrophysiological recordings of SK2 channels in membrane patches from oocytes coexpressing mutant calmodulins revealed that channel gating is mediated by Ca2+ binding to the first and second E-F hand motifs in the N-terminal domain of calmodulin. Taken together, the results support a calmodulin- and Ca2+-calmodulin-dependent conformational change in the channel α subunits, in which different domains of calmodulin are responsible for Ca2+-dependent and Ca2+-independent interactions. In addition, calmodulin is associated with each α subunit and must bind at least one Ca2+ ion for channel gating. Based on these results, a state model for Ca2+ gating was developed that simulates alterations in SK channel Ca2+sensitivity and cooperativity associated with mutations in CaM.
228 citations
TL;DR: It is proposed that the conformational change facilitates cross-links among mucin macromolecules through hydrophobic interactions at low pH, which in turn leads to a sol-gel transition when the mucin solution is sufficiently concentrated.
207 citations
TL;DR: The structural changes observed in HIV-1 IN may serve to recruit the catalytic machinery in this enzyme to a conformation that is native for ASV IN.
Abstract: Publisher Summary This chapter focuses on the human immunodeficiency virus type 1 (HIV-1) integrase–– its structural organization, conformational changes, and catalysis. The integrase (IN) protein from retroviruses catalyzes one of the critical steps in the establishment of a viral infection: the integration of the viral DNA into the genome of the host. The protein has evolved to catalyze two separate reactions that are required to accomplish this goal, a hydrolytic reaction termed “processing” and a transesterification reaction referred to as “joining” or “strand transfer.” The IN proteins from HIV-1 and avian sarcoma virus (ASV) have been studied most carefully with respect to the structural basis of catalysis. Although the active site of ASV IN does not undergo significant conformational changes on binding the required metal cofactor that of HIV-1 IN does. The reversible, metal-induced conformational change in HIV-1 IN impairs the binding of some anti-HIV-1 IN monoclonal antibodies to the enzyme and results in a differential susceptibility of the protein to proteolysis. This chapter focuses on protein structural changes that precede catalysis. Although it seems likely, it is not known if conformational changes play a significant role at later steps in the reaction, proceeding from processing to joining, and finally to the release of the product.
195 citations
TL;DR: Electron crystallography has shown monomers to consist of six membrane spanning α‐helices confirming sequence based predictions, and cytoplasmic surface exposed loops are the least conserved regions, allowing differentiation of aquaporins.
Abstract: Aquaporins form a large family of membrane channels involved in osmoregulation. Electron crystallography has shown monomers to consist of six membrane spanning alpha-helices confirming sequence based predictions. Surface exposed loops are the least conserved regions, allowing differentiation of aquaporins. Atomic force microscopy was used to image the surface of aquaporin Z, the water channel of Escherichia coli. Recombinant protein with an N-terminal fragment including 10 histidines was isolated as a tetramer by Ni-affinity chromatography, and reconstituted into two-dimensional crystals with p42(1)2 symmetry. Small crystalline areas with p4 symmetry were found as well. Imaging both crystal types before and after cleavage of the N-termini allowed the cytoplasmic surface to be identified; a drastic change of the cytoplasmic surface accompanied proteolytic cleavage, while the extracellular surface morphology did not change. Flexibility mapping and volume calculations identified the longest loop at the extracellular surface. This loop exhibited a reversible force-induced conformational change.
190 citations
TL;DR: Fluorescence measurements indicate that a bimane label on the inner face of the helix detects changes in the polarity of its environment and accessibility to a fluorescence quenching agent upon MII formation, providing further direct evidence that rhodopsin activation involves a conformational change at helix F.
172 citations
TL;DR: By defining the residues present in the epitope of a mAb against the human Mac-1 integrin that binds only the active receptor, this work provides biochemical evidence that the I domain itself undergoes a conformational change with activation and suggests that conformational differences in the Idomain are physiologically relevant and not merely a consequence of different crystal lattice interactions.
Abstract: For efficient ligand binding, integrins must be activated. Specifically, a conformational change has been proposed in a ligand binding domain present within some integrins, the inserted (I) domain [Lee, J., Bankston, L., Arnaout, M. & Liddington, R. C. (1995) Structure (London) 3, 1333–1340]. This proposal remains controversial, however, despite extensive crystal structure studies on the I domain [Lee, J., Bankston, L., Arnaout, M. & Liddington, R. C. (1995) Structure (London) 3, 1333–1340; Liddington, R. & Bankston, L. (1998) Structure (London) 6, 937–938; Qu, A. & Leahy, D. J. (1996) Structure (London) 4, 931–942; and Baldwin, E. T., Sarver, R. W., Bryant, G. L., Jr., Curry, K. A., Fairbanks, M. B., Finzel, B. C., Garlick, R. L., Heinrikson, R. L., Horton, N. C. & Kelly, L. L. (1998) Structure (London) 6, 923–935]. By defining the residues present in the epitope of a mAb against the human Mac-1 integrin (αMβ2, CD11b/CD18) that binds only the active receptor, we provide biochemical evidence that the I domain itself undergoes a conformational change with activation. This mAb, CBRM1/5, binds the I domain very close to the ligand binding site in a region that is widely exposed regardless of activation as judged by reactivity with other antibodies. The conformation of the epitope differs in two crystal forms of the I domain, previously suggested to represent active and inactive receptor. Our data suggests that conformational differences in the I domain are physiologically relevant and not merely a consequence of different crystal lattice interactions. We also demonstrate that the transition between the two conformational states depends on species-specific residues at the bottom of the I domain, which are proposed to be in an interface with another integrin domain, and that this transition correlates with functional activity.
154 citations
TL;DR: Comparison of the structures of the substrate-free enzyme and the complex with a bisubstrate analog demonstrates that acetyl coenzyme A (AcCoA) binding is accompanied by a large conformational change that in turn leads to the formation of the serotonin-binding site.
TL;DR: Kinetic and equilibrium data show that the single chain antibody fragment fragment selects monomeric peptides from the population in equilibrium with the leucine zipper dimer.
TL;DR: The position of Tyralpha89 close to the substrate explains the stereochemical selectivity of the enzyme for (2R)-methylmalonyl-CoA.
Abstract: X-ray crystal structures of methylmalonyl-CoA mutase in complexes with substrate methylmalonyl-CoA and inhibitors 2-carboxypropyl-CoA and 3-carboxypropyl-CoA (substrate and product analogues) show that the enzyme-substrate interactions change little during the course of the rearrangement reaction, in contrast to the large conformational change on substrate binding. The substrate complex shows a 5'-deoxyadenine molecule in the active site, bound weakly and not attached to the cobalt atom of coenzyme B12, rotated and shifted from its position in the substrate-free adenosylcobalamin complex. The position of Tyralpha89 close to the substrate explains the stereochemical selectivity of the enzyme for (2R)-methylmalonyl-CoA.
TL;DR: By flow cytometry, a conformational change in mouse cytochrome c of apoptotic and necrotic T hybridoma cells was detected using a monoclonal antibody that recognizes the region around amino acid residue 44 on a non-native form of the protein.
Abstract: By flow cytometry, a conformational change in mouse cytochrome c (cyt c) of apoptotic and necrotic T hybridoma cells was detected using a monoclonal antibody (mAb) that recognizes the region around amino acid residue 44 on a non-native form of the protein. The conformational change in cyt c is an early event in apoptosis, which can be identified in pre-apoptotic cells that are negative for other indicators of apoptosis. Since the mAb did not bind fixed and permeabilized live cells and did not immunoprecipitate soluble cyt c extracted with detergent from dead cells, it appears to recognize cyt c bound in a detergent-sensitive complex to other cellular components. Coincidentally, the mAb was also shown by competitive enzyme-linked immunosorbent assay to bind cyt c associated with synthetic phosphatidic acid vesicles. This suggests that the conformational change of cyt c in dying cells could be due to its association with intracellular membranes that are, perhaps, altered in cell death. By immunofluorescent ...
TL;DR: Covalent binding of L-methionine as an external aldimine to the pyridoxal 5'-phosphate-cofactor in the K41A mutant of O-acetylserine sulfhydrylase from Salmonella typhimurium induces a large conformational change in the protein.
TL;DR: A mechanism to explain how the stimulation of ATPase activity afforded by binding of single-stranded DNA stabilises the activated conformation favouring Mg2+binding and a consequent repositioning of the gamma-phosphate group which promotes ATP hydrolysis is proposed.
TL;DR: Different conformational states were adopted by P-glycoprotein upon the addition of the anthracycline derivatives in the absence and presence of MgATP or MgatPγS, and these conformational changes are shown to be related to the nature of the antitumor agents and more precisely to their capacity to accumulate in resistant cells.
TL;DR: The results obtained demonstrate the utility of FRET to investigate conformational adaptations of exchangeable apolipoproteins and are consistent with the hypothesis that, upon lipid binding, apoE3(1-183) undergoes conformational opening, repositioning helix 1 and 3 to adopt a receptor-active conformation.
TL;DR: The structure of a soluble version of the human class II MHC protein HLA-DR1 in empty and peptide-loaded forms is probed, indicating a condensation of the protein around the bound peptide, and the conformational change involves the beta-subunit helical region.
Abstract: To investigate a conformational change accompanying peptide binding to class II MHC proteins, we probed the structure of a soluble version of the human class II MHC protein HLA-DR1 in empty and peptide-loaded forms. Peptide binding induced a large decrease in the effective radius of the protein as determined by gel filtration, dynamic light scattering, and analytical ultracentrifugation. It caused a substantial increase in the cooperativity of thermal denaturation and induced alterations in MHC polypeptide backbone structure as determined by circular dichroism. These changes suggest a condensation of the protein around the bound peptide. An antibody specific for ‚58-69 preferentially bound the empty protein, indicating that the peptide-induced conformational change involves the ‚-subunit helical region. The conformational change may have important implications for the mechanisms of intracellular antigen presentation pathways.
TL;DR: Comparison of the chemical shifts of the individual amino acids in histatin-5 with those recently reported in other solvents indicates that trifluoroethanol/water has a structuring capability somewhere between water and dimethyl sulfoxide.
Abstract: The salivary antimicrobial peptide histatin-5 is able to aggregate and fuse negatively charged small unilamellar vesicles, and this fusogenic activity is selectively induced by the presence of zinc ions. Circular dichroism spectroscopy shows that histatin-5, in the presence of negatively charged vesicles and zinc ions, undergoes a conformational change leading to the stabilization of an alpha-helical secondary structure. We attribute the specific action of the zinc ions to the presence of a consensus sequence, HEXXH, located in the C-terminal functional domain of histatin-5, a recognized zinc-binding motif in many proteins. Two-dimensional proton NMR spectroscopy of histatin-5 in a trifluoroethanol/water mixture (a membrane mimetic environment) has been performed and the results analyzed by means of distance geometry and restrained molecular dynamics simulations. Our results reveal that the peptide chain, including the Zn-binding consensus sequence corresponding to residues 15-19, is in a helicoidal conformation. Comparison of the chemical shifts of the individual amino acids in histatin-5 with those recently reported in other solvents indicates that trifluoroethanol/water has a structuring capability somewhere between water and dimethyl sulfoxide. The mechanism of action of this antimicrobial peptide is discussed on the basis of its structural characteristics with particular attention to the Zn-binding motif.
TL;DR: Although the Gly293Ala and Pro295Thr substitutions do not disturb the apoenzyme structure, molecular modeling shows that the new side chains cannot be accommodated in the closed native holoenzyme complex without steric alterations.
Abstract: When horse liver alcohol dehydrogenase binds coenzyme, a rotation of about 10 degrees brings the catalytic domain closer to the coenzyme binding domain and closes the active site cleft. The conformational change requires that a flexible loop containing residues 293-298 in the coenzyme binding domain rearranges so that the coenzyme and some amino acid residues from the catalytic domain can be accommodated. The change appears to control the rate of dissociation of the coenzyme and to be necessary for installation of the proton relay system. In this study, directed mutagenesis produced the activated Gly293Ala/Pro295Thr enzyme. X-ray crystallography shows that the conformations of both free and complexed forms of the mutated enzyme and wild-type apoenzyme are very similar. Binding of NAD(+) and 2,2, 2-trifluoroethanol do not cause the conformational change, but the nicotinamide ribose moiety and alcohol are not in a fixed position. Although the Gly293Ala and Pro295Thr substitutions do not disturb the apoenzyme structure, molecular modeling shows that the new side chains cannot be accommodated in the closed native holoenzyme complex without steric alterations. The mutated enzyme may be active in the "open" conformation. The turnover numbers with ethanol and acetaldehyde increase 1.5- and 5.5-fold, respectively, and dissociation constants for coenzymes and other kinetic constants increase 40-2,000-fold compared to those of the native enzyme. Substrate deuterium isotope effects on the steady state V or V/K(m) parameters of 4-6 with ethanol or benzyl alcohol indicate that hydrogen transfer is a major rate-limiting step in catalysis. Steady state oxidation of benzyl alcohol is most rapid above a pK of about 9 for V and V/K(m) and is 2-fold faster in D(2)O than in H(2)O. The results are consistent with hydride transfer from a ground state zinc alkoxide that forms a low-barrier hydrogen bond with the hydroxyl group of Ser48.
TL;DR: The results suggest that the two proteins P1 and P2 both interact with eEF-2 inducing a conformational transition of the factor, but have acquired some specific properties during evolution.
Abstract: The eukaryotic P1 and P2 ribosomal proteins which constitute, with P0, a pentamer forming the lateral stalk of the 60 S ribosomal subunit, exhibit several differences from their prokaryotic equivalents L7 and L12; in particular, P1 does not have the same primary structure as P2 and both of them are phosphorylated, the significance of the latter remaining unclear. Rat liver P1 and P2 were overproduced in Escherichia coli cells and their interaction with elongation factor eEF-2 was studied. Both recombinant proteins were found to be required for the ribosome-dependent GTPase activity of eEF-2, with P2 in the phosphorylated form. The surface plasmon resonance technique revealed that, in vitro, both proteins interact specifically with eEF-2, with a higher affinity for P1 (Kd = 3.8 x 10-8 m) than for P2 (Kd = 2.2 x 10-6 m). Phosphorylation resulted in a moderate increase (two- to four-fold) in these affinities. The interaction of both P1 and P2 (phosphorylated or not) with eEF-2 resulted in a conformational change in the factor, revealed by an increase in the accessibility of Glu554 to proteinase Glu-C. This increase was observed in both the presence and absence of GTP and GDP, which themselves produced marked opposite effects on the conformation of eEF-2. Our results suggest that the two proteins P1 and P2 both interact with eEF-2 inducing a conformational transition of the factor, but have acquired some specific properties during evolution.
TL;DR: These observations indicate that heparin, in addition to causing aggregation, also induces a conformational change in tau in which reactive groups are unmasked or move closer leading to the DSS cross-linking of 72- and 83-kDa species.
TL;DR: Data show that calcium binding to CaM controls the surface exposure of the Met residues, thereby providing the switch for target protein binding in CaM-dependent protein kinase I complexes.
TL;DR: A strong dependence of the conformational shift on the protonation level of the phosphoryl group could be observed, showing that phosphorylation induces the strongest effect in its dianionic, i.e., physiological, form.
TL;DR: Profactor D is the most flexible of the four, and its mature enzyme displays inactive, self‐inhibited active site conformation, and Examination of the surface properties of the N‐terminus‐binding pocket indicates that Ile16 may play the initial positioning role for the N-terminus, and Leu17 probably also helps in inducing the required conformational changes.
Abstract: The crystal structure of profactor D, determined at 2.1 A resolution with an Rfree and an R-factor of 25.1 and 20.4%, respectively, displays highly flexible or disordered conformation for five regions: N-22, 71-76, 143-152, 187-193 and 215-223. A comparison with the structure of its mature serine protease, complement factor D, revealed major conformational changes in the similar regions. Comparisons with the zymogen-active enzyme pairs of chymotrypsinogen, trypsinogen and prethrombin-2 showed a similar distribution of the flexible regions. However, profactor D is the most flexible of the four, and its mature enzyme displays inactive, self-inhibited active site conformation. Examination of the surface properties of the N-terminus-binding pocket indicates that Ile16 may play the initial positioning role for the N-terminus, and Leu17 probably also helps in inducing the required conformational changes. This process, perhaps shared by most chymotrypsinogen-like zymogens, is followed by a factor D-unique step, the re-orientation of an external Arg218 to an internal position for salt-bridging with Asp189, leading to the generation of the self-inhibited factor D.
TL;DR: In this article, an x-ray analysis of B. napus ENR A138G grown in the presence of either NAD+ or NADH was performed and the structure of the corresponding ternary complexes with thienodiazaborine obtained either by soaking the drug into the crystals or by co-crystallization of the mutant with NAD+ and diazaborines.
TL;DR: Some (mostly fusogenic) peptides induced significant increases of the order parameter of the lipid hydrocarbon chains, suggesting that the lipid bilayer becomes more ordered in the presence of these peptides, possibly as a result of dehydration at the membrane surface.
Abstract: The amino-terminal region of the membrane-anchored subunit of influenza virus hemagglutinin, the fusion peptide, is crucial for membrane fusion of this virus The peptide is extruded from the interior of the protein and inserted into the lipid bilayer of the target membrane upon induction of a conformational change in the protein by low pH Although the effects of several mutations in this region on the fusion behavior and the biophysical properties of the corresponding peptides have been studied, the structural requirements for an active fusion peptide have still not been defined To probe the sensitivity of the fusion peptide structure and function to small hydrophobic perturbations in the middle of the hydrophobic region, we have individually replaced the alanine residues in positions 5 and 7 with smaller (glycine) or bulkier (valine) hydrophobic residues and measured the extent of fusion mediated by these hemagglutinin constructs as well as some biophysical properties of the corresponding synthetic peptides in lipid bilayers We find that position 5 tolerates a smaller and position 7 a larger hydrophobic side chain All peptides contained segments of alpha-helical (33-45%) and beta-strand (13-16%) conformation as determined by CD and ATR-FTIR spectroscopy The order parameters of the peptide helices and the lipid hydrocarbon chains were determined from measurements of the dichroism of the respective infrared absorption bands Order parameters in the range of 00-06 were found for the helices of these peptides, which indicate that these peptides are most likely aligned with their alpha-helices at oblique angles to the membrane normal Some (mostly fusogenic) peptides induced significant increases of the order parameter of the lipid hydrocarbon chains, suggesting that the lipid bilayer becomes more ordered in the presence of these peptides, possibly as a result of dehydration at the membrane surface
TL;DR: The role of the active site aspartate base in the aminotransferase mechanism of the copper amine oxidase from the yeast Hansenula polymorpha has been probed by site-directed mutagenesis and a model is proposed in which a central role is proposed to position the free cofactor and several enzyme intermediates for optimal activity.
Abstract: The role of the active site aspartate base in the aminotransferase mechanism of the copper amine oxidase from the yeast Hansenula polymorpha has been probed by site-directed mutagenesis. The D319E mutant catalyzes the oxidation of methylamine and phenethylamine, but not that of benzylamine. kcat/Km for methylamine is found to be 80-fold reduced compared to that of the wild type. Viscosogen and substrate and solvent deuteration have no effect on this parameter for D319E, which is suggestive of limitation of kcat/Km by a conformational change. This conformational change is proposed to be the movement of the cofactor into a productive orientation upon the binding of substrate. In the absence of substrate, a flipped cofactor orientation is likely, on the basis of resonance Raman evidence that the C5 carbonyl of the cofactor is less solvent accessible than the C3 hydrogen. kcat for D319E methylamine oxidase is reduced 200-fold compared to that of the wild type and is unaffected by substrate deuteration, but displays a substantial solvent isotope effect. A 428 nm absorbance is evident under conditions of saturating methylamine and oxygen with D319E. The D319N mutant is observed to produce a similar absorbance at 430 nm when treated with ammonia despite the fact that this mutant has no amine oxidase activity. Resonance Raman spectroscopy indicates the formation of a covalent ammonia adduct and identifies it as the deprotonated iminoquinone. In contrast, when the D319E mutant is reacted with ammonia, it gives predominantly a 340-350 nm species. This absorbance is ascribed to a localization of the cofactor oxyanion induced by binding of the cation at the active site and not to covalent adduct formation. Resonance Raman spectroscopic examination of the steady state species of D319E methylamine oxidation, in combination with the kinetic data, indicates that the 428 nm species is the deprotonated iminoquinone produced upon reoxidation of the reduced cofactor. A model is proposed in which a central role of the active site base is to position the free cofactor and several enzyme intermediates for optimal activity.