Showing papers on "Conformational change published in 1993"

Interfacial activation of the lipase-procolipase complex by mixed micelles revealed by X-ray crystallography.

Abstract: The three-dimensional structure of the lipase-procolipase complex, co-crystallized with mixed micelles of phosphatidylcholine and bile salt, has been determined at 3 A resolution by X-ray crystallography. The lid, a surface helix covering the catalytic triad of lipase, adopts a totally different conformation which allows phospholipid to bind to the enzyme's active site. The open lid is an essential component of the active site and interacts with procolipase. Together they form the lipid-water interface binding site. This reorganization of the lid structure provokes a second drastic conformational change in an active site loop, which in its turn creates the oxyanion hole (induced fit).

Conformational Coupling in DNA Polymerase Fidelity

Abstract: The fidelity of DNA polymerases is largely attributable to a two-step nucleotide binding mechanism. In the first step, binding contacts are initially made between the template and the incoming dNTP. The selectivity of this ground-state binding is similar in magnitude to the selectivity seen in forming base pairs in solution. In the second step, a change in protein conformation occurs, which leads to rapid incorporation of the dNTP into the growing polymer. This conformational change appears to occur globally in that it is inhibited by mismatches in the dNTP or in any of the three terminal base pairs of the primer/template. The open conformation allows rapid binding of the dNTP from solution, while the closed conformation provides steric checks for the proper Watson-Crick base pair geometry. This conformational change accounts for the extraordinary fidelity of polymerization and also provides selectivity to the exonuclease by inhibiting polymerization over a mismatch in the primer/template. The overall fidelity approaches one error in 10(10) by a combination of selectivity in polymerization (10(5)-10(6)) and in proofreading (10(3)-10(4)). This paradigm provides the theoretical basis for further investigation of the structural basis for fidelity by pointing to the essential elements of the polymerization reaction that need to be examined in order to evaluate active-site-directed mutants of polymerases to test appropriate structure/function relationships.

ATP-induced protein Hsp70 complex dissociation requires K + but not ATP hydrolysis

Abstract: THE molecular chaperone proteins, particularly Hsp60 and Hsp70, have been implicated in essential cell functions under both normal and stress conditions (reviewed in refs 1–5). Members of the family of heat-shock proteins ofMr70K, Hsp70, bind to unfolded proteins and short peptides6–13. Addition of Mg–ATP results in the dissociation of the substrate polypeptides from the chaperone7–11, but as ATP-γS (an ATP analogue that is only slowly hydrolysable) cannot substitute for ATP in this reaction7,9,11, it has been concluded that ATP hydrolysis is necessary to dissociate Hsp70–substrate protein complexes. By independently measuring the rates of ATP hydrolysis and substrate protein dissociation, we show here that Mg–ATP binding but not Mg–ATP hydrolysis is essential for substrate dissociation. We also show that there is an absolute requirement for K+for the effect of Mg–ATP: only the combination of K+ and Mg–ATP will cause the conformational change in HspTO that is necessary for substrate dissociation. Moreover, in the absence of K+, Mg–ATP favours complex formation. We consider these results in terms of a G-protein-like model.

Transcriptional activation by the estrogen receptor requires a conformational change in the ligand binding domain.

Abstract: The estrogen receptor (ER) is a strong hormone-inducible transcription factor that regulates the expression of many genes. It was shown for the human progesterone receptor that the binding of hormone causes distinct conformational changes in the ligand binding domain (LBD) and that these changes in LBD conformation are crucial for events after DNA binding. We now show that conformational changes in the LBD of the human ER are a prerequisite for trans-activation. Under the appropriate conditions ER binds to its response element and activates transcription only in the presence of ligand. Binding of the ligand causes changes in the conformation of the LBD. Antihormones induce distinct conformational changes, the differences between the conformations lying in the carboxy-terminal end of the receptor. Changing the experimental conditions results in a receptor that can bind to DNA and activate transcription in a ligand-independent manner. Under these conditions the LBD has a transcriptionally active conformation in the absence of ligand. Taken together, our data indicate that the conformational change induced by ligand is required for converting a receptor to the transcriptionally active form.

Inhibition of the fusion-inducing conformational change of influenza hemagglutinin by benzoquinones and hydroquinones.

Abstract: Influenza hemagglutinin (HA) undergoes a conformational change that is required for viral entry. The rearrangement includes exposure of the fusion peptide, a hydrophobic segment buried in the trimer interface of the native protein. Since fusion peptide release triggers the membrane fusion event crucial for viral replication, inhibition of fusion peptide exposure should prevent infection. We reasoned that small molecules that bind to HA and stabilize its nonfusogenic conformation would block viral activity. A computer-assisted method was used to select putative HA ligands. One of the selected compounds, 4A,5,8,8A-tetrahydro-5,8-methano-1,4-naphthoquinone, prevented the conversion of X31 HA to a conformation recognized by alpha-fusion peptide antisera. Several derivatives of this compound, including both benzoquinones and hydroquinones, also showed inhibition. The most effective compounds tested have IC50S between 1 and 20 microM. Representative compounds also inhibited virus-induced syncytia formation, HA-mediated hemolysis, and viral infectivity in vitro. The inhibitors are attractive leads for the development of antiviral drugs and can serve as probes of the mechanism of the conformational change of HA.

A cooperative conformational change in duplex DNA induced by Zn2+ and other divalent metal ions

Abstract: Zn2+ and some other divalent metal ions bind to duplex DNA at pHs above 8 and cause a conformational change. This new structure does not bind ethidium, allowing the development of a rapid fluoresce...

DNA and redox state induced conformational changes in the DNA-binding domain of the Myb oncoprotein.

Abstract: The DNA-binding domain of the oncoprotein Myb comprises three imperfect repeats, R1, R2 and R3. Only R2 and R3 are required for sequence-specific DNA-binding. Both are assumed to contain helix-turn-helix (HTH)-related motifs, but multidimensional heteronuclear NMR spectroscopy revealed a disordered structure in R2 where the second HTH helix was predicted [Jamin et al. (1993) Eur. J. Biochem., 216, 147-154]. We propose that the disordered region folds into a 'recognition' helix and generates a full HTH-related motif upon binding to DNA. This would move Cys43 into the hydrophobic core of R2. We observed that Cys43 was accessible to N-ethylmaleimide alkylation in the free protein, but inaccessible in the DNA complex. Mutant proteins with charged (C43D) or polar (C43S) side chains in position 43 bound DNA with reduced affinity, while hydrophobic replacements (C43A, C43V and C43I) gave unaltered or improved DNA-binding. Specific DNA-binding enhanced protease resistance dramatically. Fluorescence emission spectra and quenching experiments supported a DNA-induced conformational change. Moreover, reversible oxidation of Cys43 had an effect similar to the inactivating C43D mutation. The highly oxidizable Cys43 could function as a molecular sensor for a redox regulatory mechanism turning specific DNA-binding on or off by controlling the DNA-induced conformational change in R2.

Nonexponential protein relaxation: dynamics of conformational change in myoglobin

Abstract: The picosecond evolution of the tertiary conformation of myoglobin (Mb) after photodissociation of MbCO was investigated at room temperature by probing band III, a weak iron-porphyrin charge-transfer transition near 13,110 cm-1 (763 nm) that is sensitive to the out-of-plane displacement of the iron. Upon photolysis, the iron moves out of the plane of the porphyrin, causing a blue-shift of band III and a concomitant change in the protein conformation. The dynamics for this functionally important motion are highly nonexponential, in agreement with recent molecular dynamics simulations [Kuczera, K., Lambry, J.-C., Martin, J.-L. & Karplus, M. (1993) Proc. Natl. Acad. Sci. USA 90, 5805-5807]. The conformational change likely affects the height of the barrier to ligand rebinding and may explain nonexponential NO rebinding.

Binding of 4',6-diamidino-2-phenylindole (DAPI) to AT regions of DNA: evidence for an allosteric conformational change.

Abstract: The interaction of 4',6-diamidino-2-phenylindole (DAPI) with several double-helical poly- and oligonucleotides has been studied in solution using optical spectroscopic techniques: flow linear dichroism (LD), induced circular dichroism (CD), and fluorescence spectroscopy. In AT-rich sequences, where DAPI is preferentially bound, LD indicates that the molecule is edgewise inserted into the minor groove at an angle of approximately 45-degrees to the helix axis. This binding geometry is found for very low as well as quite high binding ratios. The concluded geometry is in agreement with that of the DAPI complex in a crystal with the Drew-Dickerson dodecamer, and the DAPI complex with this dodecamer in solution is verified to have an ICD spectrum similar to that of the complex with [poly(dA-dT)]2 at low binding ratios. The observation of two types of CD spectra characteristic for the binding of DAPI to DNA, and also for the interaction with [poly(dA-dT)]2, demonstrates that the first binding mode, despite its low apparent abundance (a few percent), is not due to a specific DNA site. The effect may be explained in terms of an allosteric binding such that when DAPI molecules bind contiguously to the AT sequence the conformation of the latter is changed. The new conformation, which according to LD appears to be stiffer than normal B-form DNA, is responsible for the second type of induced CD spectrum in the DAPI chromophore. Although the spectroscopic results indicate a change of DNA conformation, consistent with an allosteric binding model, they do not explicitly require any cooperativity, but accidental neighbors could also explain the data.

Formation of the meta II photointermediate is accompanied by conformational changes in the cytoplasmic surface of rhodopsin

Abstract: Five mutations of rhodopsin have been produced, each of which contains a unique cysteine residue at positions 62, 65, 140, 240, or 316 in the cytoplasmic domain. The single reactive cysteines were derivatized with a sulfhydryl-specific nitroxide spin-label, and the electron paramagnetic resonance (EPR) spectra were analyzed in both lauryl maltoside and digitonin in the dark and after photobleaching. The collision rate of the attached nitroxides with polar and nonpolar paramagnetic agents indicated that they were all exposed to the aqueous environment. Photobleaching of the mutants in digitonin, which arrests the protein at the meta I intermediate, produced little change in mobility of the attached nitroxide. On the other hand, photobleaching in lauryl maltoside produced the meta II intermediate and significant changes in the EPR spectra of the nitroxides attached to positions 140 and 316. These data directly reveal a light-induced conformational change in the cytoplasmic loops that accompanies meta II formation.

ATPase activity and ATP/ADP-induced conformational change in the soluble domain of the bacterial protein translocator HlyB

Abstract: The haemolysin exporter HlyB and its homologues are central to the unconventional signal-peptide-independent secretion of toxins, proteases and nodulation proteins by bacteria. HlyB is a member of the ATP-binding cassette (ABC) or traffic ATPase superfamily, and resembles closely in structure and function mammalian exporters such as the multidrug-resistance P-glycoprotein, combining both integral membrane and cytosolic domains. Overproduction of the HlyB cytoplasmic domain as a C-terminal peptide fused to glutathione S-transferase allowed the direct affinity purification and concentration of 30-50 mg ml-1 of soluble protein (GST-Bctp) in an apparently dimeric form possessing both transferase and ATPase activity. GST-Bctp bound to ADP-agarose and was eluted specifically by ATP and ADP, affinity behaviour which was confirmed in both the full-length HlyB and the unfused HlyB cytoplasmic domain synthesized in vitro. The stoichiometry of binding to MgATP and MgADP was close to equimolar and both ligands induced substantial conformational change in the protein. Mg(2+)-dependent ATPase activity of GST-Bctp (Vmax 1 mumol min-1 mg-1, Km 0.2 mM) was comparable with the activity of the bacterial importer MalK and human P-glycoprotein reconstituted into proteoliposomes, and over an order of magnitude higher than in vitro measurements of disaggregated MalK purified from inclusion bodies. Activity was unaffected by inhibitors of F- and V-type ATPases, non-hydrolysable ATP analogues, or translocation substrate, but was severely inhibited by inhibitors of E1E2 (P-type) ATPases, and the acidic phospholipid phosphatidyl glycerol.

The role of conformational change in serpin structure and function

Abstract: Serpins are members of a family of structurally related protein inhibitors of serine proteinases, with molecular masses between 40 and 100kDa. In contrast to other, simpler, proteinase inhibitors, they may interact with proteinases as inhibitors, as substrates, or as both. They undergo conformational interconversions upon complex formation with proteinase, upon binding of some members to heparin, upon proteolytic cleavage at the reactive center, and under mild denaturing conditions. These conformational changes appear to be critical in determining the properties of the serpin. The structures and stabilities of these various forms may differ significantly. Although the detailed structural changes required for inhibition of proteinase have yet to be worked out, it is clear that the serpin does undergo a major conformational change. This is in contrast to other, simpler, families of protein inhibitors of serine proteinases, which bind in a substrate-like or product-like manner. Proteolytic cleavage of the serpin can result in a much more stable protein with new biological properties such as chemo-attractant behaviour. These structural transformations in serpins provide opportunities for regulation of the activity and properties of the inhibitor and are likely be important in vivo, where serpins are involved in blood coagulation, fibrinolysis, complement activation and inflammation.

Inhibitor-induced conformational change in cytochrome P-450CAM.

Abstract: The X-ray crystal structures of cytochrome P-450CAM complexed with both enantiomers of a chiral, multifunctional inhibitor have been refined to R-factors of 21.0% [(+)-enantiomer] and 19.6% [(-)-enantiomer] at approximately 2.1-A resolution. Binding of either enantiomer, both considerably larger than the natural substrate camphor, results in similar, dramatic structural changes in the enzyme. In contrast to all previous P-450CAM crystallographic structures, the Tyr96 side chain is not pointing "down" toward the heme but is rather directed "up" into the proposed substrate access channel. This conformational change is accompanied by the displacement of the Phe193 side chain out into the solvent at the enzyme surface. These changes are consistent with the assignment of this region of the enzyme as the access channel [Poulos et al. (1986) Biochemistry 25, 5314-5322] and suggest that several aromatic residues lining the channel may be involved in substrate recognition and channeling to the active site. The cation usually observed coordinated to the Tyr96 carbonyl oxygen is missing in the presence of the (+)-enantiomer but is present with the (-)-enantiomer. The Phe87 side chain, located near the inhibitor binding site, adopts different orientations depending upon which enantiomer is bound. Finally, electron density reveals that although the inhibitor enantiomers were dichlorinated as provided, when bound to P-450CAM the chlorine atoms are present at only 0-20% occupancy, probably reflecting selective binding of impurities in the samples. Coordinates of these inhibited P-450CAM complexes have been deposited in the Brookhaven Protein Data Bank [Bernstein et al. (1977) J. Mol. Biol. 112, 535-542].

Proton transfer from Asp-96 to the bacteriorhodopsin Schiff base is caused by a decrease of the pKa of Asp-96 which follows a protein backbone conformational change

Abstract: In the bacteriorhodopsin photocycle the transported proton crosses the major part of the hydrophobic barrier during the M to N reaction; in this step the Schiff base near the middle of the protein is reprotonated from D96 located near the cytoplasmic surface. In the recombinant D212N protein at pH > 6, the Schiff base remains protonated throughout the photocycle [Needleman, Chang, Ni, Varo, Fornes, White, & Lanyi (1991) J. Biol. Chem. 266, 11478-11484]. Time-resolved difference spectra in the visible and infrared are described by the kinetic scheme BR-->K L N (-->N')-->BR. As evidenced by the large negative 1742-cm-1 band of the COOH group of the carboxylic acid, deprotonation of D96 in the N state takes place in spite of the absence of the unprotonated Schiff base acceptor group of the M intermediate. Instead of internal proton transfer to the Schiff base, the proton is released to the bulk, and can be detected with the indicator dye pyranine during the accumulation of N'. The D212N/D96N protein has a similar photocycle, but no proton is released. As in wild-type, deprotonation of D96 in the N state is accompanied by a protein backbone conformational change indicated by characteristic amide I and II bands. In D212N the residue D96 can thus deprotonate independent of the Schiff base, but perhaps dependent on the detected protein conformational change. This could occur through increased charge interaction between D96 and R227 and/or increased hydration near D96. We suggest that the proton transfer from D96 to the Schiff base in the wild-type photocycle is driven also by such a decrease in the pKa of D96.

Conformational changes at the active site of creatine kinase at low concentrations of guanidinium chloride

Abstract: It has been previously reported that, during denaturation of creatine kinase by guanidinium chloride (GdmCl) or urea [Tsou (1986), Trends Biochem. Sci. 11, 427-429], inactivation occurs before noticeable conformational change can be detected, and it is suggested that the conformation at the active site is more easily perturbed and hence more flexible than the molecule as a whole. In this study, the thiol and amino groups at or near the active site of creatine kinase are labelled with o-phthalaldehyde to form a fluorescent probe. Both the emission intensity and anisotropy decrease during denaturation indicating exposure of this probe and increased mobility of the active site. The above conformational changes take place together with enzyme inactivation at lower GdmCl concentrations than required to bring about intrinsic fluorescence changes of the enzyme. At the same GdmCl concentration, the rate of exposure of the probe is comparable with that of inactivation and is several orders of magnitude faster than that for the unfolding of the molecule as a whole.

Role of spike protein conformational changes in fusion of Semliki Forest virus.

Abstract: The alphavirus Semliki Forest virus (SFV) and a number of other enveloped animal viruses infect cells via a membrane fusion reaction triggered by the low pH within endocytic vesicles. In addition to having a low pH requirement, SFV fusion and infection are also strictly dependent on the presence of cholesterol in the host cell membrane. A number of conformational changes in the SFV spike protein occur following low-pH treatment, including dissociation of the E1-E2 dimer, conformational changes in the E1 and E2 subunits, and oligomerization of E1 to a homotrimer. To allow the ordering of these events, we have compared the kinetics of these conformational changes with those of fusion, using pH treatment near the fusion threshold and low-temperature incubation to slow the fusion reaction. Dimer dissociation, the E1 conformational change, and E1 trimerization all occur prior to the mixing of virus and cell membranes. Studies of cells incubated at 20 degrees C showed that as with virus fusion, E1 trimerization occurred in the endosome before transport to lysosomes. However, unlike the strictly cholesterol-dependent membrane fusion reaction, the E1 homotrimer was produced in vivo during virus uptake by cholesterol-depleted cells or in vitro by low-pH treatment of virus in the presence of artificial liposomes with or without cholesterol. Purified, lipid-free spike protein rosettes were assayed to determine the requirement for virus membrane cholesterol in E1 homotrimer formation. Spike protein rosettes were found to undergo E1 oligomerization upon exposure to low pH and target liposomes and showed an enhancement of oligomerization with cholesterol-containing membranes. The E1 homotrimer may represent a perfusion complex that requires cholesterol to carry out the final coalescence of the viral and target membranes.

Conformational changes in chicken thyroid hormone receptor alpha 1 induced by binding to ligand or to DNA.

Abstract: A classic model of steroid/thyroid hormone receptor activation postulates that a conformational change or "transformation" occurs upon ligand binding as a first step toward regulation of gene transcription. In order to test this model, physical studies have been carried out using purified full-length chicken thyroid hormone receptor alpha 1 (cT3R-alpha 1) expressed in Escherichia coli. Circular dichroism spectroscopic studies reveal that cT3R-alpha 1 adopts a different conformation upon specific binding to a cognate ligand triiodothyroacetic acid as well as to a thyroid hormone response element, an idealized inverted repeat AGGTCA TGACCT. These results suggest that cT3R-alpha 1 may adopt distinct conformations whether free or bound to ligand or to DNA. These states may reflect the changes in the conformation of steroid/thyroid hormone receptors in the signal transduction pathway.

Transmission of conformational change from the heparin binding site to the reactive center of antithrombin.

Abstract: Heparin greatly increases the rates at which antithrombin inhibits target proteinases. An important part of this rate acceleration is a heparin-induced conformational change in antithrombin. To answer the question of whether or not this change is transmitted to the reactive center, we have prepared a recombinant P1 mutant of antithrombin, R393C, labeled the cysteine with nitrobenzofuran (NBD) fluorophore, and examined the perturbation of NBD fluorescence intensity as a function of bound sulfated oligosaccharide. Two high-affinity heparins, low-affinity heparin, and dextran sulfate were used. We found (i) that binding to antithrombin of all these oligosaccharides resulted in transmission of conformational change to P1 in the reactive center, (ii) that these oligosaccharides all gave enhancements of the rate of inhibition of factor Xa beyond any contribution from surface approximation, and (iii) that the degree of perturbation of P1 correlated with the enhancement of the rate of factor Xa inhibition that was not due to surface approximation.