Conformational changes of the H+-ATPase from Escherichia coli upon nucleotide binding detected by single molecule fluorescence

BACKGROUND: An antiendomysium antibody test using human umbilical cord as antigen has recently been introduced. METHODS: We determined IgA- and IgG-class antihuman umbilical cord (HUC-ab), antireticulin (ARA), and antigliadin antibodies (AGA) in 92 untreated adult coeliac patients, in 95 non-coeliac subjects, and in 4 coeliac patients with selective IgA deficiency. Tissue antibodies were measured with an indirect immunofluorescence method and AGA with an enzyme-linked immunosorbent assay. RESULTS: Of adult coeliac patients 85% were positive for IgA-class HUC-ab, 78% were positive for ARA, and 80% for AGA; the specificity for HUC-ab and ARA was 100%, and for AGA 86%. Combination of HUC-ab, ARA, and high-titre AGA increased the sensitivity to 96% without loss of specificity. IgG-class HUC-ab was positive in 12% of coeliac patients, in all four coeliac patients with IgA deficiency, and in none of the controls. CONCLUSIONS: The HUC-ab test is highly specific but not 100% sensitive for detecting adult coeliac disease. A combination of the IgA-class HUC-ab, ARA, and high-titre AGA tests is recommended. In selective IgA deficiency the IgG-class HUC-ab test seems to work well.     

Conformational changes of the mitochondrial F1-ATPase epsilon-subunit induced by nucleotide binding as observed by phosphorescence spectroscopy

Changes in conformation of the epsilon-subunit of the bovine heart mitochondrial F1-ATPase complex as a result of nucleotide binding have been demonstrated from the phosphorescence emission of tryptophan. The triplet state lifetime shows that whereas nucleoside triphosphate binding to the enzyme in the presence of Mg2+ increases the flexibility of the protein structure surrounding the chromophore, nucleoside diphosphate acts in an opposite manner, enhancing the rigidity of this region of the macromolecule. Such changes in dynamic structure of the epsilon-subunit are evident at high ligand concentration added to both the nucleotide-depleted F1 (Nd-F1) and the F1 preparation containing the three tightly bound nucleotides (F1(2,1)). Since the effects observed are similar in both the F1 forms, the binding to the low affinity sites must be responsible for the conformational changes induced in the epsilon-subunit. This is partially supported by the observation that the Trp lifetime is not significantly affected by adding an equimolar concentration of adenine nucleotide to Nd-F1. The effects on protein structure of nucleotide binding to either catalytic or noncatalytic sites have been distinguished by studying the phosphorescence emission of the F1 complex prepared with the three noncatalytic sites filled and the three catalytic sites vacant (F1(3,0)). Phosphorescence lifetime measurements on this F1 form demonstrate that the binding of Mg-NTP to catalytic sites induces a slight enhancement of the rigidity of the epsilon-subunit. This implies that the binding to the vacant noncatalytic site of F1(2,1) must exert the opposite and larger effect of enhancing the flexibility of the protein structure observed in both Nd-F1 and F1(2,1). The observation that enhanced flexibility of the protein occurs upon addition of adenine nucleotides to F1(2,1) in the absence of Mg2+ provides direct support for this suggestion. The connection between changes in structure and the possible functional role of the epsilon-subunit is discussed.     

Conformational changes in conantokin-G induced upon binding of calcium and magnesium as revealed by NMR structural analysis

The apo- and metal-bound solution conformations of synthetic conantokin-G (con-G, G1Egamma gammaL5Q gamma NQgamma 10LIRgamma K15SN-CONH2, gamma = gamma-carboxyglutamic acid), an antagonist of N-methyl-D-aspartate receptor-derived neuronal ion channels, have been examined by one- and two-dimensional 1H NMR at neutral pH. A complete structure for the Mg2+-loaded peptide was defined by use of distance geometry calculations and was found to exist as an alpha-helix that spans the entire peptide. The alpha-helical nature of Mg2+/con-G was also supported by the small values (<5.5 Hz) of the 3JHNalpha coupling constants measured for amino acid residues 3-5, 8, 9, and 11-16, and the small values (<4 ppb/K) of the temperature coefficients observed for the alphaNH protons of residues 5-17. This conformation contrasted with that obtained for apo-con-G, which was nearly structureless in solution. Docking of Mg2+ into con-G was accomplished by use of the genetic algorithm/molecular dynamics simulation method, employing the NMR-derived Mg2+-loaded structure for initial coordinates in the midpoint calculations. For the 3 Mg2+/con-G model, it was found that binding of one Mg2+ ion is stabilized by oxygen atoms from three gamma-carboxylates of Gla3, Gla4, and Gla7; another Mg2+ is coordinated by two oxygen atoms, one from each of the gamma-carboxylates of Gla7; and a third metal ion through three donor oxygen atoms of gamma-carboxylates from Gla10 and Gla14. As shown from direct metal binding measurements to mutant con-G peptides, these latter two Gla residues probably stabilized the tightest binding Mg2+ ion. Circular dichroism studies of these same peptide variants demonstrated that all Gla residues contribute to the adoption of the Mg2+-dependent alpha-helical conformation in con-G. The data obtained in this investigation provide a molecular basis for the large conformational alteration observed in apo-con-G as a result of divalent cation binding and allow assessment of the roles of individual Gla residues in defining certain of the structure-function properties of con-G.     

Structure changes in hemoglobin upon deletion of C-terminal residues, monitored by resonance Raman spectroscopy

Loss of C-terminal residues in hemoglobin raises oxygen affinity and reduces both cooperativity and the Bohr effect. These functional changes are expected from the loss of C-terminal salt bridges, which are seen crystallographically to stabilize the T quaternary structure. Ultraviolet resonance Raman (UVRR) difference spectroscopy confirms that the strength of the T state contacts is diminished when the C-terminal and also the penultimate residues are removed chemically. Deoxy minus CO difference signals arising from the Trpbeta37-Aspalpha94 and Tyralpha42-Aspbeta99 H bonds at the alpha1 beta2 subunit interface are diminished, and at pH 9, the difference spectra reveal a shift to the R quaternary structure. These effects are small for desHisbeta146 Hb and large for desArgalpha141 Hb, consistent with the order of functional changes. In addition, the H bond between the A and E helices is strengthened by removal of Argalpha141 and is further strengthened when the effector molecule IHP (inositol hexaphosphate) is added to deoxy-desArgalpha141 Hb or when its pH is lowered to 5.8. This effect is attributed to the loss of the C-terminal anchor of the alpha chain H helix, which supports the F and A helices. The beta chain is not as sensitive because it has extra F-H interhelix H bonds. Removal of both Hisbeta146 and Tauyrbeta145 produce UVRR changes which are intermediate between desHisbeta146 and desArgalpha141 Hb, although the functional consequences are greater than for desArgalpha141 Hb. Removal of Tyralpha140 as well as Argalpha141 abolishes cooperative binding as well as the Bohr effect, and the UVRR difference signals are also lost, suggesting that quaternary constraints are removed in both the T and the R states. When the approximately 220 cm-1 iron-histidine stretching vibration of the deoxy-proteins is examined, using Raman excitation in resonance with the heme Soret band, the frequency is observed to diminish toward that of deoxyHb A (215 cm-1) as the pH is lowered and IHP is added and to increase toward a completely relaxed value (223 cm-1) as the pH is raised to 9. The relaxation is in the same order as the functional perturbations: desHisbeta146 < desArgalpha141 < desHisbeta146-Tyrbeta145 < desArgalpha141-Tyralpha140. However, even desArgalpha141-Tyralpha140 Hb shows significant reduction in the Fe-His frequency as IHP is added at low pH. The Fe-His frequency is sensitive to both tertiary and quaternary structure changes and is a global indicator of forces at the heme. The order of affinity changes can be understood on the basis of the number of stabilizing H bonds between the F and H helices. Titration curves of the Fe-His frequency against pH are not sigmoidal, consistent with a multiplicity of contributions to the Bohr effect.     

Real-time enzyme kinetics monitored by dual-color fluorescence cross-correlation spectroscopy

A method for sensitively monitoring enzyme kinetics and activities by using dual-color fluorescence cross-correlation spectroscopy is described. This universal method enables the development of highly sensitive and precise assays for real-time kinetic analyses of any catalyzed cleavage or addition reaction, where a chemical linkage is formed or cleaved through an enzyme's action between two fluorophores that can be discriminated spectrally. In this work, a homogeneous assay with restriction endonuclease EcoRI and a 66-bp double-stranded DNA containing the GAATTC recognition site and fluorophores at each 5' end is described. The enzyme activity can be quantified down to the low picomolar range (>1.6 pM) where the rate constants are linearly dependent on the enzyme concentrations over two orders of magnitude. Furthermore, the reactions were monitored on-line at various initial substrate concentrations in the nanomolar range, and the reaction rates were clearly represented by the Michaelis-Menten equation with a KM of 14 +/- 1 nM and a kcat of 4.6 +/- 0.2 min-1. In addition to kinetic studies and activity determinations, it is proposed that enzyme assays based on the dual-color fluorescence cross-correlation spectroscopy will be very useful for high-throughput screening and evolutionary biotechnology.     

Inhibition of insulin receptor activation by insulin-like growth factor binding proteins

The insulin-like growth factors (IGFs) are transported by a family of high-affinity binding proteins (IGFBPs) that protect IGFs from degradation, limit their binding to IGF receptors, and modulate IGF actions. The six classical IGFBPs have been believed to have no affinity for insulin. We now demonstrate that IGFBP-7/mac25, a newly identified member of the IGFBP superfamily that binds IGFs specifically with low affinity is a high-affinity insulin binding protein. IGFBP-7 blocks insulin binding to the insulin receptor and thereby inhibiting the earliest steps in insulin action, such as autophosphorylation of the insulin receptor beta subunit and phosphorylation of IRS-1, indicating that IGFBP-7 is a functional insulin-binding protein. The affinity of other IGFBPs for insulin can be enhanced by modifications that disrupt disulfide bonds or remove the conserved COOH terminus. Like IGFBP-7, an NH2-terminal fragment of IGFBP-3 (IGFBP-3((1-87))), also binds insulin with high affinity and blocks insulin action. IGFBPs with enhanced affinity for insulin might contribute to the insulin resistance of pregnancy, type II diabetes mellitus, and other pathological conditions.     

Conformational changes affect binding and catalysis by ester-hydrolysing antibodies

D2.3, D2.4 and D2.5 are ester-hydrolysing antibodies raised against a phosphonate transition state analogue (TSA). All three antibody-TSA binding kinetics, as monitored by fluorescence quenching, indicate an "induced-fit" mechanism: fast bimolecular association followed by a unimolecular isomerisation (k=1-7 s-1). Isomerisation leads to a 30-170-fold increase in affinity towards the TSA and, consequently, to higher catalytic rates. Antibody D2.3 exhibits a complex three-step binding mechanism, in which the last step is a "very slow" isomerisation (k<0.02 s-1). This very slow isomerisation is limiting the rate of catalysis by D2.3, as indicated by the kinetics of product release which show characteristics of enzyme "conformational memory" or "hysteresis". The results support a mechanism consisting of pre-equilibrium between "nether-active" (low affinity) and "active" (high affinity) antibody conformers (prior to ligand addition) as well as induced-fit, i.e. isomerisation of the nether-active ligand-antibody complex to give the active complex. Crystal structures of these antibodies, free and complexed, have previously indicated that their conformation does not change upon binding. Here, we show that the buffer used to crystallise the antibodies, and in particular its polyethylene glycol component, alters the pre-equilibrium in favour of the active conformer, leading to its crystallisation both in the presence and in the absence of the TSA.     

Conformational changes occurring upon reduction and NO binding in nitrite reductase from Pseudomonas aeruginosa

Nitrite reductase (NiR) from Pseudomonas aeruginosa (EC 1.9.3.2) (NiR-Pa) is a soluble enzyme catalyzing the reduction of nitrite (NO2-) to nitric oxide (NO). The enzyme is a 120 kDa homodimer, in which each monomer carries one c and one d1 heme. The oxidized and reduced forms of NiR from Paracoccus denitrificans GB17 (previously called Thiosphaera pantotropha) (NiR-Pd) have been described [Fülop, V., et al. (1995) Cell 81, 369-377; Williams, P. A., et al. (1997) Nature 389, 406-412], and we recently reported on the structure of oxidized NiR-Pa at 2.15 A [Nurizzo, D., et al. (1997) Structure 5, 1157-1171]. Although the domains carrying the d1 heme are almost identical in both NiR-Pa and NiR-Pd oxidized and reduced structures, the c heme domains show a different pattern of c heme coordination, depending on the species and the redox state. The sixth d1 heme ligand in oxidized NiR-Pd was found to be Tyr25, whereas in NiR-Pa, the homologuous Tyr10 does not interact directly with Fe3+, but via a hydroxide ion. Furthermore, upon reduction, the axial ligand of the c heme of NiR-Pd changes from His17 to Met108. Finally, in the oxidized NiR-Pa structure, the N-terminal stretch of residues (1-29) of one monomer interacts with the other monomer (domain swapping), which does not occur in NiR-Pd. Here the structure of reduced NiR-Pa is described both in the unbound form and with the physiological product, NO, bound at the d1 heme active site. Although both structures are similar to that of reduced NiR-Pd, significant differences with respect to oxidized NiR-Pd were observed in two regions: (i) a loop in the c heme domain (residues 56-62) is shifted 6 A away and (ii) the hydroxide ion, which is the sixth coordination ligand of the heme, is removed upon reduction and NO binding and the Tyr10 side chain rotates away from the position adopted in the oxidized form. The conformational changes observed in NiR-Pa as the result of reduction are less extensive than those occurring in NiR-Pd. Starting with oxidized structures that differ in many respects, the two enzymes converge, yielding reduced conformations which are very similar to each other, which indicates that the conformational changes involved in catalysis are considerably diverse.     

Conformational changes in the cytoplasmic domain of the Escherichia coli aspartate receptor upon adaptive methylation

By using targeted disulfide cross-linking, we have characterized structural changes that the Escherichia coli aspartate receptor undergoes upon modification of the four specific residues that are reversibly methylated during sensory adaptation. Cysteine residues were introduced at specific positions either in the cytoplasmic domain or in the periplasmic domain, and the rates of disulfide cross-linking were used to probe for conformational changes upon covalent modification. Conversion of the methylation sites from glutamates to glutamines greatly reduced the rate of disulfide formation between residues 265 and 265' and residues 250 and 250' in the cytoplasmic domain but not between residues 36 and 36' in the periplasmic domain. (Primes are used to indicate the second of the two identical subunits in the homologous dimer.) The covalent modification of the cytoplasmic domain induces conformational changes that are detectable in the cytoplasmic domain but none that are detectable in the periplasmic domain.     

Conformational changes in the 20S proteasome upon macromolecular ligand binding analyzed with monoclonal antibodies

Proteasomes interact with a variety of macromolecular ligands that modulate their ability to degrade peptide and protein substrates. The effector PA28 increases the peptidase activities of proteasomes whereas HSP90 and alpha-crystallin inhibit a peptide-hydrolyzing activity. Four monoclonal antibodies were used as probes to detect conformational changes of proteasome subunits. Conformational changes in alpha- or beta-subunits were found upon binding PA28, HSP90, alpha-crystallin, and the substrate casein but not with the peptide substrate analogs calpain inhibitor 1 (Ac-Leu-Leu-norleucinal), calpain inhibitor 2 (Ac-Leu-Leu-methioninal), or MG 132 (N-Cbz-Leu-Leu-leucinal).     

Conformational dynamics of cytochrome P-450cam as monitored by photoacoustic calorimetry

Conformational transitions of cytochrome P-450cam following the dissociation of CO from the ferrous heme were investigated by using photoacoustic calorimetry. The effect of substrate association on the acoustic signal was also examined. Results show that the conformational dynamics of cytochrome P-450cam substrate-free protein occur faster than 10 ns, which is the time scale of the instrument response. The enthalpy and volume change for the dissociation reaction are 2.2 kcal mol-1 and 1.8 mL mol-1, respectively. Upon addition of camphor, the reaction is markedly slowed. An intermediate is formed whose lifetime is 130 ns at 17 degrees C. The overall enthalpy and volume changes are -15.9 kcal mol-1 and 10.3 mL mol-1, respectively. These results, together with published transient Raman spectra [Wells, A. V., Pusheng, L., Champion, P. M., Martinis, S. A., & Sligar, S. G. (1992) Biochemistry 31, 4384-4393] suggest that camphor leaves the heme pocket concomitant with the photoinduced expulsion of CO into the solvent and induces a considerable conformational change in the protein.     

Partial activation of the insulin receptor kinase domain by juxtamembrane autophosphorylation

Increased enzymatic activity of receptor tyrosine kinases occurs after trans-phosphorylation of one or two tyrosines in the activation loop, located near the catalytic cleft. Partial activation of the insulin receptor's kinase domain was observed at dilute concentrations of kinase, suggesting that cis-autophosphorylation was occurring. Autophosphorylation during partial activation mapped to the juxtamembrane (JM) tyrosines and not to activation loop tyrosines. Furthermore, a double JM Tyr-to-Phe mutant kinase (JMY2F) did not undergo partial activation but catalyzed substrate phosphorylation at a very low rate. Steady-state kinetics of peptide phosphorylation were determined with and without JM autophosphorylation. The JMY2F mutant was used to prevent concurrent cis-autophosphorylation and therefore to approximate the basal state apoenzyme in the kinetic analysis. Partial activation was dominated by a decreased Michaelis constant for peptide substrate, from KM,PEP >/= 2.5 mM in the basal state to 0.2 mM in the partially activated state; the KM,ATP remained virtually unchanged at approximately 1 mM, and kcat increased from 180 to 600 min-1. The high KM,PEP suggests weak binding of peptide substrates to the apoenzyme. This was confirmed by Ki > 1 mM for peptide substrates used as inhibitors of JM autophosphorylation. The absence of comparably large changes in kcat and KM,ATP suggests that the JM region is primarily a strong barrier to the peptide entry step of trans-phosphorylation reactions. The JM region therefore functions as an intrasteric inhibitor in the basal state of the insulin receptor's kinase domain.     

Conformational changes of the leukocyte NADPH oxidase subunit p47(phox) during activation studied through its intrinsic fluorescence

The leukocyte NADPH oxidase of neutrophils is a membrane-bound enzyme that catalyzes the production of O-2 from oxygen using NADPH as the electron donor. Dormant in resting neutrophils, the enzyme acquires catalytic activity when the cells are exposed to appropriate stimuli. During activation, the cytosolic oxidase components p47phox and p67phox migrate to the plasma membrane, where they associate with cytochrome b558, a membrane-integrated flavohemoprotein, to assemble the active oxidase. Oxidase activation can be mimicked in a cell-free system using an anionic amphiphile, such as sodium dodecyl sulfate or arachidonic acid, as an activating agent. In whole cells and under certain circumstances in the cell-free system the phosphorylation of p47phox mediates the activation process. It has been proposed that conformational changes in the protein structure of cytosolic factor p47phox may be an important part of the activation mechanism. We show here that the total protein steady-state intrinsic fluorescence (an emission maximum of 338 nm) exhibited by the tryptophan residues of p47phox substantially decreased when p47phox was treated with anionic amphiphiles. A similar decrease in fluorescence was also observed when p47phox was phosphorylated with protein kinase C. Furthermore, a red shift of emission maximum and an increase of quenching by ionic quenchers and acrylamide were observed in the presence of activators. These results indicate the occurrence of a conformational change in the protein structure of p47phox. We propose that this alteration in conformation results in the appearance of a binding site through which p47phox interacts with cytochrome b558 during the activation process.     

Structural changes of sarcoplasmic reticulum Ca(2+)-ATPase upon Ca2+ binding studied by simultaneous measurement of infrared absorbance changes and changes of intrinsic protein fluorescence

Ca2+ binding to sarcoplasmic reticulum Ca(2+)-ATPase was investigated by Fourier transform infrared (FTIR) spectroscopy using the photolytic release of Ca2+ from the photolabile Ca2+ chelator 1-(2-nitro-4,5-dimethoxy)-N,N,N',N',- tetrakis[(oxycarbonyl)]methyl-1,2-ethandiamine (DM-nitrophen). IR absorbance changes in 1H2O and 2H2O were detected in the spectral region from 1800 cm-1 to 1200 cm-1, reflecting photolysis of DM-nitrophen and Ca2+ binding to the Ca(2+)-ATPase. As an independent probe for protein conformational changes, intrinsic fluorescence changes upon Ca2+ release were monitored simultaneously to the FTIR measurements. Both the IR absorbance changes and the fluorescence intensity changes correlated well with the Ca2+ binding activity of the ATPase in this specific step. Ca2+ binding caused IR difference bands mainly in the region of amide I absorption of the polypeptide backbone, reflecting conformational changes of the protein. The small amplitude of the signals indicates that only a few residues perform local structural changes such as changes of bond angles or hydrogen bonding. Other absorbance changes appearing above 1700 cm-1 can be assigned to Ca2+ binding to Glu or Asp side chain carboxyl groups and concomitant deprotonation of these residues. This assignment is strengthened by downshifts of these bands by 4 cm-1 to 6 cm-1 upon 1H2O/2H2O exchange. This is in line with results of mutagenesis studies where such residues containing carboxyl groups were associated with the high affinity Ca2+ binding site (Clarke, D.M., Loo, T.W. and MacLennan, D.H. (1990) J. Biol. Chem. 265, 6262-6267).     

Conformational changes of purine repressor DNA-binding domain upon complexation with DNA

The purine repressor (PurR) consists of two functional domains: an N-terminal DNA-binding domain and a C-terminal corepressor-binding domain. Recently, the structure of PurR-corepressor-operator ternary complex was determined by X-ray crystallography. In the complex the DNA-binding domain, consisting of 56 amino acids, was composed of four helices. Here, we have determined the solution structure of the DNA-binding domain in its DNA free state by NMR. It consists of three helices and the fourth helix (the hinge helix) region is diordered. The architecture of the first three helices of its DNA free state is very similar to that of its DNA-bound form. The hinge helix is induced by the specific DNA binding and by the dimerization of PurR which is provided by the corepressor-binding domain.     

Mechanism of insulin action--signal transductions after binding to insulin receptor

Insulin binds to the alpha subunit of the insulin receptor which activates the tyrosine kinase in the beta subunit and tyrosine-phosphorylates the insulin receptor substrates-1 (IRS-1). Insulin promotes the formation of a complex between tyrosine-phosphorylated IRS-1 and several proteins including phosphoinositide(PI) 3-kinase, a heterodimer consisting of regulatory 85-kDa (p85) and catalytic 110-kDa (p110) subunits, GRB2 and Syp via the Src homology region 2 (SH2) domains. Recently, it was suggested that GRB2-Sos complex binding to IRS-1 was linked to Ras activation and that PI 3-kinase binding to IRS-1 was linked to activation of glucose transport. Since the mechanism of insulin-stimulated glucose uptake is mainly due to translocation of glucose transporters from an intracellular vesicle pool to the plasma membrane, PI 3-kinase activity may be involved in vesicle transport in mammalian cells.     

The stability and unfolding of an IgG binding protein based upon the B domain of protein A from Staphylococcus aureus probed by tryptophan substitution and fluorescence spectroscopy

The stability and unfolding of an immunoglobulin (Ig) G binding protein based upon the B domain of protein A (SpAB) from Staphylococcus aureus were studied by substituting tryptophan residues at strategic locations within each of the three alpha-helical regions (alpha 1-alpha 3) of the domain. The role of the C-terminal helix, alpha 3, was investigated by generating two protein constructs, one corresponding to the complete SpAB, the other lacking a part of alpha 3; the Trp substitutions were made in both one- and two-domain versions of each of these constructs. The fluorescence properties of each of the single-tryptophan mutants were studied in the native state and as a function of guanidine-HCl-mediated unfolding, and their IgG binding activities were determined by a competitive enzyme-linked immunosorbent assay. The free energies of folding and of binding to IgG for each mutant were compared with those for the native domains. The effect of each substitution upon the overall structure and upon the IgG binding interface was modelled by molecular graphics and energy minimization. These studies indicate that (i) alpha 3 contributes to the overall stability of the domain and to the formation of the IgG binding site in alpha 1 and alpha 2, and (ii) alpha 1 unfolds first, followed by alpha 2 and alpha 3 together.