Localization of the binding site for modified Gb3 on verotoxin 1 using fluorescence analysis.

Verotoxins (VTs) from Escherichia coli elicit human vascular disease as a consequence of specific binding to globotriaosylceramide (Gb3) receptors on endothelial cell surfaces. Molecular models based on the VT1 crystal structure were used previously to investigate the structural basis for receptor recognition by VT1 and other verotoxins. Interestingly, these model-based predictions of glycolipid binding to VT1 differ somewhat from recently published structural data from cocrystals of the VT1 B-subunit (VT1B) and an analogue of the sugar moiety of Gb3. In this study, fluorescence spectroscopy was used to test model-based predictions of the location of Gb3 binding on the B-subunit pentamer of VT1. Resonance energy transfer was used to calculate the distance from a coumarin probe used to replace the acyl tail of Gb3 and the single tryptophan residue (Trp34) present within each VT1B monomer. The observed energy transfer efficiency (greater than 95%) suggests that these two moieties are approximately 13.3 A apart when a single distance is assumed. This distance is consistent with proposed models for the fit of Gb3 within the "cleft site" of the VT1 B-subunit. When the distances from Trp34 to the other coumarinGb3 molecules (bound to each of the four remaining monomers within the VT1B pentamer) are taken into consideration, it appears likely that the coumarin-modified Gb3 analogue used in this study associates with the previously proposed receptor binding site II of VT1. This is consistent with an observed binding preference of VT2c for coumarinGb3. To provide additional information on the association of Gb3 with the VT1 B-subunit, the influence of Gb3 glycolipid binding on the accessibility of Trp34 to different quenching agents in solution was then examined. Taken together, the data suggest that coumarin-labeled Gb3 preferentially binds to site II on VT1 in a position that is consistent with the previously described molecular models.     

NMR studies of RNA dynamics and structural plasticity using NMR residual dipolar couplings

An increasing number of RNAs are being discovered that perform their functions by undergoing large changes in conformation in response to a variety of cellular signals, including recognition of proteins and small molecular targets, changes in temperature, and RNA synthesis itself. The measurement of NMR residual dipolar couplings (RDCs) in partially aligned systems is providing new insights into the structural plasticity of RNA through combined characterization of large-amplitude collective helix motions and local flexibility in noncanonical regions over a wide window of biologically relevant timescales (相似文献   

Localization of the binding site on fibrin for the secondary binding site of thrombin

Affinity chromatography of active site inhibited thrombin on immobilized fragments derived from the central (desAB-NDSK) and terminal (D1) globular domains of fibrinogen revealed that the site responsible for the binding of thrombin at its secondary fibrin binding site is located in the central domain. Chromatography of various domains of the central nodule (desAB-NDSK, fibrinogen E, and fibrin E) having nonidentical amino acid sequences showed that all of these fragments are capable of binding to PMSF-thrombin-Sepharose, suggesting that the thrombin binding site resides within the peptide regions common to all of these fragments: alpha(Gly17-Met51), beta(Val55-Met118), and gamma(Tyr1-Lys53). Competitive affinity chromatography of the same binding domains revealed that there is no detectable difference in their binding constants to PMSF-thrombin-Sepharose, indicating that the alpha(Lys52-Lys78), beta(Gly15-Lys54)/(Tyr119-Lys122), and gamma(Thr54-Met78) peptide segments do not contribute significantly to the binding of thrombin. Chromatography of the isolated chains of fibrinogen E showed that the alpha(Gly17-Lys78) peptide region itself contains a strong binding site for PMSF-thrombin-Sepharose. The location of the binding site suggests that the secondary site interaction may play an important role in determining the cleavage specificity of thrombin on fibrinogen and can affect the rate of release of the fibrinopeptides. Affinity chromatography of fragments prepared from polymerized fibrin showed that cross-linked DD (D x D) itself does not bind to thrombin, whereas the D x DE complex remained attached to the column, suggesting that the binding site on fragment E for thrombin is distinct from its binding site for D x D.(ABSTRACT TRUNCATED AT 250 WORDS)     

The importance of being ordered: improving NMR structures using residual dipolar couplings

Residual dipolar couplings arise from small degrees of alignment of molecules in a magnetic field. Most biomolecules lack sufficient intrinsic magnetic susceptibility anisotropies for practical purposes; however, alignment can be achieved using dilute aqueous phospholipid mixtures, colloidal suspensions of rod-shaped viruses, complex phases of surfactant systems and strained gels. The stability of the liquid crystalline phases varies with respect to temperature range, pH variation and time and is critically dependent on sample composition and experimental conditions. The magnitude of the residual dipolar couplings depends upon the degree of ordering and allows the determination of the corresponding inter-nuclear vectors with respect to the molecule's alignment frame. Inclusion of dipolar constraints into NMR structure calculations leads to improved precision and accuracy of the resulting structures, especially in cases where the information content provided by traditional NOE constraints is limited. In addition, rapid evaluation of backbone protein folds and determination of the relative orientations of individual components in multi-molecular complexes have become feasible. Dipolar coupling based strategies may well emerge as the most critical developments, in establishing NMR as a valuable and competitive methodology in the structural genomics initiative.     

Characterizing the relative orientation and dynamics of RNA A-form helices using NMR residual dipolar couplings

We present a protocol for determining the relative orientation and dynamics of A-form helices in 13C/15N isotopically enriched RNA samples using NMR residual dipolar couplings (RDCs). Non-terminal Watson-Crick base pairs in helical stems are experimentally identified using NOE and trans-hydrogen bond connectivity and modeled using the idealized A-form helix geometry. RDCs measured in the partially aligned RNA are used to compute order tensors describing average alignment of each helix relative to the applied magnetic field. The order tensors are translated into Euler angles defining the average relative orientation of helices and order parameters describing the amplitude and asymmetry of interhelix motions. The protocol does not require complete resonance assignments and therefore can be implemented rapidly to RNAs much larger than those for which complete high-resolution NMR structure determination is feasible. The protocol is particularly valuable for exploring adaptive changes in RNA conformation that occur in response to biologically relevant signals. Following resonance assignments, the procedure is expected to take no more than 2 weeks of acquisition and data analysis time.     

Determination of protein global folds using backbone residual dipolar coupling and long-range NOE restraints

We report the determination of the global fold of human ubiquitin using protein backbone NMR residual dipolar coupling and long-range nuclear Overhauser effect (NOE) data as conformational restraints. Specifically, by use of a maximum of three backbone residual dipolar couplings per residue (N_i-H^N _i, N_i-C_i–1, H^N _i - C_i–1) in two tensor frames and only backbone H^N-H^N NOEs, a global fold of ubiquitin can be derived with a backbone root-mean-square deviation of 1.4 Å with respect to the crystal structure. This degree of accuracy is more than adequate for use in databases of structural motifs, and suggests a general approach for the determination of protein global folds using conformational restraints derived only from backbone atoms.     

NMR experiments for the sign determination of homonuclear scalar and residual dipolar couplings

A modified version of the JHH-TOCSY experiment, `signed COSY', is presented that allows the determination of the sign of residual dipolar ¹H-¹H coupling constants with respect to the sign of one-bond ¹H-X coupling constants in linear three-spin systems X-¹H-¹H, where X = ¹³C or ¹⁵N. In contrast to the original JHH-TOCSY experiments, the signs of J _HH couplings may be determined for CH₂-CH₂ moieties and for uniformly ¹³C/¹⁵N-labelled samples. In addition, sensitivity is enhanced, diagonal peaks are suppressed and cross peaks are observed only between directly coupled protons, as in a COSY spectrum.     

Localization of the ATP binding site on alpha-tubulin

The binding site for ATP to tubulin was established by use of the photoaffinity label [gamma-32P]N3ATP. Photolysis of the analog in the presence of tubulin resulted in covalent modification of the protein as revealed by autoradiography of electropherograms. Scanning the autoradiograms showed that the ATP analog was bound mainly to the alpha subunit of the tubulin dimer; the alpha subunit was two to three times more radioactive than was the beta subunit. The location of a particular site on the alpha subunit was further defined by peptide maps. The alpha and beta subunits from affinity-labeled tubulin were separated and digested with Staphylococcus protease. Radioactivity was found predominantly in one peptide band from the alpha subunit. The location of the [gamma-32P]N3ATP binding site on the alpha subunit distinguishes it from the previously known exchangeable GTP binding site which is on the beta subunit. Moreover, excess GTP did not compete with [gamma-32P]N3ATP binding. The ATP binding site is distinct from the nonexchangeable GTP binding site. The GTP content of tubulin was the same after dialysis in 0.5 mM ATP as it was following dialysis against ATP-free buffer. Proof that the binding site for [gamma-32P]N3ATP is the same as that for ATP was obtained by competition experiments. In the presence of ATP, photolysis of the affinity analog did not label the alpha subunit preferentially.     

Refined structure of a flexible heptasaccharide using 1H-13C and 1H-1H NMR residual dipolar couplings in concert with NOE and long range scalar coupling constants

The heptasaccharide isolated from the cell wall polysaccharide of Streptococcus mitis J22 serves as an important model for the dynamics and conformation of complex polysaccharides, illustrating the nature of flexibility with rigid epitopes joined by flexible hinges. One-bond C-H residual dipolar couplings (¹D_CH) and long-range H-H residual dipolar couplings (ⁿD_HH) were measured for the heptasaccharide in a cetylpyridinium chloride/hexanol/brine lamellar liquid crystal medium. A method is proposed to determine the ⁿD_HH in natural abundance based on a ¹³C resolved ¹H TOCSY pulse sequence previously published to determine the homonuclear scalar couplings. Different methods for interpretation of the ¹D_CH and the ⁿD_HH residual dipolar coupling data obtained were compared and combined with the NOE and long-range H,C and C,C scalar couplings available for this heptasaccharide. A flexible model of the heptasaccharide was determined in which two structurally well-defined regions involving four and two sugar residues, respectively are joined by a flexible hinge which involves two 16 glycosidic linkages.     

Extended binding site of ricin B lectin for oligosaccharide recognition

The plant lectin ricin B chain binds oligosaccharide with more affinity than the mono- or disaccharide ligands. The experiments indicated that a biantennary oligosaccharide could bind itself to any of the crystallographically established 1st or 2nd binding sites. After manual docking of either terminal galactose residues of the oligosaccharide in the 1st and 2nd binding sites of Ricin B and simulating the systems over nanosecond trajectories in implicit solvent, it was observed that the protein bound the oligosaccharide strongly through both its 1st and 2nd binding sites. Not only were the terminal galactose residues, several other residues of the oligosaccharide were involved in the binding scheme. Average gas phase energies were calculated molecular mechanically, solvation energies were calculated by Generalized Born model and the normal mode analysis was used to calculate the entropic contribution of binding. The entropy/enthalpy compensation has been observed for the protein-oligosaccharide interactions. The binding was found to be enthalpically favorable and compensating for the unfavorable entropic contribution. Comparison of the calculated free energy with the experimental data clearly suggests that binding is mono-dentate rather than bi-dentate through a single Gal-containing antenna.     

Conformational studies of Lewis X and Lewis A trisaccharides using NMR residual dipolar couplings.

The conformations of the histo-blood group carbohydrate antigens Lewis X (Le(x)) and Lewis A (Le(a)) were studied by NMR measurements of one-bond C-H residual dipolar couplings in partially oriented liquid crystal solutions. A strategy for rapid calculation of the difference between theoretical and experimental dipolar couplings of a large number of model structures generated by computer simulations was developed, resulting in an accurate model structure for the compounds. Monte Carlo simulations were used to generate models for the trisaccharides, and orientations of each model were sought that could reproduce the experimental residual dipolar coupling values. For both, Le(a) and Le(x), single low energy models giving excellent agreement with experiment were found, implying a compact rigidly folded conformation for both trisaccharides. The new approach was also applied to the pentasaccharides lacto-N-fucopentaose 2 (LNF-2) and lacto-N-fucopentaose 3 (LNF-3) proving its consistency and robustness. For describing the conformation of tightly folded oligosaccharides, a definition for characterization of ring planes in pyranoside chairs is proposed and applied to the analysis of the relation between the fucose and galactose residues in the epitopes, revealing the structural similarity between them.     

Localization of the tubulin binding site for tau protein

Limited proteolysis of tubulin with subtilisin resulted in the removal of the carboxyl-terminal moiety of tubulin subunits. The remaining peptides from both alpha and beta tubulin lacking the carboxyl terminal did not bind to tau factor nor to MAP2 or MAP1. The carboxyl-terminal fragments bind to tau factor and MAP2 and both compete for the same binding sites in the tubulin molecule. Our results suggest that the carboxyl-terminal region of tubulin is a regulatory domain for the assembly of tubulin and the site for interaction with MAPs.     

Mg2+-induced variations in the conformation and dynamics of HIV-1 TAR RNA probed using NMR residual dipolar couplings

The effects of divalent Mg(2+) on the conformation and dynamics of the stem-loop transactivation response element (TAR) RNA from HIV-1 have been characterized using NMR residual dipolar couplings (RDCs). Order matrix analysis of one bond 13C-1H RDCs measured in TAR at [Mg(2+)]:[TAR] stoichiometric ratios of approximately 3:1 (TAR(3.0Mg)) and approximately 4.5:1 (TAR(4.5Mg)) revealed that Mg(2+) reduces the average inter-helical angle from 47(+/-5) degrees in TAR(free) to 5(+/-7) degrees in TAR(4.5Mg). In contrast to the TAR(free) state, the generalized degree of order for the two stems in TAR(4.5Mg) is found to be identical within experimental uncertainty, indicating that binding of Mg(2+) leads to an arrest of inter-helical motions in TAR(free). Results demonstrate that RDC-NMR methodology can provide new insight into the effects of Mg(2+) on both the conformation and dynamics of RNA.     

Transverse relaxation optimised spin-state selective NMR experiments for measurement of residual dipolar couplings

Three transverse relaxation optimised NMR experiments (TROSY) for the measurement of scalar and dipolar couplings suitable for proteins dissolved in aqueous iso- and anisotropic solutions are described. The triple-spin-state-selective experiments yield couplings between ¹H^N-¹³C, ¹⁵N-¹³C, ¹H^N-¹³C _i–1, ¹⁵N-¹³C _i–1, ¹H^N-¹³C_i–1, ¹⁵N-¹³C_i–1, and ¹³C_i–1-¹³C _i–1 without introducing nonessential spectral crowding compared with an ordinary two-dimensional ¹⁵N-¹H correlation spectrum and without requiring explicit knowledge of carbon assignments. This set of /-J-TROSY experiments is most useful for perdeuterated proteins in studies of structure–activity relationships by NMR to observe, in addition to epitopes for ligands, also conformational changes induced by binding of ligands.     

Conformational studies of human milk oligosaccharides using (1)H-(13)C one-bond NMR residual dipolar couplings

1H-(13)C one-bond dipolar coupling values were measured for natural abundance samples of the human milk oligosaccharides "lacto-N-fucopentaose" (LNF-1 LNF-2, and LNF-3), "lacto-N-difucohexaose" (LND-1), "lacto-N-tetraose" (LNT), and "lacto-N-neo-tetraose" (LNnT), four of which have Lewis blood group epitopes. Each oligosaccharide was dissolved in a 7.5% solution of 1, 2-dimyristoyl-sn-glycero-3-phosphocholine/1, 2-dihexanoyl-sn-glycero-3-phosphocholine (DMPC/DHPC) bicelle liquid crystals oriented in the NMR magnetic field. The dipolar coupling data and NOE were fitted to conformational models with calculations of an optimum orientation tensor which best represents the dipolar coupling values for a fragment hypothesized to adopt a single conformation. In the case of LNF-1, LNF-2, LNF-3, and LND-1, the models confirm previous conformational models for the Lewis epitopes based on NOE and molecular dynamics simulations. Extensions of the model provided new structural data for the remaining residues. In all cases, upper limits for the errors in the glycosidic angles of the models were estimated. Since residual dipolar coupling provides information on long-range order, it is a valuable complement to other types of NMR data such as NOE and scalar coupling for exploring conformations of complex oligosaccharides.     

The three-dimensional structure of the gallium complex of azoverdin,a siderophore of Azomonas macrocytogenes ATCC 12334, determined by NMR using residual dipolar coupling constants

In iron-deficient conditions, Azomonas macrocytogenes ATCC 12334 excretes a fluorescent siderophore called azoverdin, which is composed of a six-amino-acid peptide chain linked to a chromophore. Azoverdin chelates iron(III) very strongly, solubilizing it and transporting it back into the cells using an outer-membrane receptor. This compound is related to the pyoverdins, the peptidic siderophores of Pseudomonas, but differs in the site on the chromophore at which the peptide is covalently linked. This feature identifies azoverdin as a member of a new class of pyoverdins: the isopyoverdins. We report the three-dimensional structure of azoverdin-Ga(III) in solution. The use of orientational constraints obtained from the measurement of residual dipolar couplings using samples dissolved in a liquid crystalline medium allowed us to define the absolute configuration of the metal complex, which is Delta. The structure is characterized by a U-shape adopted by the peptide chain, with the N(delta)-acetyl-N(delta)-hydroxyornithine side chains adopting extended conformations in order to chelate the gallium ion. This conformation leaves a large open space permitting access to the gallium ion. The structural consequences of the particular isopyoverdin chemical structure are discussed in the context of the three-dimensional structures of other pyoverdins.     

Localization of a cryptic binding site for tenascin on fibronectin

Fibronectin and tenascin are large extracellular matrix proteins that interact with each other and with integrin receptors to regulate cell growth and movement. They are both modular proteins composed of independently folded domains (modules) that are arranged in linear fashion. Fibronectin is a covalent dimer and tenascin is a hexamer. The site on tenascin to which fibronectin binds has been localized to type III modules 3-5. In this study we use surface plasmon resonance to examine the interaction between various fragments of fibronectin and tenascin to further characterize and localize the binding sites. We found that tenascin fragments that contain type III modules 3-5 bind primarily to the N-terminal 29-kDa hep-1/fib-1 domain, which contains the first five type I modules of fibronectin. The dissociation constant, K(d), is approximately 1 microm. The binding site on fibronectin appears to be cryptic in the whole molecule in solution but is exposed on the proteolytic fragments and probably when fibronectin is in the extended conformation.     

Conformational studies of blood group A and blood group B oligosaccharides using NMR residual dipolar couplings

The conformations of two synthetic trisaccharides of blood group A and B (alpha-L-Fucp-(1-->2)-[alpha-D-GalpNAc-(1-->3)]-alpha-D-Galp and alpha-L-Fucp-(1-->2)-[alpha-D-Galp-(1-->3)]-alpha-D-Galp, respectively) and of a type A tetrasaccharide alditol, Fucp-(1-->2)-[alpha-D-GalpNAc-(1-->3)]-beta-D-Galp-(1-->3)-GalNAc-ol, were studied by NMR measurements of one-bond C-H residual dipolar couplings in partially oriented liquid crystal solutions. The conformations of the three oligosaccharides were analyzed by generating thousands of structures using a Monte-Carlo method. Two different strategies were applied to calculate theoretical dipolar couplings for these structures. In the first method, the orientation of the molecule was calculated from the optimal fit of the molecular model to the experimental data, while in the second method the orientation tensor was calculated directly from the moment of inertia of the molecular model. Both methods of analysis give similar results but with slightly better agreement with experiment for the former one. The analysis of the results implies a single unique conformation for both blood group epitopes in solution in disagreement with theoretical models suggesting the existence of two conformers in solution.