Improved simulation of NOESY spectra by RELAX-JT2 including effects of J-coupling, transverse relaxation and chemical shift anisotropy

RELAX-JT2 is an extension of RELAX, a program for the simulation of 1H 2D NOESY spectra and (15)N or (13)C edited 3D NOESY-HSQC spectra of biological macromolecules. In addition to the already existing NOE-simulation it allows the proper simulation of line shapes by the integrated calculation of T(2) times and multiplet structures caused by J-couplings. Additionally the effects of relaxation mediated by chemical shift anisotropy are taken into account. The new routines have been implemented in the program AUREMOL, which aims at the automated NMR structure determination of proteins in solution. For a manual or automatic assignment of experimental spectra that is based on the comparison with the corresponding simulated spectra, the additional line shape information now available is a valuable aid. The new features have been successfully tested with the histidine-containing phosphocarrier protein HPr from Staphylococcus carnosus.     

Glucosylation of Ras by Clostridium sordellii lethal toxin: consequences for effector loop conformations observed by NMR spectroscopy

The lethal toxin (LT) from Clostridium sordellii, which belongs to the family of large clostridial cytotoxins, acts as a monoglucosyltransferase for the Rho subfamily GTPase Rac and also modifies Ras. In the present study we investigated structural changes of H-Ras in its di- and triphosphate form that occur upon glucosylation of the effector domain amino acid threonine-35 by LT. (31)P NMR experiments recorded during the enzymatic glucosylation process, using UDP-glucose as a cosubstrate, show that the modification of the threonine side chain influences the chemical shifts of the phosphate groups of the bound nucleotides. In the diphosphate-bound form (Ras.GDP) glucosylation of Thr35 induces only small changes in the chemical environment of the active center. In the triphosphate form with the GTP analogue GppNHp bound (Ras.GppNHp) Ras shows at least two different conformations in the active center that exchange on a medium-range time scale (10 to 0.1 ms). Glucosylation selectively stabilizes one distinct conformation of the effector loop (state 1) with tyrosine-32 probably apart from the nucleotide and threonine-35 not involved in magnesium ion coordination. This conformation is known to have a low affinity to effector proteins such as Raf-1, AF-6, or Byr2 and thus prevents the transduction of the activation signal in the Ras-mediated pathway. NMR correlation spectra of Ras(T35glc).GDP and denaturation experiments with urea indicate that the glucose is bound in the alpha-anomeric form to the hydroxyl group of the threonine-35 side chain. Inhibition of the glucosylation reaction by 1,5-gluconolactone suggests a stereospecific reaction mechanism with a glucosyl oxonium ion transition state for the enzymatic activity of LT.     

Structure determination and ligand interactions of the PDZ2b domain of PTP-Bas (hPTP1E): splicing-induced modulation of ligand specificity

Two versions of the PDZ2 domain of the protein tyrosine phosphatase PTP-Bas/human PTP-BL are generated by alternative splicing. The domains differ by the insertion of five amino acid residues and their affinity to the tumour suppressor protein APC. Whereas PDZ2a is able to bind APC in the nanomolar range, PDZ2b shows no apparent interaction with APC. Here the solution structure of the splicing variant of PDZ2 with the insertion has been determined using 2D and 3D heteronuclear NMR experiments. The structural reason for the changed binding specificity is the reorientation of the loop with extra five amino acid residues, which folds back onto beta-strands two and three. In addition the side-chain of Lys32 closes the binding site of the APC binding protein and the two helices, especially alpha-helix 2, change their relative position to the protein core. Consecutively, the binding site is sterically no longer fully accessible. From the NMR-titration studies with a C-terminal APC-peptide the affinity of the peptide with the protein can be estimated as 540(+/-40)microM. The binding site encompasses part of the analogous binding site of PDZ2a as already described previously, yet specific interaction sites are abolished by the insertion of amino acids in PDZ2b. As shown by high-affinity chromatography, GST-PDZ2b and GST-PDZ2a bind to phosphatidylinositol 4,5-bisphosphate (PIP(2)) micelles with a dissociation constant K(D) of 21 microM and 55 microM, respectively. In line with these data PDZ2b binds isolated, dissolved PIP(2) and PIP(3) (phosphatidylinositol 3,4,5-trisphosphate) molecules specifically with a lower K(D) of 230(+/-20)microM as detected by NMR spectroscopy. The binding site could be located by our studies and involves the residues Ile24, Val26, Val70, Asn71, Gly77, Ala78, Glu85, Arg88, Gly91 and Gln92. PIP(2) and PIP(3) binding takes place in the groove of the PDZ domain that is normally part of the APC binding site.     

Molecular alignment of proteins in bicellar solutions: quantitative evaluation of effects induced in 2D COSY spectra

Partial molecular alignment leads to an incomplete averaging of anisotropic magnetic interactions such as magnetic dipole interaction or chemical shift anisotropy. In the present contribution we quantitatively describe and evaluate the effects induced by the addition of magnetically oriented lipid bicelles in homonuclear two-dimensional (2D) NMR correlation (COSY) spectra of proteins. It is shown that 2D COSY experiments allow the measurement of H(N)-H(alpha) residual dipole couplings of positive sign which can be used for structure refinement. In contrast to the double- and triple-resonance experiments previously proposed, these measurements can be carried out even on nonisotope-enriched samples.     

Pressure-induced local unfolding of the Ras binding domain of RalGDS

The reliable prediction of the precise three-dimensional structure of proteins from their amino acid sequence is a major, still unresolved problem in biochemistry. Pressure is a parameter that controls folding/unfolding transitions of proteins through the volume change DeltaV of the protein-solvent system. By varying the pressure from 30 to 2,000 bar we detected using 15N/ 1H 2D NMR spectroscopy a unique equilibrium unfolding intermediate I in the Ras binding domain of the Ral guanine nucleotide dissociation stimulator (Ral GDS). It is characterized by a local melting of specific structural elements near hydrophobic cavities while the overall folded structure is maintained.     

AUREMOL-RFAC-3D,combination of R-factors and their use for automated quality assessment of protein solution structures

We present here the computer program AUREMOL-RFAC-3D that is a generalization of the previously published program RFAC for the fully automated estimation of residual indices (R-factors) from 2D NOESY spectra. It is part of the larger AUREMOL software package (www.auremol.de). RFAC-3D calculates R-factors directly from two-dimensional homonuclear NOESY spectra as well as from three-dimensional ¹⁵N or ¹³C edited NOESY-HSQC spectra and thus extends the application range to larger proteins. The fully automated method includes automated peak picking and integration, a Bayesian noise and artifact recognition and the use of the complete relaxation matrix formalism. To enhance the reliability of the calculated R-factors the method is also generalized to calculate combined R-factors from a set of 2D and 3D-spectra. For an optimal combination of the information derived from different sources a plausible formalism had to be derived. In addition, we present a novel direct R-factors based measure that correlates an R-factors as defined in this paper to the root mean square deviation of the actual structure from the optimal structure. The new program has been successfully tested on the histidine containing phosphocarrier protein (HPr) from Staphylococcus carnosus and on the Ras-binding domain (RBD) of the Ral guanine-nucleotide dissociation stimulation factor (RalGDS).     

Molecular dynamics simulations of HPr under hydrostatic pressure

The histidine-containing protein (HPr) plays an important role in the phosphotransferase system (PTS). The deformations induced on the protein structure at high hydrostatic pressure values (4, 50, 100, 150, and 200 MPa) were previously (H. Kalbitzer, A. G?rler, H. Li, P. Dubovskii, A. Hengstenberg, C. Kowolik, H. Yamada, and K. Akasaka, Protein Science 2000, Vol. 9, pp. 693-703) analyzed by NMR experiments: the nonlinear variations of the amide chemical shifts at high pressure values were supposed to arise from induced shifts in the protein conformational equilibrium. Molecular dynamics (MD) simulations are here performed, to analyze the protein internal mobility at 0.1 MPa, and to relate the nonlinear variations of chemical shifts observed at high pressure, to variations in conformational equilibrium. The global features of the protein structure are only slightly modified along the pressure. Nevertheless, the values of the Voronoi residues volumes show that the residues of alpha-helices are more compressed that those belonging to the beta-sheet. The alpha-helices are also displaying the largest internal mobility and deformation in the simulations. The nonlinearity of the 1H chemical shifts, computed from the MD simulation snapshots, is in qualitative agreement with the nonlinearity of the experimentally observed chemical shifts.     

Stereochemistry and lifetime of the GTP hydrolysis intermediate at the active site of elongation factor Tu from Bacillus stearothermophilus as inferred from the 17O-55Mn superhyperfine interaction

Electron paramagnetic resonance spectroscopy has been used to obtain information on the structure and stability of the products of GTP cleavage at the active site of elongation factor Tu (EF-Tu) from Bacillus stearothermophilus. Using stereospecifically labelled (Sp)-(Rp)-[beta-17O]GTP (prepared by modification of a previously published procedure which is now also suitable for guanine nucleotides), it was found that only one of the two possible diastereomers (Sp) led to detectable line-broadening of the EPR spectrum of Mn2+ at the active site of EF-Tu (linewidth 1.5 mT), whereas the Rp isomer caused the same linewidth as unlabelled nucleotide (1.3 mT). From our earlier work and from a demonstration that the lifetime of the state giving the broadened spectrum is too long to be assigned to the EF-Tu.GDP.Mn complex [the rate constant for decay as measured by displacement of GDP by the fluorescent 2'(3')-O-(N-methylanthraniloyl)-GDP is 6.2 x 10(-3) s-1 at 25 degrees C and pH 6.8], we conclude that the broadened signal arises from the EF-Tu.Mn.GDP.Pi complex, the predominant steady-state species. During the hydrolysis of GTP the Mn2+ remains bound to the beta-phosphate oxygen of GDP which arises from the beta pro-S oxygen of GTP, possibly until GDP dissociates and certainly until Pi dissociates. Addition of elongation factor Ts (EF-Ts) to this intermediate leads to rapid reduction of the linewidth to that expected for random distribution of interactions of one 17O and two 16O atoms of GDP with Mn2+, and is not distinguishable from that exhibited by (Rp)-[beta-17O]GTP in the corresponding complex in the presence of EF-Ts.     

A general method for the unbiased improvement of solution NMR structures by the use of related X-Ray data, the AUREMOL-ISIC algorithm

Background  

Rapid and accurate three-dimensional structure determination of biological macromolecules is mandatory to keep up with the vast progress made in the identification of primary sequence information. During the last few years the amount of data deposited in the protein data bank has substantially increased providing additional information for novel structure determination projects. The key question is how to combine the available database information with the experimental data of the current project ensuring that only relevant information is used and a correct structural bias is produced. For this purpose a novel fully automated algorithm based on Bayesian reasoning has been developed. It allows the combination of structural information from different sources in a consistent way to obtain high quality structures with a limited set of experimental data. The new ISIC (Intelligent Structural Information Combination) algorithm is part of the larger AUREMOL software package.     

Automated assignment of NOESY NMR spectra using a knowledge based method (KNOWNOE)

Automated assignment of NOESY spectra is a prerequisite for automated structure determination of biological macromolecules. With the program KNOWNOE we present a novel, knowledge based approach to this problem. KNOWNOE is devised to work directly with the experimental spectra without interference of an expert. Besides making use of routines already implemented in AUREMOL, it contains as a central part a knowledge driven Bayesian algorithm for solving ambiguities in the NOE assignments. These ambiguities mainly arise from chemical shift degeneration which allows multiple assignments of cross peaks. Using a set of 326 protein NMR structures, statistical tables in the form of atom-pairwise volume probability distributions (VPDs) were derived. VPDs for all assignment possibilities relevant to the assignments of interproton NOEs were calculated. With these data for a given cross peak with N possible assignments A _i(i = 1,...,N) the conditional probabilities P(A _i, a|V ₀) can be calculated that the assignment A _idetermines essentially all (a-times) of the cross peak volume V ₀. An assignment A _kwith a probability P(A _k, a|V ₀) higher than 0.8 is transiently considered as unambiguously assigned. With a list of unambiguously assigned peaks a set of structures is calculated. These structures are used as input for a next cycle of iteration where a distance threshold D _maxis dynamically reduced. The program KNOWNOE was tested on NOESY spectra of a medium size protein, the cold shock protein (TmCsp) from Thermotoga maritima. The results show that a high quality structure of this protein can be obtained by automated assignment of NOESY spectra which is at least as good as the structure obtained from manual data evaluation.