Determination of the complete three-dimensional structure of the trypsin inhibitor from squash seeds in aqueous solution by nuclear magnetic resonance and a combination of distance geometry and dynamical simulated annealing

The complete three-dimensional structure of the trypsin inhibitor from seeds of the squash Cucurbita maxima in aqueous solution was determined on the basis of 324 interproton distance constraints, 80 non-nuclear Overhauser effect distances, and 22 hydrogen-bonding constraints, supplemented by 27 phi backbone angle constraints derived from nuclear magnetic resonance measurements. The nuclear magnetic resonance input data were converted to the distance constraints in a semiquantitative manner after a sequence specific assignment of 1H spectra was obtained using two-dimensional nuclear magnetic resonance techniques. Stereospecific assignments were obtained for 17 of the 48 prochiral centers of the squash trypsin inhibitor using the floating chirality assignment introduced at the dynamical simulated annealing stage of the calculations. A total of 34 structures calculated by a hybrid distance geometry-dynamical simulated annealing method exhibit well-defined positions for both backbone and side-chain atoms. The average atomic root-mean-square difference between the individual structures and the minimized mean structure is 0.35(+/- 0.08) A for the backbone atoms and 0.89(+/- 0.17) A for all heavy atoms. The precision of the structure determination is discussed and correlated to the experimental input data.     

Structural heterogeneity in the core oligosaccharide of the S-layer glycoprotein from Aneurinibacillus thermoaerophilus DSM 10155.

The surface layer glycoprotein of Aneurinibacillus thermoaerophilus DSM 10155 has a total carbohydrate content of 15% (by mass), consisting of O-linked oligosaccharide chains. After proteolytic digestion of the S-layer glycoprotein byPronase E and subsequent purification of the digestion products by gel permeation chromatography, chromatofocusing and high-performance liquid chromatography two glycopeptide pools A and B with identical glycans and the repeating unit structure -->4)-alpha-l-Rha p -(1-->3)-beta-d- glycero -d- manno -Hep p -(1--> (Kosma et al., 1995b, Glycobiology, 5, 791-796) were obtained. Combined evidence from modified Edman-degradation in combination with liquid chromatography electrospray mass-spectrometry and nuclear magnetic resonance spectroscopy revealed that both glycopeptides contain equal amounts of the complete core structure alpha-l-Rha p -(1-->3)-alpha-l-Rha p -(1-->3)-beta-d-Gal p NAc-(1-->O)-Thr/Ser and the truncated forms alpha-l-Rha p -(1-->3)-beta-d-Gal p NAc-(1-->O)-Thr/Ser and beta-d-Gal p NAc-(1-->O)-Thr/Ser. All glycopeptides possessed the novel linkage types beta-d-Gal p NAc-(1-->O)-Thr/Ser. The different cores were substituted with varying numbers of disaccharide repeating units. By 300 MHz proton nuclear magnetic resonance spectroscopy the complete carbohydrate core structure of the fluorescently labeled glyco-peptide B was determined after Smith-degradation of its glycan chain. The NMR data confirmed and complemented the results of the mass spectroscopy experiments. Based on the S-layer glycopeptide structure, a pathway for its biosynthesis is suggested.     

Quantitative determination of conformations of flexible molecules in solution using lanthanide ions as nuclear magnetic resonance probes: application to adenosine-5'-monophosphate

A procedure is described here whereby the conformation, of a flexible molecule in solution can be found. The method depends on the study of the nuclear magnetic resonance spectrum of the molecule in the presence of perturbations due to specifically bound lanthanide cations. The magnetic perturbations are of two kinds: shifts of nuclear magnetic resonance spectral lines in the presence of cations such as Eu³⁺ and changes in relaxation rates of the nuclear magnetic resonance excitations in the presence of cations such as Gd³⁺. Suitable expressions are given for the relation between the magnitude of the perturbations and the geometry of the lanthanide complex in the absence of through-bond perturbations and for an axially symmetric system. It is proved that the spectral changes described here are not due to through-bond (contact) effects. The circumstances, in which the anisotropy of the magnetic susceptibility tensor, as seen in the nuclear magnetic resonance spectra, is of axial symmetry, are defined. The experimental systems described are of this kind. A computer program has been devised that searches for the conformations of the molecule which fit the nuclear magnetic resonance data.We outline here the principles of the method and how we have used a combination of relaxation and shift probes to obtain the conformation of adenosine-5′-monophosphate at pH 2. It is shown that a small family of closely related conformations fit the nuclear magnetic resonance data. These conformations are very similar to that of the crystal structure of AMP.     

NMR-derived folate-bound structure of dihydrofolate reductase 1 from the halophile Haloferax volcanii

Folate binds to dihydrofolate reductase (DHFR) to form a binary complex whose structure maintains the overall configuration of the enzyme; however, some significant changes are evident when a comparison is made to the enzyme. The structure of DHFR1 from the halophilic Halopherax volcanii was solved in its folate-bound form using nuclear magnetic resonance spectroscopy. NOE data obtained from the (15)N-NOESY-HSQC and (13)C-NOESY-HSQC experiments of the triply labeled ((1)H, (13)C, and (15)N) binary complex were used as input for the structure calculation with the Crystallography and Nuclear Magnetic Resonance System program. The resulting family of structures was compared with the enzyme solved by both nuclear magnetic resonance and X-ray crystallography and also to the mesophilic folate-bound enzyme from Escherichia coli. The binary complex exhibited less convergence of structure in the alpha2-helix and differences in the hinge residues D39 and A94. In comparison to the previously reported mesophilic binary complex solved by X-ray crystallography, the halophilic binary complex reported here does not agree with the convergence of the M20 loop to a single structure. The corresponding L21 loop of the halophilic binary complex family of structures solved by nuclear magnetic resonance indicates variability in this region.     

Three-dimensional structure of porcine C5adesArg from 1H nuclear magnetic resonance data

Two-dimensional nuclear magnetic resonance spectra of porcine C5adesArg (73 residues) have been used to construct a list of 34 hydrogen bonds, 27 dihedral angle constraints, and 151 distance constraints, derived from nuclear Overhauser effect data. These constraints were used in restrained molecular dynamics calculations on residues 1-65 of C5a, starting from a folded structure modeled on the crystal structure of a homologous protein, C3a. Forty-one structures have been calculated, which fall into three similar families with few violations of the imposed constraints. Structures in the most populated family have a root-mean-square deviation from the average structure of 1.02 A for the C alpha atoms, with good definition of the internal residues. There is good agreement between the calculated structures and other nuclear magnetic resonance data. The structure is very similar to that recently reported for human C5a [Zuiderweg et al. (1989) Biochemistry 28, 172-185]. Some biological implications of these structures are discussed.     

Solution structure of the kringle 4 domain from human plasminogen by 1H nuclear magnetic resonance spectroscopy and distance geometry

Kringle 4 is an autonomous structural and folding domain within the proenzyme plasminogen. Homologous domains are found throughout the blood clotting and fibrinolytic proteins. In this paper, we present the almost complete assignment of the 1H nuclear magnetic resonance (n.m.r.) spectrum of the kringle 4 domain of human plasminogen. A detailed structural analysis has been completed. The sequential pattern of nuclear Overhauser enhancements indicated little regular secondary structure but rather a series of turns and loops connecting beta-strands. A small stretch of antiparallel beta-sheet was identified between the residues 61 to 63 and 71 to 73 and the close proximity of other strands was determined from two-dimensional nuclear Overhauser enhancement spectra. Slowly exchanging amide (NH) resonances were found to be associated with residues of the beta-sheet and neighbouring strands that support the hydrophobic core of the domain. A total of 526 interproton distance constraints and two hydrogen bonds were specified as input to the distance geometry program DISGEO. Tertiary structures were produced that were consistent with the n.m.r. data. The structures were compared with that of our earlier model based on n.m.r. studies and with that of prothrombin fragment 1 determined crystallographically.     

Solution conformation of a heptadecapeptide comprising the DNA binding helix F of the cyclic AMP receptor protein of Escherichia coli. Combined use of 1H nuclear magnetic resonance and restrained molecular dynamics

A nuclear magnetic resonance study on a heptadecamer (17-mer) peptide comprising the DNA binding helix F of the cyclic AMP receptor protein of Escherichia coli is presented under solution conditions (viz. 40% (v/v) trifluorethanol) where it adopts an ordered helical structure as judged by circular dichroism. Using a combination of two-dimensional nuclear magnetic resonance techniques, complete resonance assignments are obtained in a sequential manner. From the two-dimensional nuclear Overhauser enhancement spectra, a set of 87 approximate distance restraints is derived and used as the basis for three-dimensional structure determination with a restrained molecular dynamics algorithm in which the interproton distances are incorporated into the total energy function of the system in the form of an additional effective potential term. The convergence properties of this approach are tested by starting from three different initial structures, namely an alpha-helix, a beta-strand and a 3-10 helix. In all three cases, convergence to an alpha-helical structure is achieved with a root mean square difference of less than 3 A for all atoms and less than 2 A for the backbone atoms.     

Protein structure determination from pseudocontact shifts using ROSETTA

Paramagnetic metal ions generate pseudocontact shifts (PCSs) in nuclear magnetic resonance spectra that are manifested as easily measurable changes in chemical shifts. Metals can be incorporated into proteins through metal binding tags, and PCS data constitute powerful long-range restraints on the positions of nuclear spins relative to the coordinate system of the magnetic susceptibility anisotropy tensor (Δχ-tensor) of the metal ion. We show that three-dimensional structures of proteins can reliably be determined using PCS data from a single metal binding site combined with backbone chemical shifts. The program PCS-ROSETTA automatically determines the Δχ-tensor and metal position from the PCS data during the structure calculations, without any prior knowledge of the protein structure. The program can determine structures accurately for proteins of up to 150 residues, offering a powerful new approach to protein structure determination that relies exclusively on readily measurable backbone chemical shifts and easily discriminates between correctly and incorrectly folded conformations.     

Structure of an isolated gramicidin A double helical species by high-resolution nuclear magnetic resonance.

A conformational species of gramicidin A has been isolated in dioxane by high pressure liquid chromatography and characterized by circular dichroism and two-dimensional proton nuclear magnetic resonance. Double-quantum filtered two-dimensional correlation spectroscopy, two-dimensional homonuclear Hartman Hahn spectroscopy and two-dimensional nuclear Overhauser effect spectra at 500 MHz were used to obtain virtually complete proton assignments and produce 192 distance constraints. Protocols to determine the state of aggregation, monomer-specific assignment of nuclear Overhauser enhancement values, hydrogen bonding pattern and helix handedness are described. A distance geometry/simulated annealing routine was used to generate well-defined backbone and side-chain structures. The species isolated is a right-handed intertwined double helix, with approximately 5.7 residues per turn. Unique values for helical dimensions are also specified.     

Sequential resonance assignments as a basis for determination of spatial protein structures by high resolution proton nuclear magnetic resonance

A general scheme is proposed for the determination of spatial protein structures by proton nuclear magnetic resonance. The scheme relies on experimental observation by two-dimensional nuclear magnetic resonance techniques of complete throughbond and through-space proton-proton connectivity maps. These are used to obtain sequential resonance assignments for the individual residues in the amino acid sequence and to characterize the spatial polypeptide structure by a tight network of semi-quantitative, intramolecular distance constraints.     

Solution structure of O-glycosylated C-terminal leucine zipper domain of human salivary mucin (MUC7)

Solution structures of a 23 residue glycopeptide II (KIS* RFLLYMKNLLNRIIDDMVEQ, where * denotes the glycan Gal-beta-(1-3)-alpha-GalNAc) and its deglycosylated counterpart I derived from the C-terminal leucine zipper domain of low molecular weight human salivary mucin (MUC7) were studied using CD, NMR spectroscopy and molecular modeling. The peptide I was synthesized using the Fmoc chemistry following the conventional procedure and the glycopeptide II was synthesized incorporating the O-glycosylated building block (Nalpha-Fmoc-Ser-[Ac4-beta-D-Gal-(1,3)-Ac2-alpha-D-GalN3+ ++]-OPfp) at the appropriate position in stepwise assembly of peptide chain. Solution structures of these glycosylated and nonglycosylated peptides were studied in water and in the presence of 50% of an organic cosolvent, trifluoroethanol (TFE) using circular dichroism (CD), and in 50% TFE using two-dimensional proton nuclear magnetic resonance (2D 1H NMR) spectroscopy. CD spectra in aqueous medium indicate that the apopeptide I adapts, mostly, a beta-sheet conformation whereas the glycopeptide II assumes helical structure. This transition in the secondary structure, upon glycosylation, demonstrates that the carbohydrate moiety exerts significant effect on the peptide backbone conformation. However, in 50% TFE both the peptides show pronounced helical structure. Sequential and medium range NOEs, CalphaH chemical shift perturbations, 3JNH:CalphaH couplings and deuterium exchange rates of the amide proton resonances in water containing 50% TFE indicate that the peptide I adapts alpha-helical structure from Ile2-Val21 and the glycopeptide II adapts alpha-helical structure from Ser3-Glu22. The observation of continuous stretch of helix in both the peptides as observed by both NMR and CD spectroscopy strongly suggests that the C-terminal domain of MUC7 with heptad repeats of leucines or methionine residues may be stabilized by dimeric leucine zipper motif. The results reported herein may be invaluable in understanding the aggregation (or dimerization) of MUC7 glycoprotein which would eventually have implications in determining its structure-function relationship.     

Sequence determination of oligosaccharides and regular polysaccharides using NMR spectroscopy and a novel Web-based version of the computer program CASPER

A WWW-interface to a program for structure elucidation of oligo- and polysaccharides using NMR data, CASPER, is presented. The interface and the underlying program have been extensively tested using published data and it was able to simulate 13C NMR spectra of >200 structures with an average error of about 0.3 ppm/resonance. When applied to the repeating units of Escherichia coli O-antigens the published structures were found among the five highest ranked structures in 75% of the cases. The average deviation between calculated and experimental 13C chemical shifts was 0.45 ppm. Oligosaccharide spectra were calculated with even better accuracy (0.23 ppm/resonance) and the correct structure was ranked 1st or 2nd in all the cases examined. Additional NMR experiments that may be required to distinguish between candidate structures are aided by the assignments provided by the program. This computational approach is also suitable for use in structural confirmation of chemically or enzymatically synthesized oligosaccharides. The program is found at http://www.casper.organ.su.se/casper.     

The program XEASY for computer-supported NMR spectral analysis of biological macromolecules

Summary A new program package, XEASY, was written for interactive computer support of the analysis of NMR spectra for three-dimensional structure determination of biological macromolecules. XEASY was developed for work with 2D, 3D and 4D NMR data sets. It includes all the functions performed by the precursor program EASY, which was designed for the analysis of 2D NMR spectra, i.e., peak picking and support of sequence-specific resonance assignments, cross-peak assignments, cross-peak integration and rate constant determination for dynamic processes. Since the program utilizes the X-window system and the Motif widget set, it is portable on a wide range of UNIX workstations. The design objective was to provide maximal computer support for the analysis of spectra, while providing the user with complete control over the final resonance assignments. Technically important features of XEASY are the use and flexible visual display of strips, i.e., two-dimensional spectral regions that contain the relevant parts of 3D or 4D NMR spectra, automated sorting routines to narrow down the selection of strips that need to be interactively considered in a particular assignment step, a protocol of resonance assignments that can be used for reliable bookkeeping, independent of the assignment strategy used, and capabilities for proper treatment of spectral folding and efficient transfer of resonance assignments between spectra of different types and different dimensionality, including projected, reduced-dimensionality triple-resonance experiments.Abbreviations 1D, 2D, 3D, 4D one-, two-, three-, four-dimensional - NOE nuclear Overhauser enhancement - NOESY nuclear Overhauser enhancement spectroscopy - TOCSY total correlation spectroscopy - COSY correlation spectroscopy - TPPI time-proportional phase incrementation     

13C NMR or coenzyme A and its constituent moieties.

Natural abundance 13C nuclear magnetic resonance spectra, in D2O, were obtained for the following: pantoyl lactone, beta-alanine, cysteamine hydrochloride, cystamine dihydrochloride, calcium pantothenate, beta-aletheine oxalate, pantetheine, pantethine, pantetheine 4'-phosphate, oxypantetheine 4'-phosphate, desulfopantetheine 4'-phosphate, N-acetyl-aminodesthiopantetheine 4'-phosphate, adenosine 2',5'-diphosphate, adenosine 3',5'-diphosphate, and coenzyme A. A complete assignment of the 13C chemical shifts in the NMR spectrum of CoA is reported. Comparison of spectra indicates that CoA most likely exists in an extended conformation.     

Determination of the complete three-dimensional structure of the alpha-amylase inhibitor tendamistat in aqueous solution by nuclear magnetic resonance and distance geometry

The complete three-dimensional structure of the alpha-amylase inhibitor Tendamistat in aqueous solution was determined by 1H nuclear magnetic resonance and distance geometry calculations using the program DISMAN. Compared to an earlier, preliminary determination of the polypeptide backbone conformation, stereo-specific assignments were obtained for 41 of the 89 prochiral groups in the protein, and a much more extensive set of experimental constraints was collected, including 842 distance constraints from nuclear Overhauser effects and over 100 supplementary constraints from spin-spin coupling constants and the identification of intramolecular hydrogen bonds. The complete protein molecule, including the amino acid side-chains is characterized by a group of nine structures corresponding to the results of the nine DISMAN calculations with minimal residual error functions. The average of the pairwise minimal root-mean-square distances among these nine structures is 0.85 A for the polypeptide backbone, and 1.52 A for all the heavy atoms. The procedures used for the structure determination are described and a detailed analysis is presented of correlations between the experimental input data and the precision of the structure determination.     

Three-dimensional structure of the wild-type lac Pribnow promoter DNA in solution. Two-dimensional nuclear magnetic resonance studies and distance geometry calculations

The solution structure of a 12 base-pair DNA duplex containing the wt-lac promoter Pribnow sequence TATGTT has been studied by two-dimensional nuclear magnetic resonance spectroscopy. Proton assignments for the 24 sugar and base residues were obtained from two-dimensional correlated nuclear magnetic resonance and two-dimensional nuclear Overhauser effect spectra in both 2H2O and H2O, and by two-dimensional relayed coherence transfer nuclear magnetic resonance spectroscopy experiments. Time-dependent, two-dimensional nuclear Overhauser effect spectra were used to determine the initial cross-relaxation rates between 212 pairs of assigned protons, leading to 212 interproton distances in the double helix (8 to 9 per nucleotide). These distance constraints, and known bond lengths and angles, were entered into a distance matrix. After smoothing the bounds of the distance matrix, 12 trial matrices within the bounds constraints were independently generated and embedded in three-dimensional space using a distance geometry algorithm, to generate 12 trial structures. These trial structures were then refined until they no longer violated the distance matrix. The resulting structures are very similar at the local base-pair and nearest-neighbor base-pair level, but exhibit increasing variation at more distant and global levels. At the nearest-neighbor level, the A to T step and the G to T step within the Pribnow hexamer, as well as the G to T step preceding the hexamer, all exhibit very low screw pitch, i.e. 5(+/- 6) degrees. Conversely, the T to G step in the center of the promoter has a large screw pitch (47(+/- 2) degrees) and the T to G step at the 3' end of the promoter has a very large screw pitch (60(+/- 3) degrees). The limitations of nuclear magnetic resonance spectroscopy distance determination of structure are discussed in terms of resolution and spectral overlap of two-dimensional nuclear Overhauser effect crosspeaks. In the present duplex, the inability to measure several 1'-2' and 1'-2" distances resulted in underdetermination of the precise local sugar conformation for seven of the 24 residues, although the spatial position of all sugars was well defined.     

Chemical characterization of the regularly arranged surface layer glycoprotein of Clostridium thermosaccharolyticum D120-70

Clostridium thermosaccharolyticum D120-70 possesses as its outermost cell envelope layer a square-arranged array of glycoprotein molecules. SDS/polyacrylamide gel electrophoresis of the purified surface layer showed a broadened band in the molecular mass range of about 115 kDa which, upon periodic acid/Schiff staining, gave a positive reaction. After proteolytic degradation of this material, two glycopeptide fractions were obtained. One- and two-dimensional nuclear magnetic resonance studies, together with methylation analysis and periodate oxidation, were used to determine the structures of the polysaccharide portions of these glycopeptides. The combined chemical and spectroscopic evidence suggests the following structures: (formula; see text).     

Determination of the three-dimensional structure of the Antennapedia homeodomain from Drosophila in solution by 1H nuclear magnetic resonance spectroscopy

The determination of the three-dimensional structure of the Antennapedia homeodomain from Drosophila in solution is described. The techniques used are 1H nuclear magnetic resonance spectroscopy for the data collection, and calculation of the protein structure with the program DISMAN followed by restrained energy minimization with a modified version of the program AMBER. A group of 19 conformers characterizes a well-defined structure for residues 7 to 59, with an average root-mean-square distance from the backbone atoms of 0.6 A relative to the mean of the 19 structures. The structure contains a helix from residues 10 to 21, a helix-turn-helix motif from residues 28 to 52, which is similar to those reported for several prokaryotic repressor proteins, and a somewhat flexible fourth helix from residues 53 to 59, which essentially forms an extension of the presumed recognition helix, residues 42 to 52. The helices enclose a structurally well-defined molecular core of hydrophobic amino acid side-chains.     

A distance geometry program for determining the structures of small proteins and other macromolecules from nuclear magnetic resonance measurements of intramolecular1H−1H proximities in solution

DISGEO is a new implementation of a distance geometry algorithm which has been specialized for the calculation of macromolecular conformation from distance measurements obtained by two-dimensional nuclear Overhauser enhancement spectroscopy. The improvements include (1) a decomposition of the complete embedding process into two successive, more tractable calculations by the use of “substructures”, (2) a compact data structure for storing incomplete distance information on a structure, (3) a more efficient shortest-path algorithm for computing the triangle inequality limits on all distances from this information, (4) a new algorithm for selecting random metric spaces from within these limits, (5) the use of chirality constraints to obtain good covalent geometry without the use ofad hoc weights or excessive optimization. The utility of the resultant program with nuclear magnetic resonance data is demonstrated by embedding complete spatial structures for the protein basic pancreatic trypsin inhibitor vs all 508 intramolecular, interresidue proton-proton contacts shorter than 4.0 Å that were present in its crystal structure. The crystal structure could be reproduced from this data set to within 1.3 Å minimum root mean square coordinate difference between the backbone atoms. We conclude that the information potentially available from nuclear magnetic resonance experiments in solution is sufficient to define the spatial structure of small proteins.     

Relaxation matrix refinement of the solution structure of squash trypsin inhibitor

The structure of the small squash trypsin inhibitor CMTI-I is refined by directly minimizing the difference between the observed two-dimensional nuclear Overhauser enhancement (NOE) intensities and those calculated by the full relaxation matrix approach. To achieve this, a term proportional to this difference was added to the potential energy function of the molecular dynamics program X-PLOR. Derivatives with respect to atomic co-ordinates are calculated analytically. Spin diffusion effects are thus accounted for fully during the refinement. Initial structures for the refinement were those determined recently by solution nuclear magnetic resonance using the isolated two-spin approximation to derive distance range estimates. The fits to the nuclear magnetic resonance data improve significantly with only small shifts in the refined structures during a few cycles of conjugate gradient minimization. However, larger changes (approximately 1 A) in the conformation occur during simulated annealing, which is accompanied by a further reduction of the difference between experimental and calculated two-dimensional NOE intensities. The refined structures are closer to the X-ray structure of the inhibitor complexed with trypsin than the initial structures. The root-mean-square difference for backbone atoms between the initial structures and the X-ray structure is 0.96 A, and that between the refined structures and the X-ray structure 0.61 A.