1H and 15N NMR assignment and solution structure of the SH3 domain of spectrin: Comparison of unrefined and refined structure sets with the crystal structure

The assignment of the ¹H and ¹⁵Nnuclear magnetic resonance spectra of the Src-homology region 3 domain ofchicken brain -spectrin has been obtained. A set of solutionstructures has been determined from distance and dihedral angle restraints,which provide a reasonable representation of the protein structure insolution, as evaluated by a principal component analysis of the globalpairwise root-mean-square deviation (rmsd) in a large set of structuresconsisting of the refined and unrefined solution structures and the crystalstructure. The solution structure is well defined, with a lower degree ofconvergence between the structures in the loop regions than in the secondarystructure elements. The average pairwise rmsd between the 15 refinedsolution structures is 0.71 ± 0.13 Å for the backbone atoms and1.43 ± 0.14 Å for all heavy atoms. The solution structure isbasically the same as the crystal structure. The average rmsd between the 15refined solution structures and the crystal structure is 0.76 Å forthe backbone atoms and 1.45 ± 0.09 Å for all heavy atoms. Thereare, however, small differences probably caused by intermolecular contactsin the crystal structure.     

Determination of a high-quality nuclear magnetic resonance solution structure of the bovine pancreatic trypsin inhibitor and comparison with three crystal structures.

A high-quality three-dimensional structure of the bovine pancreatic trypsin inhibitor (BPTI) in aqueous solution was determined by 1H nuclear magnetic resonance (n.m.r.) spectroscopy and compared to the three available high-resolution X-ray crystal structures. A newly collected input of 642 distance constraints derived from nuclear Overhauser effects and 115 dihedral angle constraints was used for the structure calculations with the program DIANA, followed by restrained energy minimization with the program AMBER. The BPTI solution structure is represented by a group of 20 conformers with an average root-mean-square deviation (RMSD) relative to the mean solution structure of 0.43 A for backbone atoms and 0.92 A for all heavy atoms of residues 2 to 56. The pairwise RMSD values of the three crystal structures relative to the mean solution structure are 0.76 to 0.85 A for the backbone atoms and 1.24 to 1.33 A for all heavy atoms of residues 2 to 56. Small local differences in backbone atom positions between the solution structure and the X-ray structures near residues 9, 25 to 27, 46 to 48 and 52 to 58, and conformational differences for individual amino acid side-chains were analyzed for possible correlations with intermolecular protein-protein contacts in the crystal lattices, using the pairwise RMSD values among the three crystal structures as a reference.     

Determination of a high precision structure of a novel protein, Linum usitatissimum trypsin inhibitor (LUTI), using computer-aided assignment of NOESY cross-peaks

The solution structure of a novel 69 residue proteinase inhibitor, Linum usitatissimum trypsin inhibitor (LUTI), was determined using a method based on computer aided assignment of nuclear Overhauser enhancement spectroscopy (NOESY) data. The approach applied uses the program NOAH/DYANA for automatic assignment of NOESY cross-peaks. Calculations were carried out using two unassigned NOESY peak lists and a set of determined dihedral angle restraints. In addition, hydrogen bonds involving amide protons were identified during calculations using geometrical criteria and values of HN temperature coefficients. Stereospecific assignment of beta-methylene protons was carried out using a standard procedure based on nuclear Overhauser enhancement intensities and 3J(alpha)(beta) coupling constants. Further stereospecific assignment of methylene protons and diastereotopic methyl groups were established upon structure-based method available in the program GLOMSA and chemical shift calculations. The applied algorithm allowed us to assign 1968 out of 2164 peaks (91%) derived from NOESY spectra recorded in H2O and 2H2O. The final experimental data input consisted of 1609 interproton distance restraints, 88 restraints for 44 hydrogen bonds, 63 torsion angle restraints and 32 stereospecifically assigned methylene proton pairs and methyl groups. The algorithm allowed the calculation of a high precision protein structure without the laborious manual assignment of NOESY cross-peaks. For the 20 best conformers selected out of 40 refined ones in the program CNS, the calculated average pairwise rmsd values for residues 3 to 69 were 0.38 A (backbone atoms) and 1.02 A (all heavy atoms). The three-dimensional LUTI structure consists of a mixed parallel and antiparallel beta-sheet, a single alpha-helix and shows the fold of the potato 1 family of proteinase inhibitors. Compared to known structures of the family, LUTI contains Arg and Trp residues at positions P6' and P8', respectively, instead of two Arg residues, involved in the proteinase binding loop stabilization. A consequence of the ArgTrp substitution at P8' is a slightly more compact conformation of the loop relative to the protein core.     

NMR solution structure of plastocyanin from the photosynthetic prokaryote, Prochlorothrix hollandica

The solution structure of a divergent plastocyanin (PC) from the photosynthetic prokaryote Prochlorothrix hollandica was determined by homonuclear 1H NMR spectroscopy. Nineteen structures were calculated from 1222 distance restraints, yielding a family of structures having an average rmsd of 0.42 +/- 0.08 A for backbone atoms and 0.71 +/- 0.07 A for heavy atoms to the mean structure. No distance constraint was violated by more than 0.26 A in the structure family. Despite the low number of conserved residues shared with other PC homologues, the overall folding pattern of P. hollandica PC is similar to other PCs, in that the protein forms a two-sheet beta-barrel tertiary structure. The greatest variability among the backbone structures is seen in the loop region from residues 47-60. The differences seen in the P. hollandica PC homologue likely arise due to a small deletion of 2-4 residues compared to the PC consensus; this yields a less extended loop containing a short alpha-helix from residues Ala52-Leu55. Additionally, the protein has an altered hydrophobic patch thought to be important in binding reaction partners. Whereas the backbone structure is very similar within the loops of the hydrophobic region, the presence of two unique residues (Tyr12 and Pro14) yields a structurally different hydrophobic surface likely important in binding P. hollandica Photosystem I.     

Solution structure of the PDZ2 domain from human phosphatase hPTP1E and its interactions with C-terminal peptides from the Fas receptor

The solution structure of the second PDZ domain (PDZ2) from human phosphatase hPTP1E has been determined using 2D and 3D heteronuclear NMR experiments. The binding of peptides derived from the C-terminus of the Fas receptor to PDZ2 was studied via changes in backbone peptide and protein resonances. The structure is based on a total of 1387 nonredundant experimental NMR restraints including 1261 interproton distance restraints, 45 backbone hydrogen bonds, and 81 torsion angle restraints. Analysis of 30 lowest-energy structures resulted in rmsd values of 0.41 +/- 0.09 A for backbone atoms (N, Calpha, C') and 1.08 +/- 0.10 A for all heavy atoms, excluding the disordered N- and C-termini. The hPTP1E PDZ2 structure is similar to known PDZ domain structures but contains two unique structural features. In the peptide binding domain, the first glycine of the GLGF motif is replaced by a serine. This serine appears to replace a bound water observed in PDZ crystal structures that hydrogen bonds to the bound peptide's C-terminus. The hPTP1E PDZ2 structure also contains an unusually large loop following strand beta2 and proximal to the peptide binding site. This well-ordered loop folds back against the PDZ domain and contains several residues that undergo large amide chemical shift changes upon peptide binding. Direct observation of peptide resonances demonstrates that as many as six Fas peptide residues interact with the PDZ2 domain.     

NMR structure of cysteinyl-phosphorylated enzyme IIB of the N,N'-diacetylchitobiose-specific phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli

The determination by NMR of the solution structure of the phosphorylated enzyme IIB (P-IIB(Chb)) of the N,N'-diacetylchitobiose-specific phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli is presented. Most of the backbone and side-chain resonances were assigned using a variety of mostly heteronuclear NMR experiments. The remaining resonances were assigned with the help of the structure calculations.NOE-derived distance restraints were used in distance geometry calculations followed by molecular dynamics and simulated annealing protocols. In addition, combinations of ambiguous restraints were used to resolve ambiguities in the NOE assignments. By combining sets of ambiguous and unambiguous restraints into new ambiguous restraints, an error function was constructed that was less sensitive to information loss caused by assignment uncertainties. The final set of structures had a pairwise rmsd of 0.59 A and 1.16 A for the heavy atoms of the backbone and side-chains, respectively.Comparing the P-IIB(Chb) solution structure with the previously determined NMR and X-ray structures of the wild-type and the Cys10Ser mutant shows that significant differences between the structures are limited to the active-site region. The phosphoryl group at the active-site cysteine residue is surrounded by a loop formed by residues 10 through 16. NOE and chemical shift data suggest that the phosphoryl group makes hydrogen bonds with the backbone amide protons of residues 12 and 15. The binding mode of the phosphoryl group is very similar to that of the protein tyrosine phosphatases. The differences observed are in accordance with the presumption that IIB(Chb) has to be more resistant to hydrolysis than the protein tyrosine phosphatases. We propose a proton relay network by which a transfer occurs between the cysteine SH proton and the solvent via the hydroxyl group of Thr16.     

Determination of the three-dimensional solution structure of barnase using nuclear magnetic resonance spectroscopy

The solution conformation of the ribonuclease barnase has been determined by using 1H nuclear magnetic resonance (NMR) spectroscopy. The 20 structures were calculated by using 853 interproton distance restraints obtained from analyses of two-dimensional nuclear Overhauser spectra, 72 phi and 53 chi 1 torsion angle restraints, and 17 hydrogen-bond distance restraints. The calculated structures contain two alpha-helices (residues 6-18 and 26-34) and a five-stranded antiparallel beta-sheet (residues 50-55, 70-75, 85-91, 94-101, and 105-108). The core of the protein is formed by the packing of one of the alpha-helices (residues 6-18) onto the beta-sheet. The average RMS deviation between the calculated structures and the mean structure is 1.11 A for the backbone atoms and 1.75 A for all atoms. The protein is least well-defined in the N-terminal region and in three large loops. When these regions are excluded, the average RMS deviation between the calculated structures and the mean structure for residues 5-34, 50-56, 71-76, 85-109 is 0.62 A for the backbone atoms and 1.0 A for all atoms. The NMR-derived structure has been compared with the crystal structure of barnase [Mauguen et al. (1982) Nature (London) 297, 162-164].     

Solution structure of neurotoxin I from the sea anemone Stichodactyla helianthus. A nuclear magnetic resonance, distance geometry, and restrained molecular dynamics study

The three-dimensional structure of the sea anemone polypeptide Stichodactyla helianthus neurotoxin I in aqueous solution has been determined using distance geometry and restrained molecular dynamics simulations based on NMR data acquired at 500 MHz. A set of 470 nuclear Overhauser enhancement values was measured, of which 216 were used as distance restraints in the structure determination along with 15 dihedral angles derived from coupling constants. After restrained molecular dynamics refinement, the eight structures that best fit the input data form a closely related family. They describe a structure that consists of a core of twisted, four-stranded, antiparallel beta-sheet encompassing residues 1-3, 19-24, 29-34, and 40-47, joined by three loops, two of which are well defined by the NMR data. The third loop, encompassing residues 7-16, is poorly defined by the data and is assumed to undergo conformational averaging in solution. Pairwise root mean square displacement values for the backbone heavy atoms of the eight best structures are 1.3 +/- 0.2A when the poorly defined loop is excluded and 3.6 +/- 1.0A for all backbone atoms. Refinement using restrained molecular dynamics improved the quality of the structures generated by distance geometry calculations with respect to the number of nuclear Overhauser enhancements violated, the size of the total distance violations and the total potential energies of the structures. The family of structures for S. heliathus neurotoxin I is compared with structures of related sea anemone proteins that also bind to the voltage-gated sodium channel.     

Solution structure of a sweet protein single-chain monellin determined by nuclear magnetic resonance and dynamical simulated annealing calculations

Single-chain monellin (SCM), which is an engineered 94-residue polypeptide, has proven to be as sweet as native two-chain monellin. SCM is more stable than the native monellin for both heat and acidic environments. Data from gel filtration HPLC and NMR indicate that the SCM exists as a monomer in aqueous solution. The solution structure of SCM has been determined by nuclear magnetic resonance (NMR) spectroscopy and dynamical simulated annealing calculations. A stable alpha-helix spanning residues Phe11-Ile26 and an antiparallel beta-sheet formed by residues 2-5, 36-38, 41-47, 54-64, 69-75, and 83-88 have been identified. The sheet was well defined by backbone-backbone NOEs, and the corresponding beta-strands were further confirmed by hydrogen bond networks based on amide hydrogen exchange data. Strands beta2 and beta3 are connected by a small bulge comprising residues Ile38-Cys41. A total of 993 distance and 56 dihedral angle restraints were used for simulated annealing calculations. The final simulated annealing structures (k) converged well with a root-mean-square deviation (rmsd) between backbone atoms of 0.49 A for secondary structural regions and 0.70 A for backbone atoms excluding two loop regions. The average restraint energy-minimized (REM) structure exhibited root-mean-square deviations of 1.19 A for backbone atoms and 0.85 A for backbone atoms excluding two loop regions with respect to 20 k structures. The solution structure of SCM revealed that the long alpha-helix was folded into the concave side of a six-stranded antiparallel beta-sheet. The side chains of Tyr63 and Asp66 which are common to all sweet peptides showed an opposite orientation relative to H1 helix, and they were all solvent-exposed. Residues at the proposed dimeric interface in the X-ray structure were observed to be mostly solvent-exposed and demonstrated high degrees of flexibility.     

A refined model for the solution structure of oxidized putidaredoxin

A refined model for the solution structure of oxidized putidaredoxin (Pdxo), a Cys4Fe2S2 ferredoxin, has been determined. A previous structure (Pochapsky et al. (1994) Biochemistry 33, 6424-6432; PDB entry ) was calculated using the results of homonuclear two-dimensional NMR experiments. New data has made it possible to calculate a refinement of the original Pdxo solution structure. First, essentially complete assignments for diamagnetic 15N and 13C resonances of Pdxo have been made using multidimensional NMR methods, and 15N- and 13C-resolved NOESY experiments have permitted the identification of many new NOE restraints for structural calculations. Stereospecific assignments for leucine and valine CH3 resonances were made using biosynthetically directed fractional 13C labeling, improving the precision of NOE restraints involving these residues. Backbone dihedral angle restraints have been obtained using a combination of two-dimensional J-modulated 15N,1H HSQC and 3D (HN)CO(CO)NH experiments. Second, the solution structure of a diamagnetic form of Pdx, that of the C85S variant of gallium putidaredoxin, in which a nonligand Cys is replaced by Ser, has been determined (Pochapsky et al. (1998) J. Biomol. NMR 12, 407-415), providing information concerning structural features not observable in the native ferredoxin due to paramagnetism. Third, a crystal structure of a closely related ferredoxin, bovine adrenodoxin, has been published (Müller et al. (1998) Structure 6, 269-280). This structure has been used to model the metal binding site structure in Pdx. A family of fourteen structures is presented that exhibits an rmsd of 0.51 A for backbone heavy atoms and 0.83 A for all heavy atoms. Exclusion of the modeled metal binding loop region reduces overall the rmsd to 0.30 A for backbone atoms and 0.71 A for all heavy atoms.     

Three-dimensional solution structure of an insulin dimer. A study of the B9(Asp) mutant of human insulin using nuclear magnetic resonance, distance geometry and restrained molecular dynamics.

The solution structure of the B9(Asp) mutant of human insulin has been determined by two-dimensional 1H nuclear magnetic resonance spectroscopy. Thirty structures were calculated by distance geometry from 451 interproton distance restraints based on intra-residue, sequential and long-range nuclear Overhauser enhancement data, 17 restraints on phi torsional angles obtained from 3JH alpha HN coupling constants, and the restraints from 17 hydrogen bonds, and the three disulphide bridges. The distance geometry structures were optimized using restrained molecular dynamics (RMD) and energy minimization. The average root-mean-square deviation for the best 20 RMD refined structures is 2.26 A for the backbone and 3.14 A for all atoms if the less well-defined N and C-terminal residues are excluded. The helical regions are better defined, with root-mean-square deviation values of 1.11 A for the backbone and 2.03 A for all atoms. The data analysis and the calculations show that B9(Asp) insulin, in water solution at the applied pH (1.8 to 1.9), is a well-defined dimer with no detectable difference between the two monomers. The association of the two monomers in the solution dimer is relatively loose as compared with the crystal dimer. The overall secondary and tertiary structures of the monomers in the 2Zn crystal hexamer is found to be preserved. The conformation-averaged NMR structures obtained for the monomer is close to the structure of molecule 1 in the hexamer of the 2Zn insulin crystal. However, minor, but significant deviations from this structure, as well as from the structure of monomeric insulin in solution, exist and are ascribed to the absence of the hexamer and crystal packing forces, and to the presence of monomer-monomer interactions, respectively. Thus, the monomer in the solution dimer shows a conformation similar to that of the crystal monomer in molecular regions close to the monomer-monomer interface, whereas it assumes a conformation similar to that of the solution structure of monomeric insulin in other regions, suggesting that B9(Asp) insulin adopts a monomer-like conformation when this is not inconsistent with the monomer-monomer arrangement in the dimer.     

Determination of the three-dimensional solution structure of the antihypertensive and antiviral protein BDS-I from the sea anemone Anemonia sulcata: a study using nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing

The three-dimensional solution structure of the antihypertensive and antiviral protein BDS-I from the sea anemone Anemonia sulcata has been determined on the basis of 489 interproton and 24 hydrogen-bonding distance restraints supplemented by 23 phi backbone and 21 chi 1 side-chain torsion angle restraints derived from nuclear magnetic resonance (NMR) measurements. A total of 42 structures is calculated by a hybrid metric matrix distance geometry-dynamical simulated annealing approach. Both the backbone and side-chain atom positions are well defined. The average atomic rms difference between the 42 individual SA structures and the mean structure obtained by averaging their coordinates is 0.67 +/- 0.12 A for the backbone atoms and 0.90 +/- 0.17 A for all atoms. The core of the protein is formed by a triple-stranded antiparallel beta-sheet composed of residues 14-16 (strand 1), 30-34 (strand 2), and 37-41 (strand 3) with an additional mini-antiparallel beta-sheet at the N-terminus (residues 6-9). The first and second strands of the triple-stranded antiparallel beta-sheet are connected by a long exposed loop (residues 17-30). A number of side-chain interactions are discussed in light of the structure.     

The structure and dynamics of rat apo-cellular retinol-binding protein II in solution: comparison with the X-ray structure

The structure and dynamics of rat apo-cellular retinol binding protein II (apo-CRBP II) in solution has been determined by multidimensional NMR analysis of uniformly enriched recombinant rat 13C, 15N-apo-CRBP II and 15N-apo-CRBP II. The final ensemble of 24 NMR structures has been calculated from 3274 conformational restraints or 24.4 restraints/residue. The average root-mean-square deviation of the backbone atoms for the final 24 structures relative to their mean structure is 1.06 A. Although the average solution structure is very similar to the crystal structure, it differs at the putative entrance to the binding cavity, which is formed by the helix-turn-helix motif, the betaC-betaD turn and the betaE-betaF turn. The mean coordinates of the main-chain atoms of amino acid residues 28-38 are displaced in the solution structure relative to the crystal structure. The side-chain of F58, located on the betaC-betaD turn, is reoriented such that it interacts with L37 and no longer blocks entry into the ligand-binding pocket. Residues 28-35, which form the second helix of the helix-turn-helix motif in the crystal structure, do not exhibit a helical conformation in the solution structure. The solution structure of apo-CRBP II exhibits discrete regions of backbone disorder which are most pronounced at residues 28-32, 37-38 and 73-76 in the betaE-betaF turn as evaluated by the consensus chemical shift index, the root-mean-square deviation, amide 1H exchange rates and 15N relaxation studies. These studies indicate that fluctuations in protein conformation occur on the microseconds to ms time-scale in these regions of the protein. Some of these exchange processes can be directly observed in the three-dimensional 15N-resolved NOESY spectrum. These results suggest that in solution, apo-CRBP II undergoes conformational changes on the microseconds to ms time-scale which result in increased access to the binding cavity.     

NMR structure of tissue inhibitor of metalloproteinases-1 implicates localized induced fit in recognition of matrix metalloproteinases

A high quality solution structure of the matrix metalloproteinase inhibitory N-terminal domain of recombinant human tissue inhibitor of metalloproteinases-1 (N-TIMP-1) has been determined. For the rigidly packed residues, the average RMSD to the mean structure is 0. 57 A for the backbone atoms and 1.00 A for all heavy atoms. Comparison of the solution structure of free N-TIMP-1 with the crystal structure of TIMP-1 bound to the catalytic domain of MMP-3 ( Gomis-R]uth et al., 1997 ) shows that the structural core of the beta barrel flanked by helices is nearly unchanged by the association with MMP-3, evident from a backbone RMSD of 1.15 A. However, clear differences in the conformation of the MMP-binding ridge of free and MMP-bound TIMP-1 suggest induced fit throughout the ridge. The MMP-dependent conformational changes in the ridge include a dramatic bending of AB loop residues Glu28 through Leu34, moderate hinge bending of the CD-loop about residues Ala65 and Cys70, and modest bending of the Cys1 through Pro6 segment. A large number of interresidue Nuclear Overhauser enhancements (NOEs) augmented by stereospecific assignments, torsion restraints, and dipolar couplings (an average of 18 non-trivial restraints per residue) engender confidence in these structural inferences. A tight cluster of three lysine residues and one arginine residue atop beta-strands A and B, and identical among TIMP sequences, form the heart of a highly conserved electropositive patch that may interact with anionic components of the extracellular matrix.     

Structure of crambin in solution, crystal and in the trajectories of molecular dynamics simulations

The mechanisms of the three-dimensional crambin structure alterations in the crystalline environments and in the trajectories of the molecular dynamics simulations in the vacuum and crystal surroundings have been analyzed. In the crystalline state and in the solution the partial regrouping of remote intramolecular packing contacts, involved in the formation and stabilization of the tertiary structure of the crambin molecule, occurs in NMR structures. In the crystalline state it is initiated by the formation of the intermolecular contacts, the conformational influence of its appearance is distributed over the structure. The changes of the conformations and positions of the residues of the loop segments, where the intermolecular contacts of the crystal surroundings are preferably concentrated, are most observable. Under the influence of these contacts the principal change of the regular secondary structure of crambin is taking place: extension of the two-strand β structure to the three-strand structure with the participation of the single last residue N46 of the C-terminal loop. In comparison with the C-terminal loop the more profound changes are observed in the conformation and the atomic positions of the backbone atoms and in the solvent accessibility of the residues of the interhelical loop. In the solution of the ensemble of the 8 NMR structures relative accessibility to the solvent differs more noticeably also in the region of the loop segments and rather markedly in the interhelical loop. In the crambin cryogenic crystal structures the positions of the atoms of the backbone and/or side chain of 14–18 of 46 residues are discretely disordered. The disorganizations of at least 8 of 14 residues occur directly in the regions of the intermolecular contacts and another 5 residues are disordered indirectly through the intramolecular contacts with the residues of the intermolecular contacts. Upon the molecular dynamics simulation in the vacuum surrounding as in the solution of the crystalline structure of crambin the essential changes of the backbone conformation are caused by the intermolecular contacts absence, but partly masked by the structure changes owing to the nonpolar H atoms absence on the simulated structure. The intermolecular contact absence is partly manifested upon the molecular dynamics simulation of the crambin crystal with one protein molecule. Compared to the crystal structure the lengths of the interpeptide hydrogen bonds and other interresidue contacts in an average solution NMR structure are somewhat shorter and accordingly the energy of the interpeptide hydrogen bonds is better. This length shortening can occur at the stage of the refinement of the NMR structures of the crambin and other proteins by its energy minimizations in the vacuum surroundings and not exist in the solution protein structures.     

Solution structure of PAFP-S: a new knottin-type antifungal peptide from the seeds of Phytolacca americana

The three-dimensional solution structure of PAFP-S, an antifungal peptide extracted from the seeds of Phytolacca americana, was determined using 1H NMR spectroscopy. This cationic peptide contains 38 amino acid residues. Its structure was determined from 302 distance restraints and 36 dihedral restraints derived from NOEs and coupling constants. The peptide has six cysteines involved in three disulfide bonds. The previously unassigned parings have now been determined from NMR data. The solution structure of PAFP-S is presented as a set of 20 structures using ab initio dynamic simulated annealing, with an average RMS deviation of 1.68 A for the backbone heavy atoms and 2.19 A for all heavy atoms, respectively. For the well-defined triple-stranded beta-sheet involving residues 8-10, 23-27, and 32-36, the corresponding values were 0.39 and 1.25 A. The global fold involves a cystine-knotted three-stranded antiparallel beta-sheet (residues 8-10, 23-27, 32-36), a flexible loop (residues 14-19), and four beta-reverse turns (residues 4-8, 11-14, 19-22, 28-32). This structure features all the characteristics of the knottin fold. It is the first structural model of an antifungal peptide that adopts a knottin-type structure. PAFP-S has an extended hydrophobic surface comprised of residues Tyr23, Phe25, Ile27, Tyr32, and Val34. The side chains of these residues are well-defined in the NMR structure. Several hydrophilic and positively charged residues (Arg9, Arg38, and Lys36) surround the hydrophobic surface, giving PAFP-S an amphiphilic character which would be the main structural basis of its biological function.     

Nuclear magnetic resonance solution structure of hirudin(1-51) and comparison with corresponding three-dimensional structures determined using the complete 65-residue hirudin polypeptide chain.

The three-dimensional structure of the N-terminal 51-residue domain of recombinant hirudin in aqueous solution was determined by 1H nuclear magnetic resonance (NMR) spectroscopy, and the resulting high-quality solution structure was compared with corresponding structures obtained from studies with the intact, 65-residue polypeptide chain of hirudin. On the basis of 580 distance constraints derived from nuclear Overhauser effects and 109 dihedral angle constraints, a group of 20 conformers representing the solution structure of hirudin(1-51) was computed with the program DIANA and energy-minimized with a modified version of the program AMBER. Residues 3 to 30 and 37 to 48 form a well-defined molecular core with two antiparallel beta-sheets composed of residues 14 to 16 and 20 to 22, and 27 to 31 and 36 to 40, and three reverse turns at residues 8 to 11 (type II), 17 to 20 (type II') and 23 to 26 (type II). The average root-mean-square deviation of the individual NMR conformers relative to their mean co-ordinates is 0.38 A for the backbone atoms and 0.77 A for all heavy atoms of these residues. Increased structural disorder was found for the N-terminal dipeptide segment, the loop at residues 31 to 36, and the C-terminal tripeptide segment. The solution structure of hirudin(1-51) has the same molecular architecture as the corresponding polypeptide segment in natural hirudin and recombinant desulfatohirudin. It is also closely similar to the crystal structure of the N-terminal 51-residue segment of hirudin in a hirudin-thrombin complex, with root-mean-square deviations of the crystal structure relative to the mean solution structure of 0.61 A for the backbone atoms and 0.91 A for all heavy atoms of residues 3 to 30 and 37 to 48. Further coincidence is found for the loop formed by residues 31 to 36, which shows increased structural disorder in all available solution structures of hirudin, and of which residues 32 to 35 are not observable in the electron density map of the thrombin complex. Significant local structural differences between hirudin(1-51) in solution and hirudin in the crystalline thrombin complex were identified mainly for the N-terminal tripeptide segment and residues 17 to 21. These are further analyzed in an accompanying paper.     

Refinement of the three-dimensional solution structure of barley serine proteinase inhibitor 2 and comparison with the structures in crystals.

The three-dimensional structure of barley serine proteinase inhibitor, CI-2, has been determined using nuclear magnetic resonance spectroscopy. The present structure determination is a refinement of the structure previously determined by us, using in the present case stereo-specific assignments, and a virtually complete set of assignments of the two-dimensional nuclear Overhauser spectrum. The structure determination is based on the identification of more than 1300 nuclear Overhauser effects, of which 961 were used in the structure calculation as distance restraints, and on 94 dihedral angle restraints, of which 31 are for chi 1 angles in defined chiral centers. These have been used to calculate a series of 20 three-dimensional structures using a combination of distance geometry, simulated annealing and restrained molecular dynamics. Each of the 20 structures was in agreement within less than 0.5 A of each of the distance restraints and with all dihedral angle restraints. When compared to the geometric average structure of the 20 refined structures the root-mean-square differences for the backbone atoms were 0.8 (+/- 0.2) A and for all atoms were 1.6 (+/- 0.2) A. By comparison, the values obtained for the structures determined previously were 1.4 (+/- 0.2) A and 2.1 (+/- 0.1) A, respectively. The structures were also compared to the structure determined in the crystalline state by X-ray diffraction showing root-mean-square differences of 1.6 (+/- 0.2) A and 2.8 (+/- 0.2) A for the backbone and all atoms, respectively. Common features of the solution structure and the two crystal structures are the four-stranded beta-structure, composed of a pair of parallel strands, and three pairs of antiparallel beta-strands flanked on one side by a 12-residue alpha-helix and on the other side by a loop containing the serine proteinase binding site. The new analysis of the structure has revealed an additional pair of antiparallel beta-strands, consisting of residues 65 to 67 and 81 to 83, that was not seen in either of the crystal structures or the previous solution structure. Identification of this was based on nuclear magnetic resonance evidence for the hydrogen bond (67HN to 81CO) not reported previously. Also the presence of a bifurcated hydrogen bond involving Phe69 CO and HN atoms of Ala77 and Gln78 was observed in solution but not in crystals. Minor differences between the two structures were observed in the phi-angles of residues Met59 and Glu60 in the inhibitory site.     

Nuclear magnetic resonance solution structure of truncated human GRObeta [5-73] and its structural comparison with CXC chemokine family members GROalpha and IL-8

The three-dimensional structure of a novel four amino acid truncated form of the CXC chemokine GRObeta [5-73] isolated from bone marrow stromal cells with potent hematopoietic and anti-infective activities has been determined by two-dimensional (1)H nuclear magnetic resonance (NMR) spectroscopy in solution. On the basis of 1878 upper distance constraints derived from nuclear Overhauser effects (NOE) and 314 dihedral angle constraints, a group of 20 conformers representing the solution structure of the human GRObeta [5-73] was computed with the program DYANA. At the concentrations used for NMR study, GRObeta [5-73] forms a dimer in solution that is architectured by a six-stranded antiparallel beta-sheet (residues 25 to 29, 39 to 44, 49 to 52) and a pair of helices (residues 58 to 68) with 2-fold symmetry, while the C terminus of the protein is disordered. The average of the pairwise root-mean-square deviations of individual NMR conformers relative to the mean coordinates for the backbone atoms N, C(alpha) and C' of residues 5 to 68 is 0.47 A. Overall, the global fold of GRObeta [5-73] is similar to that of the previously reported NMR structure of GROalpha and the NMR and X-ray structures of interleukin-8. Among these three CXC chemokines, GRObeta [5-73] is most similar in structure to GROalpha. Significant differences between GRObeta [5-73], GROalpha and interleukin-8 are in the N-terminal loop comprising residues 12 to 19. The N-terminal arm containing the conserved ELR motif and the loop of residues 30 to 38 containing the GPH motif are different among these three CXC chemokines. The structural differences in these two regions may be responsible for the specificity of the receptor binding and biological activity of different chemokines.