The polypeptide fold of the globular domain of histone H5 in solution. A study using nuclear magnetic resonance, distance geometry and restrained molecular dynamics.

The polypeptide fold of the 79-residue globular domain of chicken histone H5 (GH5) in solution has been determined by the combined use of distance geometry and restrained molecular dynamics calculations. The structure determination is based on 307 approximate interproton distance restraints derived from n.m.r. measurements. The structure is composed of a core made up of residues 3-18, 23-34, 37-60 and 71-79, and two loops comprising residues 19-22 and 61-70. The structure of the core is well defined with an average backbone atomic r.m.s. difference of 2.3 +/- 0.3 A between the final eight converged restrained dynamics structures and the mean structure obtained by averaging their coordinates best fitted to the core residues. The two loops are also well defined locally but their orientation with respect to the core could not be determined as no long range ([i-j[ greater than 5) proton-proton contacts could be observed between the loop and core residues in the two-dimensional nuclear Overhauser enhancement spectra. The structure of the core is dominated by three helices and has a similar fold to the C-terminal DNA binding domain of the cAMP receptor protein.     

Determination of the spatial structure of insectotoxin 15A from Buthus erpeus by (1)H-NMR spectroscopy data]

The solution structure of insectotoxin 15A (35 residues) from scorpion Buthus eupeus was determined on the basis of 386 interproton distance restraints 12 hydrogen-bonding restraints and 113 dihedral angle restraints derived from 1H NMR experiments. A group of 20 structures was calculated with the distance geometry program DIANA followed by the restrained energy minimization with the program CHARMM. The atomic RMS distribution about the mean coordinate position is 0.64 +/- 0.11 A for the backbone atoms and 1.35 +/- 0.20 A for all atoms. The structure contains an alpha-helix (residues 10-20) and a three-stranded antiparallel beta-sheet (residues 2-5, 24-28 and 29-33). A pairing of the eight cysteine residues of insectotoxin 15A was established basing on NMR data. Three disulfide bridges (residues 2-19, 16-31 and 20-33) connect the alpha-helix with the beta-sheet, and the fourth one (5-26) joins beta-strands together. The spatial fold of secondary structure elements (the alpha-helix and the beta-sheet) of the insectotoxin 15A is very similar to those of the other short and long scorpion toxins in spite of a low (about 20%) sequence homology.     

The three-dimensional structure of guanine-specific ribonuclease F1 in solution determined by NMR spectroscopy and distance geometry.

Two-dimensional 1H-NMR studies have been performed on ribonuclease F1 (RNase F1), which contains 106 amino acid residues. Sequence-specific resonance assignments were accomplished for the backbone protons of 99 amino acid residues and for most of their side-chain protons. The three-dimensional structures were constructed on the basis of 820 interproton-distance restraints derived from NOE, 64 distance restraints for 32 hydrogen bonds and 33 phi torsion-angle restraints. A total of 40 structures were obtained by distance geometry and simulated-annealing calculations. The average root-mean-square deviation (residues 1-106) between the 40 converged structures and the mean structure obtained by averaging their coordinates was 0.116 +/- 0.018 nm for the backbone atoms and 0.182 +/- 0.015 nm for all atoms including the hydrogen atoms. RNase F1 was determined to be an alpha/beta-type protein. A well-defined structure constitutes the core region, which consists of a small N-terminal beta-sheet (beta 1, beta 2) and a central five-stranded beta-sheet (beta 3-beta 7) packed on a long helix. The structure of RNase F1 has been compared with that of RNase T1, which was determined by X-ray crystallography. Both belong to the same family of microbial ribonucleases. The polypeptide backbone fold of RNase F1 is basically identical to that of RNase T1. The conformation-dependent chemical shifts of the C alpha protons are well conserved between RNase F1 and RNase T1. The residues implicated in catalysis are all located on the central beta-sheet in a geometry similar to that of RNase T1.     

Global folds of proteins with low densities of NOEs using residual dipolar couplings: application to the 370-residue maltodextrin-binding protein

The global fold of maltose-binding protein in complex with the substrate beta-cyclodextrin was determined by solution NMR methods. The two-domain protein is comprised of a single polypeptide chain of 370 residues, with a molecular mass of 42 kDa. Distance information in the form of H(N)-H(N), H(N)-CH(3) and CH(3)-CH(3) NOEs was recorded on (15)N, (2)H and (15)N, (13)C, (2)H-labeled proteins with methyl protonation in Val, Leu, and Ile (C(delta1) only) residues. Distances to methyl protons, critical for the structure determination, comprised 77 % of the long-range restraints. Initial structures were calculated on the basis of 1943 NOEs, 48 hydrogen bond and 555 dihedral angle restraints. A global pair-wise backbone rmsd of 5.5 A was obtained for these initial structures with rmsd values for the N and C domains of 2.4 and 3.8 A, respectively. Direct refinement against one-bond (1)H(N)-(15)N, (13)C(alpha)-(13)CO, (15)N-(13)CO, two-bond (1)H(N)-(13)CO and three-bond (1)H(N)-(13)C(alpha) dipolar couplings resulted in structures with large numbers of dipolar restraint violations. As an alternative to direct refinement against measured dipolar couplings we have developed an approach where discrete orientations are calculated for each peptide plane on the basis of the dipolar couplings described above. The orientation which best matches that in initial NMR structures calculated from NOE and dihedral angle restraints exclusively is used to refine further the structures using a new module written for CNS. Modeling studies from four different proteins with diverse structural motifs establishes the utility of the methodology. When applied to experimental data recorded on MBP the precision of the family of structures generated improves from 5.5 to 2.2 A, while the rmsd with respect to the X-ray structure (1dmb) is reduced from 5.1 to 3.3 A.     

Determination of the three-dimensional solution structure of the C-terminal domain of cellobiohydrolase I from Trichoderma reesei. A study using nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing

The solution structure of a synthetic 36-residue polypeptide comprising the C-terminal cellulose binding domain of cellobiohydrolase I (CT-CBH I) from Trichoderma reesei was investigated by nuclear magnetic resonance (NMR) spectroscopy. The 1H NMR spectrum was completely assigned in a sequential manner by two-dimensional NMR techniques. A large number of stereospecific assignments for beta-methylene protons, as well as ranges for the phi, psi, and chi 1 torsion angles, were obtained on the basis of sequential and intraresidue nuclear Overhauser enhancement (NOE) and coupling constant data in combination with a conformational data base search. The structure calculations were carried out in an iterative manner by using the hybrid distance geometry-dynamical simulated annealing method. This involved computing a series of initial structures from a subset of the experimental data in order to resolve ambiguities in the assignments of some NOE cross-peaks arising from chemical shift degeneracy. Additionally, this permitted us to extend the stereospecific assignments to the alpha-methylene protons of glycine using information on phi torsion angles derived from the initial structure calculations. The final experimental data set consisted of 554 interproton distance restraints, 24 restraints for 12 hydrogen bonds, and 33 phi, 24 psi, and 25 chi 1 torsion angle restraints. CT-CBH I has two disulfide bridges whose pairing was previously unknown. Analysis of structures calculated with all three possible combinations of disulfide bonds, as well as without disulfide bonds, indicated that the correct disulfide bridge pairing was 8-25 and 19-35. Forty-one structures were computed with the 8-25 and 19-35 disulfide bridges, and the average atomic rms difference between the individual structures and the mean structure obtained by averaging their coordinates was 0.33 +/- 0.04 A for the backbone atoms and 0.52 +/- 0.06 A for all atoms. The protein has a wedgelike shape with an amphiphilic character, one face being predominantly hydrophilic and the other mainly hydrophobic. The principal element of secondary structure is made up of an irregular triple-stranded antiparallel beta-sheet composed of residues 5-9 (beta 1), 24-28 (beta 2), and 33-36 (beta 3) in which strand beta 3 is hydrogen bonded to the other two strands.(ABSTRACT TRUNCATED AT 400 WORDS)     

The NMR structure of the activation domain isolated from porcine procarboxypeptidase B.

The three-dimensional structure of the activation domain isolated from porcine pancreatic procarboxypeptidase B was determined using 1H NMR spectroscopy. A group of 20 conformers is used to describe the solution structure of this 81 residue polypeptide chain, which has a well-defined backbone fold from residues 11-76 with an average root mean square distance for the backbone atoms of 1.0 +/- 0.1 A relative to the mean of the 20 conformers. The molecular architecture contains a four-stranded beta-sheet with the polypeptide segments 11-17, 36-39, 50-56 and 75-76, two well defined alpha-helices from residues 20-30 and 60-70, and a 3(10) helix from residues 43-46. The three helices are oriented almost exactly antiparallel to each other, are all on the same side of the beta-sheet, and the helix axes from an angle of approximately 45 degrees relative to the direction of the beta-strands. Three segments linking beta-strands and helical secondary structures, with residues 32-35, 39-43 and 56-61, are significantly less well ordered than the rest of the molecule. In the three-dimensional structure two of these loops (residues 32-35 and 56-61) are located close to each other near the protein surface, forming a continuous region of increased mobility, and the third disordered loop is separated from this region only by the peripheral beta-strand 36-39 and precedes the short 3(10) helix.     

Solution structure of recombinant hirudin and the Lys-47----Glu mutant: a nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing study

The solution structure of recombinant wild-type hirudin and of the putative active site mutant Lys-47----Glu has been investigated by nuclear magnetic resonance (NMR) spectroscopy at 600 MHz. The 1H NMR spectra of the two hirudin variants are assigned in a sequential manner with a combination of two-dimensional NMR techniques. Some assignments made in our previous paper [Sukumaran, D. K., Clore, G. M., Preuss, A., Zarbock, J., & Gronenborn, A. M. (1987) Biochemistry 26, 333-338] were found to be incorrect and are now corrected. Analysis of the NOE data indicates that hirudin consists of an N-terminal compact domain (residues 1-49) held together by three disulfide linkages and a disordered C-terminal tail (residues 50-65) which does not fold back on the rest of the protein. This last observation corrects conclusions drawn by us previously on hirudin extracted from its natural source, the leech Hirudo medicinalis. The improved sensitivity of the 600-MHz spectrometer relative to that of our old 500-MHz spectrometer, the availability of two variants with slightly different chemical shifts, and the additional information arising from stereospecific assignments of methylene beta-protons and methyl protons of valine have permitted the determination of the solution structure of hirudin with much greater precision than before. Structure calculations on the N-terminal domain using the hybrid distance geometry-dynamical simulated annealing method were based on 685 and 661 approximate interproton distance restraints derived from nuclear Overhauser enhancement (NOE) data for the wild-type and mutant hirudin, respectively, together with 16 distance restraints for 8 backbone hydrogen bonds identified on the basis of NOE and amide NH exchange data and 26 phi backbone and 18 chi 1 side-chain torsion angle restraints derived from NOE and three-bond coupling constant data. A total of 32 structures were computed for both the wild-type and mutant hirudin. The structure of residues 2-30 and 37-48 which form the core of the N-terminal domain is well determined in both cases with an average atomic rms difference between the individual structures and the respective mean structures of approximately 0.7 A for the backbone atoms and approximately 1 A for all atoms. As found previously, the orientation of the exposed finger of antiparallel beta-sheet (residues 31-36) with respect to the core could not be determined on the basis of the present data due to the absence of any long-range NOEs between the exposed finger and the core.(ABSTRACT TRUNCATED AT 250 WORDS)     

Three-dimensional structure of acyl carrier protein in solution determined by nuclear magnetic resonance and the combined use of dynamical simulated annealing and distance geometry

The solution conformation of acyl carrier protein from Escherichia coli (77 residues) has been determined on the basis of 423 interproton-distance restraints and 32 hydrogen-bonding restraints derived from NMR measurements. A total of nine structures were computed using a hybrid approach combining metric matrix distance geometry and dynamic simulated annealing. The polypeptide fold is well defined with an average backbone atomic root-mean-square difference of 0.20 +/- 0.03 nm between the final nine converged structures and the mean structure obtained by averaging their coordinates. The principal structural motif is composed of three helices: 1 (residues 3-12), 2 (residues 37-47) and 4 (residues 65-75) which line a hydrophobic cavity. Helices 2 and 4 are approximately parallel to each other and anti-parallel at an angle of approximately equal to 150 degrees to helix 1. The smaller helix 3 (residues 56-63) is at an angle of approximately equal to 100 degrees to helix 4.     

The solution conformation of the antibacterial peptide cecropin A: a nuclear magnetic resonance and dynamical simulated annealing study

The solution conformation of the antibacterial polypeptide cecropin A from the Cecropia moth is investigated by nuclear magnetic resonance (NMR) spectroscopy under conditions where it adopts a fully ordered structure, as judged by previous circular dichroism studies [Steiner, H. (1982) FEBS Lett. 137, 283-287], namely, 15% (v/v) hexafluoroisopropyl alcohol. By use of a combination of two-dimensional NMR techniques the 1H NMR spectrum of cecropin A is completely assigned. A set of 243 approximate interproton distance restraints is derived from nuclear Overhauser enhancement (NOE) measurements. These, together with 32 distance restraints for the 16 intrahelical hydrogen bonds identified on the basis of the pattern of short-range NOEs, form the basis of a three-dimensional structure determination by dynamical simulated annealing [Nilges, M., Clore, G.M., & Gronenborn, A.M. (1988) FEBS Lett. 229, 317-324]. The calculations are carried out starting from three initial structures, an alpha-helix, an extended beta-strand, and a mixed alpha/beta structure. Seven independent structures are computed from each starting structure by using different random number seeds for the assignments of the initial velocities. All 21 calculated structures satisfy the experimental restraints, display very small deviations from idealized covalent geometry, and possess good nonbonded contacts. Analysis of the 21 converged structure indicates that there are two helical regions extending from residues 5 to 21 and from residues 24 to 37 which are very well defined in terms of both atomic root mean square differences and backbone torsion angles. For the two helical regions individually the average backbone rms difference between all pairs of structures is approximately 1 A. The long axes of the two helices lie in two planes, which are at an angle of 70-100 degrees to each other. The orientation of the helices within these planes, however, cannot be determined due to the paucity of NOEs between the two helices.     

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.     

C-terminal retroviral-type zinc finger domain from the HIV-1 nucleocapsid protein is structurally similar to the N-terminal zinc finger domain

Two-dimensional NMR spectroscopic and computational methods were employed for the structure determination of an 18-residue peptide with the amino acid sequence of the C-terminal retroviral-type (r.t.) zinc finger domain from the nucleocapsid protein (NCP) of HIV-1 [Zn(HIV1-F2)]. Unlike results obtained for the first retroviral-type zinc finger peptide, Zn(HIV1-F1), [Summers et al. (1990) Biochemistry 29, 329], broad signals indicative of conformational lability were observed in the 1H NMR spectrum of Zn-(HIV1-F2) at 25 degrees C. The NMR signals narrowed upon cooling to -2 degrees C, enabling complete 1H NMR signal assignment via standard two-dimensional (2D) NMR methods. Distance restraints obtained from qualitative analysis of 2D nuclear Overhauser effect (NOESY) data were used to generate 30 distance geometry (DG) structures with penalties (penalty = sum of the squared differences between interatomic distances defined in the restraints file and in the DG structures) in the range 0.02-0.03 A2. All structures were qualitatively consistent with the experimental NOESY spectrum based on comparisons with 2D NOESY back-calculated spectra. Superposition of the backbone atoms (C, C alpha, N) for residues C(1)-C(14) gave pairwise RMSD values in the range 0.16-0.75 A. The folding of Zn(HIV1-F2) is very similar to that observed for Zn(HIV1-F1). Small differences observed between the two finger domains are localized to residues between His(9) and Cys(14), with residues M(11)-C(14) forming a 3(10) helical corner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequence-specific 1H NMR assignments and secondary structure in the sea anemone polypeptide Stichodactyla helianthus neurotoxin I

Sequence-specific assignments are reported for the 500-MHz 1H nuclear magnetic resonance (NMR) spectrum of the 48-residue polypeptide neurotoxin I from the sea anemone Stichodactyla helianthus (Sh I). Spin systems were first identified by using two-dimensional relayed or multiple quantum filtered correlation spectroscopy, double quantum spectroscopy, and spin lock experiments. Specific resonance assignments were then obtained from nuclear Overhauser enhancement (NOE) connectivities between protons from residues adjacent in the amino acid sequence. Of a total of 265 potentially observable resonances, 248 (i.e., 94%) were assigned, arising from 39 completely and 9 partially assigned amino acid spin systems. The secondary structure of Sh I was defined on the basis of the pattern of sequential NOE connectivities, NOEs between protons on separate strands of the polypeptide backbone, and backbone amide exchange rates. Sh I contains a four-stranded antiparallel beta-sheet encompassing residues 1-5, 16-24, 30-33, and 40-46, with a beta-bulge at residues 17 and 18 and a reverse turn, probably a type II beta-turn, involving residues 27-30. No evidence of alpha-helical structure was found.     

Three-dimensional structure of potato carboxypeptidase inhibitor in solution. A study using nuclear magnetic resonance, distance geometry, and restrained molecular dynamics

The solution conformation of potato carboxypeptidase inhibitor (CPI) has been investigated by 1H NMR spectroscopy. The spectrum is assigned in a sequential manner by using two-dimensional NMR techniques to identify through-bond and through-space (less than 5 A) connectivities. A set of 309 approximate interproton distance restraints is derived from the two-dimensional nuclear Overhauser enhancement spectra and used as the basis of a three-dimensional structure determination by a combination of metric matrix distance geometry and restrained molecular dynamics calculations. A total of 11 converged distance geometry structures were computed and refined by using restrained molecular dynamics. The average atomic root mean square (rms) difference between the final 11 structures and the mean structure obtained by averaging their coordinates is 1.4 +/- 0.3 A for residues 2-39 and 0.9 +/- 0.2 A for residues 5-37. The corresponding values for all atoms are 1.9 +/- 0.3 and 1.4 +/- 0.2 A, respectively. The larger values for residues 2-38 relative to those for residues 5-37 arise from the fact that the positions of the N- (residues 1-4) and C- (residues 38-39) terminal tails are rather poorly determined, whereas those of the core of the protein (residues 5-37) are well determined by the experimental interproton distance data. The computed structures are very close to the X-ray structure of CPI in its complex with carboxypeptidase, and the backbone atomic rms difference between the mean of the computed structures and the X-ray structure is only 1.2 A. Nevertheless, there are some real differences present which are evidenced by significant deviations between the experimental upper interproton distance limits and the corresponding interproton distances derived from the X-ray structure. These principally occur in two regions, residues 18-20 and residues 28-30, the latter comprising part of the region of secondary contacts between CPI and carboxypeptidase in the X-ray structure.     

High-resolution three-dimensional structure of reduced recombinant human thioredoxin in solution

The solution structure of recombinant human thioredoxin (105 residues) has been determined by nuclear magnetic resonance (NMR) spectroscopy combined with hybrid distance geometry-dynamical simulated annealing calculations. Approximate interproton distance restraints were derived from nuclear Overhauser effect (NOE) measurements. In addition, a large number of stereospecific assignments for beta-methylene protons and torsion angle restraints for phi, psi, and chi 1 were obtained by using a conformational grid search on the basis of the intraresidue and sequential NOE data in conjunction with 3JHN alpha and 3J alpha beta coupling constants. The structure calculations were based on 1983 approximate interproton distance restraints, 52 hydrogen-bonding restraints for 26 hydrogen bonds, and 98 phi, 71 psi, and 72 chi 1 torsion angle restraints. The 33 final simulated annealing structures obtained had an average atomic rms distribution of the individual structures about the mean coordinate positions of 0.40 +/- 0.06 A for the backbone atoms and 0.78 +/- 0.05 A for all atoms. The solution structure of human thioredoxin consists of a five-stranded beta-sheet surrounded by four alpha-helices, with an active site protrusion containing the two redox-active cysteines. The overall structure is similar to the crystal and NMR structures of oxidized [Katti, S. K., LeMaster, D. M., & Eklund, H. (1990) J. Mol. Biol. 212, 167-184] and reduced [Dyson, J. H., Gippert, G. P., Case, D. A., Holmgren, A., & Wright, P. (1990) Biochemistry 29, 4129-4136] Escherichia coli thioredoxin, respectively, despite the moderate 25% amino acid sequence homology. Several differences, however, can be noted. The human alpha 1 helix is a full turn longer than the corresponding helix in E. coli thioredoxin and is characterized by a more regular helical geometry. The helix labeled alpha 3 in human thioredoxin has its counterpart in the 3(10) helix of the E. coli protein and is also longer in the human protein. In contrast to these structural differences, the conformation of the active site loop in both proteins is very similar, reflecting the perfect sequence identity for a stretch of eight amino acid residues around the redox-active cysteines.     

The influence of stereospecific assignments on the determination of three-dimensional structures of proteins by nuclear magnetic resonance spectroscopy. Application to the sea anemone protein BDS-I

The influence of the stereospecific assignments of beta-methylene protons and the classification of chi 1 torsion angles on the definition of the three-dimensional structures of proteins determined from NMR data is investigated using the sea anemone protein BDS-I (43 residues) as a model system. Two sets of structures are computed. The first set comprises 42 converged structures (denoted STEREO structures) calculated on the basis of the complete list of restraints derived from the NMR data, consisting of 489 interproton and 24 hydrogen bonding distance restraints, supplemented by 23 phi backbone and 21 chi 1 side chain torsion angle restraints. The second set comprises 31 converged structures (denoted NOSTEREO structures) calculated from a reduced data set in which those restraints arising from stereospecific assignments, and the corresponding chi 1 torsion angle restraints, are explicitly omitted. The results show that the inclusion of the stereospecific restraints leads to a significant improvement in the definition of the structure of BDS-I, both with respect to the backbone and the detailed arrangement of the side chains. Average atomic rms differences between the individual structures and the mean structures for the backbone atoms are 0.67 +/- 0.12 A and 0.93 +/- 0.16 A for the STEREO and NOSTEREO structures, respectively; the corresponding values for all atoms are 0.90 +/- 0.17 A and 1.17 +/- 0.17 A, respectively. In addition, while the overall fold remains unchanged, there is a small but significant atomic displacement between the two sets of structures.     

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].     

Low resolution structure of interleukin-1 beta in solution derived from 1H-15N heteronuclear three-dimensional nuclear magnetic resonance spectroscopy

A low resolution solution structure of the cytokine interleukin-1 beta, a 153 residue protein of molecular weight 17,400, has been determined on the basis of 446 nuclear Overhauser effect (NOE) derived approximate interproton distance restraints involving solely NH, C alpha H and C beta H protons, supplemented by 90 distance restraints for 45 hydrogen bonds, and 79 phi torsion angle restraints. With the exception of 27 C alpha H-C alpha H NOEs, all the NOEs were assigned from a three-dimensional 1H-1H NOE 15N-1H heteronuclear multiple quantum coherence (HMQC) spectrum. The torsion angle restraints were obtained from accurate 3JHN alpha coupling constants measured from a HMQC-J spectrum, while the hydrogen bonds were derived from a qualitative analysis of the NOE, coupling constant and amide exchange data. A total of 20 simulated annealing (SA) structures was computed using the hybrid distance geometry-dynamical simulated annealing method. The solution structure of IL-1 beta comprises 12 beta-strands arranged in three pseudo-symmetrical topological units (each consisting of 5 anti-parallel beta-strands), joined by turns, short loops and long loops. The core of the structure, which is made up of the 12 beta-strands, together with the turns joining strands I and II, strands VIII and IX and strands X and XI, is well determined with a backbone atomic root-mean-square (r.m.s.) distribution about the mean co-ordinate positions of 1.2(+/- 0.1) A. The loop conformations, on the other hand, are poorly determined by the current data. A comparison of the core of the low resolution solution structure of IL-1 beta with that of the X-ray structure indicates that they are similar, with a backbone atomic r.m.s. difference of only 1.5 A between the co-ordinates of the restrained minimized mean of the SA structures and the X-ray structure.     

1H NMR studies of human C3a anaphylatoxin in solution: sequential resonance assignments, secondary structure, and global fold

The spin systems that comprise the 1H nuclear magnetic resonance (NMR) spectrum of the complement fragment C3a (Mr 8900) have been completely identified by an approach which integrates data from a wide range of two-dimensional NMR experiments. Both relayed and multiple quantum experiments play an essential role in the analysis. After the first stage of analysis the spin systems of 60 of the 77 residues were assigned to the appropriate residue type, providing an ample basis for subsequent sequence-specific assignments. Elements of secondary structure were identified on the basis of networks of characteristic sequential and medium-range nuclear Overhauser effects (NOEs), values of 3JHN alpha, and locations of slowly exchanging backbone amide protons. Three well-defined helical segments are found. Gradients of increasing mobility in distinct segments of the C3a polypeptide are observed, with very high mobilities for several residues near the C- and N-termini, including the complete C-terminal receptor binding site pentapeptide LGLAR. The NMR data, combined with known disulfide linkages and a small number of critical long-range NOEs, provide the global folding pattern of C3a in solution. Identical solution structures were found for both the intact active protein and the largely inactive physiologic product des-Arg77-C3a.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.