The NMR solution structure of the pheromone Er-11 from the ciliated protozoan Euplotes raikovi.

The NMR solution structure of the pheromone Er-11, a 39-residue protein from the ciliated protozoan Euplotes raikovi, was calculated with the distance geometry program DIANA from 449 NOE upper distance constraints and 97 dihedral angle constraints, and the program OPAL was employed for structure refinement by molecular mechanics energy minimization in a water bath. For a group of 20 conformers used to characterize the solution structure, the average of the pairwise RMS deviations from the mean structure calculated for the backbone heavy atoms N, C alpha, and C' of residues 2-38 was 0.30 A. The molecular architecture is dominated by an up-down-up bundle of three short helices with residues 2-9, 12-19, and 22-32, which is closely similar to the previously determined structures of the homologous pheromones Er-1, Er-2, and Er-10. This finding provides structural evidence for the capability shown by these pheromones to compete with each other in binding reactions to their cell-surface receptors.     

Three-dimensional structure of the neurotoxin ATX Ia from Anemonia sulcata in aqueous solution determined by nuclear magnetic resonance spectroscopy

With the aid of 1H nuclear magnetic resonance (NMR) spectroscopy, the three-dimensional structure in aqueous solution was determined for ATX Ia, which is a 46 residue polypeptide neurotoxin of the sea anemone Anemonia sulcata. The input for the structure calculations consisted of 263 distance constraints from nuclear Overhauser effects (NOE) and 76 vicinal coupling constants. For the structure calculation several new or ammended programs were used in a revised strategy consisting of five successive computational steps. First, the program HABAS was used for a complete search of all backbone and chi 1 conformations that are compatible with the intraresidual and sequential NMR constraints. Second, using the program DISMAN, we extended this approach to pentapeptides by extensive sampling of all conformations that are consistent with the local and medium-range NMR constraints. Both steps resulted in the definition of additional dihedral angle constraints and in stereospecific assignments for a number of beta-methylene groups. In the next two steps DISMAN was used to obtain a group of eight conformers that contain no significant residual violations of the NMR constraints or van der Waals contacts. Finally, these structures were subjected to restrained energy refinement with a modified version of the molecular mechanics module of AMBER, which in addition to the energy force field includes potentials for the NOE distance constraints and the dihedral angle constraints. The average of the pairwise minimal RMS distances between the resulting refined conformers calculated for the well defined molecular core, which contains the backbone atoms of 35 residues and 20 interior side chains, is 1.5 +/- 0.3 A. This core is formed by a four-stranded beta-sheet connected by two well-defined loops, and there is an additional flexible loop consisting of the eleven residues 8-18. The core of the protein is stabilized by three disulfide bridges, which are surrounded by hydrophobic residues and shielded on one side by hydrophilic residues.     

The NMR solution structure of the pheromone Er-2 from the ciliated protozoan Euplotes raikovi.

The NMR structure of the pheromone Er-2 from the ciliated protozoan Euplotes raikovi has been determined in aqueous solution. The structure of this 40-residue protein was calculated with the distance geometry program DIANA from 621 distance constraints and 89 dihedral angle constraints; the program OPAL was employed for the energy minimization. For a group of 20 conformers used to characterize the solution structure, the average pairwise RMS deviation from the mean structure calculated for the backbone heavy atoms N, C alpha, and C' of residues 3-37 was 0.31 A. The molecular architecture is dominated by an up-down-up bundle of 3 short helices of residues 5-11, 14-20, and 23-33, which is similar to the structures of the homologous pheromones Er-1 and Er-10. Novel structural features include a well-defined N-cap on the first helix, a 1-residue deletion in the second helix resulting in the formation of a 3(10)-helix rather than an alpha-helix as found in Er-1 and Er-10, and the simultaneous presence of 2 different conformations for the C-terminal tetrapeptide segment, i.e., a major conformation with the Leu 39-Pro 40 peptide bond in the trans form and a minor conformation with this peptide bond in the cis form.     

Nuclear magnetic resonance solution structure of the alpha-neurotoxin from the black mamba (Dendroaspis polylepis polylepis).

The three-dimensional structure in solution of the alpha-neurotoxin from the black mamba (Dendroaspis polylepis polylepis) has been determined by nuclear magnetic resonance spectroscopy. A high quality structure for this 60-residue protein was obtained from 656 NOE distance constraints and 143 dihedral angle constraints, using the distance geometry program DIANA for the structure calculation and AMBER for restrained energy minimization. For a group of 20 conformers used to represent the solution structure, the average root-mean-square deviation value calculated for the polypeptide backbone heavy atoms relative to the mean structure was 0.45 A. The protein consists of a core region from which three finger-like loops extend outwards. It includes a short, two-stranded antiparallel beta-sheet of residues 1-5 and 13-17, a three-stranded antiparallel beta-sheet involving residues 23-31, 34-42 and 51-55, and four disulfide bridges in the core region. There is also extensive non-regular hydrogen bonding between the carboxy-terminal tail of the polypeptide chain and the rest of the core region. Comparison with the crystal structure of erabutoxin-b indicates that the structure of alpha-neurotoxin is quite similar to other neurotoxin structures, but that local structural differences are seen in regions thought to be important for binding of neurotoxins to the acetylcholine receptor. For two regions of the alpha-neurotoxin structure there is evidence for an equilibrium between multiple conformations, which might be related to conformational rearrangements upon binding to the receptor. Overall, the alpha-neurotoxin presents itself as a protein with a stable core and flexible surface areas that interact with the acetylcholine receptor in such a way that high affinity binding is achieved by conformational rearrangements of the deformable regions of the neurotoxin structure.     

The NMR solution structure of the pheromone Er-1 from the ciliated protozoan Euplotes raikovi.

The 3-dimensional structure of the pheromone Er-1 isolated from the ciliated protozoan Euplotes raikovi has been determined in aqueous solution by 1H NMR spectroscopy. The structure of this 40-residue protein was calculated with the distance geometry program DIANA on the basis of 503 upper distance constraints derived from nuclear Overhauser effects and 77 dihedral angle constraints derived from spin-spin coupling constants, and refined by restrained energy minimization with the program OPAL. The Er-1 solution structure is represented by a group of 20 conformers with an average RMS deviation relative to the mean structure of 0.55 A for the backbone atoms N, C alpha, and C', and 0.93 A for all heavy atoms of the complete polypeptide chain, residues 1-40. The molecular architecture is dominated by an up-down-up bundle of 3 alpha-helices formed by residues 2-9, 12-19, and 24-33. Although this core part coincides closely with the previously determined structure of the homologous pheromone Er-10, the C-terminal peptide segment adopts a novel conformation. This is of interest in view of previous suggestions, based on sequence comparisons, that this molecular region may be important for the different specificity of receptor recognition by different pheromones.     

NMR solution structure of alpha-conotoxin ImI and comparison to other conotoxins specific for neuronal nicotinic acetylcholine receptors

Alpha-Conotoxins, peptides produced by predatory species of Conus marine snails, are potent antagonists of nicotinic acetylcholine receptors (nAChRs), ligand-gated ion channels involved in synaptic transmission. We determined the NMR solution structure of the smallest known alpha-conotoxin, ImI, a 12 amino acid peptide that binds specifically to neuronal alpha7-containing nAChRs in mammals. Calculation of the structure was based on a total of 80 upper distance constraints and 31 dihedral angle constraints resulting in 20 representative conformers with an average pairwise rmsd of 0.44 A from the mean structure for the backbone atoms N, Calpha, and C' of residues 2-11. The structure of ImI is characterized by two compact loops, defined by two disulfide bridges, which form distinct subdomains separated by a deep cleft. Two short 310-helical regions in the first loop are followed by a C-terminal beta-turn in the second. The two disulfide bridges and Ala 9 form a rigid hydrophobic core, orienting the other amino acid side chains toward the surface. Comparison of the three-dimensional structure of ImI to those of the larger, 16 amino acid alpha-conotoxins PnIA, PnIB, MII, and EpI-also specific for neuronal nAChRs-reveals remarkable similarity in local backbone conformations and relative solvent-accessible surface areas. The core scaffold is conserved in all five conotoxins, whereas the residues in solvent-exposed positions are highly variable. The second helical region, and the specific amino acids that the helix exposes to solvent, may be particularly important for binding and selectivity. This comparative analysis provides a three-dimensional structural basis for interpretation of mutagenesis data and structure-activity relationships for ImI as well other neuronal alpha-conotoxins.     

A structurally deviant member of the Euplotes raikovi pheromone family: Er-23

Pheromones of Euplotes raikovi form a homologous family of proteins with 37- to 40-amino acid residues, including six cysteines that form three strictly conserved disulfide bridges. The determination of the primary structure of the pheromone Er-23, which was isolated from cells derived from natural populations of E. raikovi that secrete the other known pheromones, has now revealed a novel structure type. The polypeptide chain of this pheromone contains 51 residues, 10 of which are cysteines presumably involved in the formation of five disulfide bridges, and lacks a carboxyl-terminal tail following the last cysteine of the sequence. The elongation of the Er-23 molecule is presumed to result from multiple events of gene duplication starting from an ancestral motif Xxx(2-4)-Cys-Xxx(5-7)-Cys.     

Solution conformation of proteinase inhibitor IIA from bull seminal plasma by 1H nuclear magnetic resonance and distance geometry

A determination of the solution conformation of the proteinase inhibitor IIA from bull seminal plasma (BUSI IIA) is described. Two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) was used to obtain a list of 202 distance constraints between individually assigned hydrogen atoms of the polypeptide chain, to identify the positions of the three disulfide bridges, and to locate the single cis peptide bond. Supplementary geometric constraints were derived from the vicinal spin-spin couplings and the locations of certain hydrogen bonds, as determined by nuclear magnetic resonance (n.m.r.). Using a new distance geometry program (DISGEO) which is capable of computing all-atom structures for proteins the size of BUSI IIA, five conformers were computed from the NOE distance constraints alone, and another five were computed with the supplementary constraints included. Comparison of the different structures computed from the n.m.r. data among themselves and with the crystal structures of two homologous proteins shows that the global features of the conformation of BUSI IIA (i.e. the overall dimensions of the molecule and the threading of the polypeptide chain) were well-defined by the available n.m.r. data. In the Appendix, we describe a preliminary energy refinement of the structure, which showed that the constraints derived from the n.m.r. data are compatible with a low energy spatial structure.     

Tertiary structure of conotoxin GIIIA in aqueous solution

The three-dimensional structure of conotoxin GIIIA, an important constituent of the venom from the marine hunting snail Conus geographus L., was determined in aqueous solution by two-dimensional proton nuclear magnetic resonance and simulated annealing based methods. On the basis of 162 assigned nuclear Overhauser effect (NOE) connectivities obtained at the medium field strength frequency of 400 MHz, 74 final distance constraints of sequential and tertiary ones were derived and used together with 18 torsion angle (phi, chi 1) constraints and 9 distance constraints derived from disulfide bridges. A total of 32 converged structures were obtained from 200 runs of calculations. The atomic root-mean-square (RMS) difference about the mean coordinate positions (excluding the terminal residues 1 and 22) is 0.8 A for backbone atoms (N, C alpha, C). Conotoxin GIIIA is characterized by a particular folding of the 22 amino acid peptidic chain, which is stabilized by three disulfide bridges arranged in cage at the center of a discoidal structure of approximately 20-A diameter. The seven cationic side chains of lysine and arginine residues project radially into the solvent and form potential sites of interaction with the skeletal muscle sodium channel for which the toxin is a strong inhibitor. The present results provide a molecular basis to elucidate the remarkable physiological properties of this neurotoxin.     

Three-dimensional structure of the mini-M conotoxin mr3a

Conotoxin mr3a from the venom of Conus marmoreus, a novel peptide that induces rolling seizures in mice, has the peptide sequence GCCGSFACRFGCVOCCV, where O is trans-4-hydroxyproline, and the chain is cross-linked with disulfide bonds between Cys-2 and Cys-16, Cys-3 and Cys-12, and Cys-8 and Cys-15. The tertiary structure of mr3a was determined by 2D 1H NMR in combination with a standard distance-geometry algorithm. The final set of 22 structures for the peptide had a mean global backbone RMS deviation of 0.53 +/- 0.22 A based on 51 NOE, 6 hydrogen bond, 6 phi dihedral angle, and 3 disulfide bond constraints. Conotoxin mr3a is the first example of the new mini-M branch of conopeptides in the M superfamily. Members of the maxi-M branch, whose structures are known, include the mu- and psi-conotoxins, both of which share a common disulfide bond connectivity. Although mr3a has the same arrangement of Cys residues as the mu- and psi-conotoxins, its disulfide connectivity is different. This gives mr3a a distinctive "triple-turn" backbone.     

Solution structure of neuronal bungarotoxin determined by two-dimensional NMR spectroscopy: sequence-specific assignments, secondary structure, and dimer formation

The solution structure of neuronal bungarotoxin (nBgt) has been studied by using two-dimensional 1H NMR spectroscopy. Sequence-specific assignments for over 95% of the backbone resonances and 85% of the side-chain resonances have been made by using a series of two-dimensional spectra at four temperatures. From these assignments over 75% of the NOESY spectrum has been assigned, which has in turn provided 582 distance constraints. Twenty-seven coupling constants (NH-alpha CH) were determined from the COSY spectra, which have provided dihedral angle constraints. In addition, hydrogen exchange experiments have suggested the probable position of hydrogen bonds. The NOE constraints, dihedral angle constraints, and the rates of amide proton exchange suggest that a triple-stranded antiparallel beta sheet is the major component of secondary structure, which includes 25% of the amino acid residues. A number of NOE peaks were observed that were inconsistent with the antiparallel beta-sheet structure. Because we have confirmed by sedimentation equilibrium that nBgt exists as a dimer, we have reinterpreted these NOE constraints as intermolecular interactions. These constraints suggest that the dimer consists of a six-stranded antiparallel beta sheet (three from each monomer), with residues 55-59 forming the dimer interface.     

NMR solution structure of the non-RGD disintegrin obtustatin

The solution structure of obtustatin, a novel non-RGD disintegrin of 41 residues isolated from Vipera lebetina obtusa venom, and a potent and selective inhibitor of the adhesion of integrin alpha(1)beta(1) to collagen IV, has been determined by two-dimensional nuclear magnetic resonance. Almost the whole set of chemical shifts for 1H, 13C and 15N were assigned at natural abundance from 2D homonuclear and heteronuclear 500 MHz, 600 MHz and 800 MHz spectra at pH 3.0 recorded at 298 K and 303 K. Final structural constraints consisted of 302 non-redundant NOE (95 long-range, 60 medium, 91 sequential and 56 intra-residue), four disulfide bond distances, five chi1 dihedral angles and four hydrogen bonds. The 20 conformers with lowest total energy had no NOE violations greater than 0.35A or dihedral angle violations greater than 12 degrees. The average root-mean-square deviation (RMSD) for backbone atoms of all residues among the 20 conformers was 1.1A and 0.6A for the 29 best-defined residues. Obtustatin lacks any secondary structure. Compared to all known disintegrin structures in which the RGD motif is located at the apex of an 11 residue hairpin loop, the active KTS tripeptide of obtustatin is oriented towards a side of its nine residue integrin-binding loop. The C-terminal tail is near to the active loop, and these two structural elements display the largest atomic displacements due to local conformational disorder. Double cross-peaks for W20, Y28 and H27 in the aromatic region of TOCSY spectra, local RMSD values for these residues, and positive cross-peaks in a ROESY spectrum (600 MHz, 100 ms mixing time), suggest that these residues act as a hinge allowing for the overall flexibility of the entire integrin-binding loop. These distinct structural features, along with its different electrostatic surface potential in relation to other known disintegrins, may confer to obtustatin its reported alpha(1)beta(1) integrin inhibitory selectivity.     

Solution structure of BTK-2, a novel hK(v)1.1 inhibiting scorpion toxin, from the eastern Indian scorpion Mesobuthus tamulus

The three dimensional structure of a 32 residue three disulfide scorpion toxin, BTK-2, from the Indian red scorpion Mesobuthus tamulus has been determined using isotope edited solution NMR methods. Samples for structural and electrophysiological studies were prepared using recombinant DNA methods. Electrophysiological studies show that the peptide is active against hK(v)1.1 channels. The structure of BTK-2 was determined using 373 distance restraints from NOE data, 66 dihedral angle restraints from NOE, chemical shift and scalar coupling data, 6 constraints based on disulfide linkages and 8 constraints based on hydrogen bonds. The root mean square deviation (r.m.s.d) about the averaged co-ordinates of the backbone (N, C(α), C') and all heavy atoms are 0.81 ± 0.23? and 1.51 ± 0.29? respectively. The backbone dihedral angles (? and ψ) for all residues occupy the favorable and allowed regions of the Ramachandran map. The three dimensional structure of BTK-2 is composed of three well defined secondary structural regions that constitute the α-β-β structural motif. Comparisons between the structure of BTK-2 and other closely related scorpion toxins pointed towards distinct differences in surface properties that provide insights into the structure-function relationships among this important class of voltage-gated potassium channel inhibiting peptides.     

Determination of three-dimensional solution structure of waglerin I,a toxin from Trimeresurus wagleri,using 2D-NMR and molecular dynamics simulation

The solution conformation of a synthetic snake venom toxin waglerin I, has been determined by using proton nuclear magnetic resonance spectroscopy. By y a combination of various two-dimensional NMR techniques, the ¹H-NMR spectrum of waglerin I was completely assigned. A set of 247 interproton distance restraints was derived from nuclear Overhauser enhancement (NOE) measurements. These NOE constraints, in addition to the 2 dihedral angle restraints (from coupling constant measurements) and 7 ω torsion angle restraints for prolines, formed the basis of three-dimensional structure determined by molecular dynamics techniques. The 19 structures that were obtained satisfy the experimental restraints, and display small deviation from idealized covalent geometry. Analysis of converged structures indicates that the toxin has no special secondary structure. In the solution structure of waglerin I, the central ring region is well defined but the N- and C-termini possesses more disorder.     

Solution structure of plant nonspecific lipid transfer protein-2 from rice (Oryza sativa)

The three-dimensional structure of rice nonspecific lipid transfer protein (nsLTP2) has been solved for the first time. The structure of nsLTP2 was obtained using 813 distance constraints, 30 hydrogen bond constraints, and 19 dihedral angle constraints. Fifteen of the 50 random simulated annealing structures satisfied all of the constraints and possessed good nonbonded contacts. The novel three-dimensional fold of rice nsLTP2 contains a triangular hydrophobic cavity formed by three prominent helices. The four disulfide bonds required for stabilization of the nsLTP2 structure show a different pattern of cysteine pairing compared with nsLTP1. The C terminus of the protein is very flexible and forms a cap over the hydrophobic cavity. Molecular modeling studies suggested that the hydrophobic cavity could accommodate large molecules with rigid structures, such as sterols. The positively charged residues on the molecular surface of nsLTP2 are structurally similar to other plant defense proteins.     

Solution structure of an M-1 conotoxin with a novel disulfide linkage

The M-superfamily of conotoxins has a typical Cys framework (-CC-C-C-CC-), and is one of the eight major superfamilies found in the venom of the cone snail. Depending on the number of residues located in the last Cys loop (between Cys4 and Cys5), the M-superfamily family can be divided into four branches, namely M-1, -2, -3 and -4. Recently, two M-1 branch conotoxins (mr3e and tx3a) have been reported to possess a new disulfide bond arrangement between Cys1 and Cys5, Cys2 and Cys4, and Cys3 and Cys6, which is different from those seen in the M-2 and M-4 branches. Here we report the 3D structure of mr3e determined by 2D (1)H NMR in aqueous solution. Twenty converged structures of this peptide were obtained on the basis of 190 distance constraints obtained from NOE connectivities, as well as six varphi dihedral angle, three hydrogen bond, and three disulfide bond constraints. The rmsd values about the averaged coordinates of the backbone atoms were 0.43 +/- 0.19 A. Although mr3e has the same Cys arrangement as M-2 and M-4 conotoxins, it adopts a distinctive backbone conformation with the overall molecule resembling a 'flying bird'. Thus, different disulfide linkages may be employed by conotoxins with the same Cys framework to result in a more diversified backbone scaffold.     

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.     

Solution structure of Eucommia antifungal peptide: a novel structural model distinct with a five-disulfide motif

The three-dimensional structure in aqueous solution of Eucommia antifungal peptide 2 (EAFP2) from Eucommia ulmoides Oliv was determined using (1)H NMR spectroscopy. EAFP2 is a newly discovered 41-residue peptide distinct with a five-disulfide cross-linked motif. This peptide exhibits chitin-binding activity and inhibitory effects on the growth of cell wall chitin-containing fungi and chitin-free fungi. The structure was calculated by using torsion angle dynamic simulated annealing with a total of 614 distance restraints and 16 dihedral restraints derived from NOESY and DQF-COSY spectra, respectively. The five disulfide bonds were assigned from preliminary structures using a statistical analysis of intercystinyl distances. The solution structure of EAFP2 is presented as an ensemble of 20 conformers with a backbone RMS deviation of 0.65 (+/-0.13) A for the well-defined Cys3-Cys39 segment. The tertiary structure of EAFP2 represents the first five-disulfide cross-linked structural model of the plant antifungal peptide. EAFP2 adopts a compact global fold composed of a 3(10) helix (Cys3-Arg6), an alpha-helix (Gly26-Cys30), and a three-strand antiparallel beta-sheet (Cys16-Ser18, Tyr22-Gly24, and Arg36-Cys37). The tertiary structure of EAFP2 shows a chitin-binding domain (residues 11-30) with a hydrophobic face and a characteristic sector formed by the N-terminal 10 residues and the C-terminal segment cross-linked through the unique disulfide bond Cys7-Cys37, which brings all four positively charged residues (Arg6, Arg9, Arg36, and Arg40) onto a cationic face. On the basis of such a structural feature, the possible structural basis for the functional properties of EAFP2 is discussed.     

1H nuclear-magnetic-resonance studies of the three-dimensional structure of the cardiotoxin CTXIIb from Naja mossambica mossambica in aqueous solution and comparison with the crystal structures of homologous toxins

Using the previously reported sequence-specific 1H-NMR assignments, structural constraints for the cardiotoxin CTXIIb from Naja mossambica mossambica were collected. These include distance constraints from nuclear Overhauser enhancement measurements both in the laboratory and in the rotating frame, dihedral angle constraints derived from spin-spin coupling constants, and constraints from hydrogen bonds and disulfide bridges. Structure calculations with the distance geometry program DISMAN confirmed the presence of the previously identified antiparallel beta-sheets formed by residues 1-5 and 10-14, and by 20-27, 35-39 and 49-55, and established the nature of the connections between the individual beta-strands. These include a right-handed crossover between the two peripheral strands in the triple-stranded beta-sheet, and a type I tight turn immediately preceding the beta-strand 49-55. The spatial arrangement of the polypeptide backbone in the solution structure of CTXIIb is closely similar to that in the crystal structure of the homologous cardiotoxin VII4 from the same species. In an Appendix the origin of the large pH dependence of two amide proton chemical shifts in CTXIIb is explained.     

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.