Bovine pancreatic polypeptide (bPP) undergoes significant changes in conformation and dynamics upon binding to DPC micelles

The pancreatic polypeptide (PP), a 36-residue, C-terminally amidated polypeptide hormone is a member of the neuropeptide Y (NPY) family. Here, we have studied the structure and dynamics of bovine pancreatic polypeptide (bPP) when bound to DPC-micelles as a membrane-mimicking model as well as the dynamics of bPP in solution. The comparison of structure and dynamics of bPP in both states reveals remarkable differences. The overall correlation time of 5.08ns derived from the 15N relaxation data proves unambiguously that bPP in solution exists as a dimer. Therein, intermolecular as well as intramolecular hydrophobic interactions from residues of both the amphiphilic helix and of the back-folded N terminus contribute to the stability of the PP fold. The overall rigidity is well-reflected in positive values for the heteronuclear NOE for residues 4-34.The membrane-bound species displays a partitioning into a more flexible N-terminal region and a well-defined alpha-helical region comprising residues 17-31. The average RMSD value for residues 17-31 is 0.22(+/-0.09)A. The flexibility of the N terminus is compatible with negative values of the heteronuclear NOE observed for the N-terminal residues 4-12 and low values of the generalized order parameter S(2). The membrane-peptide interface was investigated by micelle-integrating spin-labels and H,2H exchange measurements. It is formed by those residues which make contacts between the C-terminal alpha-helix and the polyproline helix. In contrast to pNPY, also residues from the N terminus display spatial proximity to the membrane interface. Furthermore, the orientation of the C terminus, that presumably contains residues involved in receptor binding, is different in the two environments. We speculate that this pre-positioning of residues could be an important requirement for receptor activation. Moreover, we doubt that the PP fold is of functional relevance for binding at the Y(4) receptor.     

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.     

Structural similarities of micelle-bound peptide YY (PYY) and neuropeptide Y (NPY) are related to their affinity profiles at the Y receptors

Here, we investigate the structure of porcine peptide YY (pPYY) both when unligated in solution at pH 4.2 and when bound to dodecylphosphocholine (DPC) micelles at pH 5.5. pPYY in solution displays the PP-fold, with the N-terminal segment being back-folded onto the C-terminal alpha-helix, which extends from residue 17 to 31. In contrast to the solution structure of Keire et al. published in the year 2000 the C-terminal helix does not display a kink around residue 23-25. The root mean square deviation (RMSD) for backbone atoms of the NMR ensemble of conformers to the mean structure is 0.99(+/-0.35) Angstrom for residues 14-31. The back-fold is supported by values of 0.60+/-0.1 for the (15)N(1)H-NOE and by generalized order parameters S(2) of 0.74+/-0.1 for residues 5-31 which indicate that the peptide is folded in that segment. We have additionally used DPC micelles as a membrane model and determined the structure of pPYY when bound to it. Therein, an alpha-helix occurs in the segment comprising residues 17-31 and the N terminus freely diffuses in solution. The hydrophobic side of the amphipathic helix forms the micelle-binding interface and hydrophobic side-chains extend into the micelle interior. A significant stabilization of helical conformation occurs in the C-terminal pentapeptide, which is important for receptor binding. The latter is supported by positive values of the heteronuclear NOE in that segment (0.52+/-0.1 compared to 0.08+/-0.4 for the unligated form) and by values of S(2) of 0.6+/-0.2 (versus 0.38+/-0.2 for the unligated form). The structures of micelle-bound pPYY and pNPY are much more similar than those of pPYY and bPP with pairwise RMSDs of 1.23(+/-0.21)A or 3.21(+/-0.39) Angstrom, respectively. In contrast to the conformational similarities in the DPC-bound state their structures in solution are very different. In fact pPYY is more similar to bPP, which with its strong preference for the Y(4) receptor displays a completely different binding profile. Considering the high degree of sequence homology of pNPY and pPYY (>80%) and the fact, that their binding affinities at all receptor subtypes are high and, more importantly, rather similar, it is much more likely that PYY and NPY are recognized by the Y receptors from the membrane-bound state. As a consequence of the latter the PP-fold is not important for recognition of PYY or NPY at the Y receptors. To our knowledge this work provides for the first time strong arguments derived from structural data that support a membrane-bound receptor recognition pathway.     

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.     

Solution structure and orientation of the transmembrane anchor domain of the HIV-1-encoded virus protein U by high-resolution and solid-state NMR spectroscopy

The structure of the membrane anchor domain (VpuMA) of the HIV-1-specific accessory protein Vpu has been investigated in solution and in lipid bilayers by homonuclear two-dimensional and solid-state nuclear magnetic resonance spectroscopy, respectively. Simulated annealing calculations, using the nuclear Overhauser enhancement data for the soluble synthetic peptide Vpu1-39 (positions Met-1-Asp-39) in an aqueous 2,2,2-trifluoroethanol (TFE) solution, afford a compact well-defined U-shaped structure comprised of an initial turn (residues 1-6) followed by a linker (7-9) and a short helix on the N-terminal side (10-16) and a further longer helix on the C-terminal side (22-36). The side chains of the two aromatic residues (Trp-22 and Tyr-29) in the longer helix are directed toward the center of the molecule around which the hydrophobic core of the folded VpuMA is positioned. As the observed solution structure is inconsistent with the formation of ion-conductive membrane pores defined previously for VpuMA in planar lipid bilayers, the isolated VpuMA domain as peptide Vpu1-27 was investigated in oriented phospholipid bilayers by proton-decoupled 15N cross polarization solid-state NMR spectroscopy. The line widths and chemical shift data of three selectively 15N-labeled peptides are consistent with a transmembrane alignment of a helical polypeptide. Chemical shift tensor calculations imply that the data sets are compatible with a model in which the nascent helices of the folded solution structure reassemble to form a more regular linear alpha-helix that lies parallel to the bilayer normal with a tilt angle of 相似文献   

Conformational flexibility in a small globular hormone: X-ray analysis of avian pancreatic polypeptide at 0.98-Å resolution

The 36-amino acid avian pancreatic polypeptide has been studied by x-ray analysis at 0.98-Å resolution and refined using a restrained least-squares technique to an agreement factor of 15.6%. The polypeptide, which has a compact globular structure with a hydrophobic core, comprises a polyproline–like helix (residues 2–8) and an α-helix (residues 14–32). The molecule forms symmetrical dimers linked through zinc atoms in the crystal lattice. The high-resolution analysis defines sequence-dependent distortions in the α-helical parameters due to hydrogen bonding of water molecules and side chains. The thermal parameters indicate an increased flexibility of the main chain at the turn between the helices and in the C-terminal residues. For the first time, six-parameter anisotropic thermal ellipsoids have been refined for each atom; these define the directions of the molecular motions in the polypeptide, indicating concerted vibrations. The physiological roles of conformation, flexibility, and dynamics of this polypeptide hormone are discussed.     

Solution structure of apamin determined by nuclear magnetic resonance and distance geometry

The solution structure of the bee venom neurotoxin apamin has been determined with a distance geometry program using distance constraints derived from NMR. Twenty embedded structures were generated and refined by using the program DSPACE. After error minimization using both conjugate gradient and dynamics algorithms, six structures had very low residual error. Comparisons of these show that the backbone of the peptide is quite well-defined with the largest rms difference between backbone atoms in these structures of 1.34 A. The side chains have far fewer constraints and show greater variability in their positions. The structure derived here is generally consistent with the qualitative model previously described, with most differences occurring in the loop between the beta-turn (residues 2-5) and the C-terminal alpha-helix (residues 9-17). Comparisons are made with previously derived models from NMR data and other methods.     

The structure of aconitase

The crystal structure of the 80,000 Da Fe-S enzyme aconitase has been solved and refined at 2.1 A resolution. The protein contains four domains; the first three from the N-terminus are closely associated around the [3Fe-4S] cluster with all three cysteine ligands to the cluster being provided by the third domain. Association of the larger C-terminal domain with the first three domains creates an extensive cleft leading to the Fe-S cluster. Residues from all four domains contribute to the active site region, which is defined by the Fe-S cluster and a bound SO4(2-) ion. This region of the structure contains 4 Arg, 3 His, 3 Ser, 2 Asp, 1 Glu, 3 Asn, and 1 Gln residues, as well as several bound water molecules. Three of these side chains reside on a three-turn 3(10) helix in the first domain. The SO4(2-) ion is bound 9.3 A from the center of the [3Fe-4S] cluster by the side chains of 2 Arg and 1 Gln residues. Each of 3 His side chains in the putative active site is paired with Asp or Glu side chains.     

Crystal structure of calmodulin

The crystal structure of calmodulin has been determined to 3.6 A resolution. At this resolution the polypeptide chain can be traced. Some of the side chains have tentatively been identified. Refinement of the structure with x-ray diffraction data measured to 1.65 A resolution is continuing. As reported by Babu et al. calmodulin is about 65 A long and 30 A in diameter. Homolog domains 1 and 2 are related by a local twofold axis, as in parvalbumin and in troponin C, and form one end of the molecule. Domains 3 and 4 form the other end. The second alpha-helix of domain 2 and a short interdomain region are continuous with the first helix of domain 3, thereby forming a single helix from residues 67-93. The central region, residues 75-84, of this long helix forms a handle connecting the two pairs of homolog domains. Exclusive of the residues, 75-84, in the handle the closet approach of side chains of pair 1, 2 to pair 3, 4 is 12 A. The spatial relationship of pair 1, 2 to pair 3, 4 is similar in calmodulin to the relationship of the corresponding pairs in troponin C. However, in troponin C there are three additional residues in the handle region of the long alpha-helix and the two pairs are about 5.0 A further apart. On the surface of pair 1, 2 in calmodulin there is one extended region with many hydrophobic side chains from both domain 1 and domain 2. This hydrophobic patch is bounded by two distinct clusters of anionic side chains, one from the beginning of the first helix of domain 1 and on the other side of the hydrophobic surface one from the beginning of the first helix of domain 2. Homologously, the hydrophobic patch on the surface of pair 3, 4 is bounded by two clusters of aspartate and glutamate residues. Either or both of these hydrophobic surfaces may be sites to which calmodulin target proteins bind.     

Three-dimensional structure of the two peptides that constitute the two-peptide bacteriocin lactococcin G

The three-dimensional structures of the two peptides, lactococcin G-alpha (LcnG-alpha; contains 39 residues) and lactococcin G-beta (LcnG-beta, contains 35 residues), that constitute the two-peptide bacteriocin lactococcin G (LcnG) have been determined by nuclear magnetic resonance (NMR) spectroscopy in the presence of DPC micelles and TFE. In DPC, LcnG-alpha has an N-terminal alpha-helix (residues 3-21) that contains a GxxxG helix-helix interaction motif (residues 7-11) and a less well defined C-terminal alpha-helix (residues 24-34), and in between (residues 18-22) there is a second somewhat flexible GxxxG-motif. Its structure in TFE was similar. In DPC, LcnG-beta has an N-terminal alpha-helix (residues 6-19). The region from residues 20 to 35, which also contains a flexible GxxxG-motif (residues 18-22), appeared to be fairly unstructured in DPC. In the presence of TFE, however, the region between and including residues 23 and 32 formed a well defined alpha-helix. The N-terminal helix between and including residues 6 and 19 seen in the presence of DPC, was broken at residues 8 and 9 in the presence of TFE. The N-terminal helices, both in LcnG-alpha and -beta, are amphiphilic. We postulate that LcnG-alpha and -beta have a parallel orientation and interact through helix-helix interactions involving the first GxxxG (residues 7-11) motif in LcnG-alpha and the one (residues 18-22) in LcnG-beta, and that they thus lie in a staggered fashion relative to each other.     

Solution conformation of human neuropeptide Y by 1H nuclear magnetic resonance and restrained molecular dynamics.

The solution structure of human neuropeptide Y has been solved by conventional two-dimensional NMR techniques followed by distance-geometry and molecular-dynamics methods. The conformation obtained is composed of two short contiguous alpha-helices comprising residues 15-26 and 28-35, linked by a hinge inducing a 100 degree angle. The first helix (15-26) is connected to a polyproline stretch (residues 1-10) by a tight hairpin (residues 11-14). The helices and the polyproline stretch are packed together by hydrophobic interactions. This structure is related to that of the homologous avian pancreatic polypeptide and bovine pancreatic polypeptide. The C- and N-terminii, known to be involved in the biological activity for respectively the receptor binding and activation, are close together in space. The side chains of residues Arg33, Arg35 and Tyr36 on the one hand, and Tyr1 and Pro2 on the other, form a continuous solvent-exposed surface of 4.9 mm2 which is supposed to interact with the receptor for neuropeptide Y.     

Backbone dynamics of the monomeric lambda repressor denatured state ensemble under nondenaturing conditions

Oxidizing two native methionine residues predominantly populates the denatured state of monomeric lambda repressor (MetO-lambdaLS) under nondenaturing conditions. NMR was used to characterize the secondary structure and dynamics of MetO-lambdaLS in standard phosphate buffer. 13Calpha and 1Halpha chemical shift indices reveal a region of significant helicity between residues 9 and 29. This helical content is further supported by the observation of medium-range amide NOEs. The remaining residues do not exhibit significant helicity as determined by NMR. We determined 15N relaxation parameters for 64 of 85 residues at 600 and 800 MHz. There are two distinct regions of reduced flexibility, residues 8-32 in the N-terminal third and residues 50-83 in the C-terminal third. The middle third, residues 33-50, has greater flexibility. We have analyzed the amplitude of the backbone motions in terms of the physical properties of the amino acids and conclude that conformational restriction of the backbone MetO-lambdaLS is due to nascent helix formation in the region corresponding to native helix 1. The bulkiness of amino acid residues in the C-terminal third leads to the potential for hydrophobic interactions, which is suggested by chemical exchange detected by the difference in spectral density J(0) at the two static magnetic fields. The more flexible middle region is the result of a predominance of small side chains in this region.     

Sequence-specific 1H NMR assignment and secondary structure of neuropeptide Y in aqueous solution

Sequence-specific assignment of the 1H NMR spectrum of the 36 amino acid polypeptide porcine neuropeptide Y (pNPY) at pH 3.1 is reported. It was achieved by use of standard two-dimensional techniques and by a combination of the sequential and main-chain-directed assignment strategies. The secondary structure was derived from inspection of the nuclear Overhauser spectra, slow hydrogen-deuterium exchange effects, chemical shifts of main-chain HA resonances, and coupling constants. These studies indicate that the C-terminal segment (residues 11-36) folds into an amphiphilic alpha-helix; the N-terminal segment, containing three prolines in both cis and trans conformations, assumes no regular structure. CD studies of pNPY at pH 3.1 and 7.4 show an increase in ordered structure at neutral pH. The difference spectrum, however, is typical of an alpha-helix and suggests a stabilization of residues 11-36, possibly via Maxfield-Scheraga pair interactions involving side-chain residues. This is supported by a comparison of the one-dimensional 1H NMR spectra of pNPY at pH 3.1 and 7.4, where no remarkable differences are observed.     

Conformational flexibility of the C terminus with implications for substrate binding and catalysis revealed in a new crystal form of deacetoxycephalosporin C synthase

Deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus catalyses the oxidative ring expansion of the penicillin nucleus into the nucleus of cephalosporins. The reaction requires dioxygen and 2-oxoglutarate as co-substrates to create a reactive iron-oxygen intermediate from a ferrous iron in the active site. The active enzyme is monomeric in solution. The structure of DAOCS was determined earlier from merohedrally twinned crystals where the last four C-terminal residues (308-311) of one molecule penetrate the active site of a neighbouring molecule, creating a cyclic trimeric structure in the crystal. Shortening the polypeptide chain from the C terminus by more than four residues diminishes activity. Here, we describe a new crystal form of DAOCS in which trimer formation is broken and the C-terminal arm is free. These crystals show no signs of twinning, and were obtained from DAOCS labelled with an N-terminal His-tag. The modified DAOCS is catalytically active. The free C-terminal arm protrudes into the solvent, and the C-terminal domain (residues 268-299) is rotated by about 16 degrees towards the active site. The last 12 residues (300-311) are disordered. Structures for various enzyme-substrate and enzyme-product complexes in the new crystal form confirm overlapping binding sites for penicillin and 2-oxoglutarate. The results support the notion that 2-oxoglutarate and dioxygen need to react first to produce an oxidizing iron species, followed by reaction with the penicillin substrate. The position of the penicillin nucleus is topologically similar in the two crystal forms, but the penicillin side-chain in the new non-twinned crystals overlaps with the position of residues 304-306 of the C-terminal arm in the twinned crystals. An analysis of the interactions between the C-terminal region and residues in the active site indicates that DAOCS could also accept polypeptide chains as ligands, and these could bind near the iron.     

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.     

Porcine pancreatic phospholipase A2: sequence-specific 1H and 15N NMR assignments and secondary structure

The solution structure of porcine pancreatic phospholipase A2 (124 residues, 14 kDa) has been studied by two-dimensional homonuclear 1H and two- and three-dimensional heteronuclear 15N-1H nuclear magnetic resonance spectroscopy. Backbone assignments were made for 117 of the 124 amino acids. Short-range nuclear Overhauser effect (NOE) data show three alpha-helices from residues 1-13, 40-58, and 90-109, an antiparallel beta-sheet for residues 74-85, and a small antiparallel beta-sheet between residues 25-26 and 115-116. A 15N-1H heteronuclear multiple-quantum correlation experiment was used to monitor amide proton exchange over a period of 22 h. In total, 61 amide protons showed slow or intermediate exchange, 46 of which are located in the three large helices. Helix 90-109 was found to be considerably more stable than the other helices. For the beta-sheets, four hydrogen bonds could be identified. The secondary structure of porcine PLA in solution, as deduced from NMR, is basically the same as the structure of porcine PLA in the crystalline state. Differences were found in the following regions, however. Residues 1-6 in the first alpha-helix are less structured in solution than in the crystal structure. Whereas in the crystal structure residues 24-29 are involved both in a beta-sheet with residues 115-117 and in a hairpin turn, the expected hydrogen bonds between residues 24-117 and 25-29 do not show slow exchange behavior. This and the absence of several expected NOEs imply that this region has a less well defined structure in solution. Finally, the hydrogen bond between residues 78-81, which is part of a beta-sheet, does not show slow exchange behavior.     

Molecular dynamics study of kaliotoxin in water

Kaliotoxin (KTX), a potassium channel blocker found in the venom of the scorpion Androctonous Mauretanicus is a 38 residue polypeptide with a well defined structure consisting of a alpha-helix and a three strand antiparallel beta-sheet interconnected by three disulfide bonds. Although the 3D structure has been determined by NMR, there is a number of features, mainly concerning the conformation and flexibility of the side chains, but also the long range order in the peptide and its fluctuations, that may have escaped the experimental study. These questions are now being addressed using molecular dynamics (MD) simulations. Accordingly, the present work reports the analysis of a 430 ps molecular dynamics trajectory of the polypeptide soaked with 4700 TIP3 water molecules inside a 56 A box. MD calculations were performed with periodic boundary conditions. Analysis of the conformational space sampled by each of the residues along the trajectory, suggests a special behavior of Pro17 and Lys19 both located on the helix. Furthermore, analysis of the relative movements of the secondary structure elements indicates that the alpha-helix and beta-sheets fluctuate in a correlated motion, preserving the tertiary structure of the polypeptide along the trajectory. Finally, analysis of the charge distribution was also examined. The direction of the dipole moment, computed from the center of masses appears to be an interesting feature of the structure probably related to the biological function of the molecule.     

Three-dimensional structure of the two peptides that constitute the two-peptide bacteriocin plantaricin EF

The three-dimensional structures of the two peptides plantaricin E (plnE; 33 residues) and plantaricin F (plnF; 34 residues) constituting the two-peptide bacteriocin plantaricin EF (plnEF) have been determined by nuclear magnetic resonance (NMR) spectroscopy in the presence of DPC micelles. PlnE has an N-terminal alpha-helix (residues 10-21), and a C-terminal alpha-helix-like structure (residues 25-31). PlnF has a long central alpha-helix (residues 7-32) with a kink of 38+/-7 degrees at Pro20. There is some flexibility in the helix in the kink region. Both helices in plnE are amphiphilic, while the helix in plnF is polar in its N-terminal half and amphiphilic in its C-terminal half. The alpha-helical content obtained by NMR spectroscopy is in agreement with CD studies. PlnE has two GxxxG motifs which are putative helix-helix interaction motifs, one at residues 5 to 9 and one at residues 20 to 24, while plnF has one such motif at residues 30 to 34. The peptides are flexible in these GxxxG regions. It is suggested that the two peptides lie parallel in a staggered fashion relative to each other and interact through helix-helix interactions involving the GxxxG motifs.     

X-ray structure of simian immunodeficiency virus integrase containing the core and C-terminal domain (residues 50-293)--an initial glance of the viral DNA binding platform

The crystal structure of simian immunodeficiency virus (SIV) integrase that contains in a single polypeptide the core and the C-terminal deoxyoligonucleotide binding domain has been determined at 3 A resolution with an R-value of 0.203 in the space group P2(1)2(1)2(1). Four integrase core domains and one C-terminal domain are found to be well defined in the asymmetric unit. The segment extending from residues 114 to 121 assumes the same position as seen in the integrase core domain of avian sarcoma virus as well as human immunodeficiency virus type-1 (HIV-1) crystallized in the absence of sodium cacodylate. The flexible loop in the active site, composed of residues 141-151, remains incompletely defined, but the location of the essential Glu152 residue is unambiguous. The residues from 210-218 that link the core and C-terminal domains can be traced as an extension from the core with a short gap at residues 214-215. The C(alpha) folding of the C-terminal domain is similar to the solution structure of this domain from HIV-1 integrase. However, the dimeric form seen in the NMR structure cannot exist as related by the non-crystallographic symmetry in the SIV integrase crystal. The two flexible loops of the C-terminal domain, residues 228-236 and residues 244-249, are much better fixed in the crystal structure than in the NMR structure with the former in the immediate vicinity of the flexible loop of the core domain. The interface between the two domains encompasses a solvent-exclusion area of 1500 A(2). Residues from both domains purportedly involved in DNA binding are narrowly distributed on the same face of the molecule. They include Asp64, Asp116, Glu152 and Lys159 from the core and Arg231, Leu234, Arg262, Arg263 and Lys264 from the C-terminal domain. A model for DNA binding is proposed to bridge the two domains by tethering the 228-236 loop of the C-terminal domain and the flexible loop of the core.