The three-dimensional structure of the Nudix enzyme diadenosine tetraphosphate hydrolase from Lupinus angustifolius L.

The solution structure of diadenosine 5',5'''-P1,P4-tetraphosphate hydrolase from Lupinus angustifolius L., an enzyme of the Nudix family, has been determined by heteronuclear NMR, using a torsion angle dynamics/simulated annealing protocol based on approximately 12 interresidue NOEs per residue. The structure represents the first Ap4A hydrolase to be determined, and sequence homology suggests that other members will have the same fold. The family of structures shows a well-defined fold comprised of a central four-stranded mixed beta-sheet, a two-stranded antiparallel beta-sheet and three helices (alphaI, alphaIII, alphaIV). The root-mean-squared deviation for the backbone (C',O,N,Calpha) of the rigid parts (residues 9 to 75, 97 to 115, 125 to 160) of the protein is 0.32 A. Several regions, however, show lower definition, particularly an isolated helix (alphaII) that connects two strands of the central sheet. This poor definition is mainly due to a lack of long-range NOEs between alphaII and other parts of the protein. Mapping conserved residues outside of the Nudix signature and those sensitive to an Ap4A analogue suggests that the adenosine-ribose moiety of the substrate binds into a large cleft above the four-stranded beta-sheet. Four conserved glutamate residues (Glu55, Glu58, Glu59 and Glu125) form a cluster that most likely ligates an essential magnesium ion, however, Gly41 also an expected magnesium ligand, is distant from this cluster.

Enhanced protein fold recognition using secondary structure information from NMR.

NMR offers the possibility of accurate secondary structure for proteins that would be too large for structure determination. In the absence of an X-ray crystal structure, this information should be useful as an adjunct to protein fold recognition methods based on low resolution force fields. The value of this information has been tested by adding varying amounts of artificial secondary structure data and threading a sequence through a library of candidate folds. Using a literature test set, the threading method alone has only a one-third chance of producing a correct answer among the top ten guesses. With realistic secondary structure information, one can expect a 60-80% chance of finding a homologous structure. The method has then been applied to examples with published estimates of secondary structure. This implementation is completely independent of sequence homology, and sequences are optimally aligned to candidate structures with gaps and insertions allowed. Unlike work using predicted secondary structure, we test the effect of differing amounts of relatively reliable data.

Redox-related conformational changes in Rhodobacter capsulatus cytochrome c2.

WEFT-NOESY and transfer WEFT-NOESY NMR spectra were used to determine the heme proton assignments for Rhodobacter capsulatus ferricytochrome c2. The Fermi contact and pseudo-contact contributions to the paramagnetic effect of the unpaired electron in the oxidized state were evaluated for the heme and ligand protons. The chemical shift assignments for the 1H and 15N NMR spectra were obtained by a combination of 1H-1H and 1H-15N two-dimensional NMR spectroscopy. The short-range nuclear Overhauser effect (NOE) data are consistent with the view that the secondary structure for the oxidized state of this protein closely approximates that of the reduced form, but with redox-related conformational changes between the two redox states. To understand the decrease in stability of the oxidized state of this cytochrome c2 compared to the reduced form, the structural difference between the two redox states were analyzed by the differences in the NOE intensities, pseudo-contact shifts and the hydrogen-deuterium exchange rates of the amide protons. We find that the major difference between redox states, although subtle, involve heme protein interactions, orientation of the heme ligands, differences in hydrogen bond networks and, possible alterations in the position of some internal water molecules. Thus, it appears that the general destabilization of cytochrome c2, which occurs on oxidation, is consistent with the alteration of hydrogen bonds that result in changes in the internal dynamics of the protein.

Pro----Ala-35 Rhodobacter capsulatus cytochrome c2 shows dynamic not structural differences. A 1H and 15N NMR study.

Comparative analysis of nuclear Overhauser effects show that the time average conformation of the wild-type and mutant Pro----Ala-35 Rhodobacter capsulatus cytochrome c2 are indistinguishable. The ring resonances of Phe-51 and Tyr-53 show that their ring flip rates increase in P35A. NH proton exchange studies show that the exchange rates of the NH of Gly-34 and the NpiH of His-17 increase by approximately 10(2) in P35A suggesting that their respective hydrogen bonds are destabilized in this protein. However, 3JchiNH 1H and 15N chemical shift data argue that these bonds are intact. These data are compatible if the replacement of a Pro with an Ala residue forms a cavity or increases local flexibility thus reducing steric hinderance and increasing solvent accessibility.

Conformational heterogeneity in polypeptide cardiac stimulants from sea anemones.

High-resolution 1H NMR spectra at 300 MHz of the polypeptide cardiac stimulants anthopleurin-A and Anemonia sulcata toxin II reveal conformational heterogeneity in both molecules. The two conformations, manifest in a number of split 1H resonances, are in slow exchange over a wide range of pH and temperature. Heterogeneity affects a region of these molecules containing the structurally and functionally important Asp residues. By comparison with a homologous polypeptide Anemonia sulcata toxin I, which does not show this type of heterogeneity, it is suggested that the heterogeneity may originate in cis-trans isomerism of the Gly-40 to Pro-41 peptide bond.

Computer simulation of the binding of naphthyridinomycin and cyanocycline A to DNA.

Cyanocycline A was found to have a pKa of 6.6. Protonation of N14 was established by 1H NMR spectroscopy. In strongly acidic solution the oxazolidine ring opened irreversibly. A model was derived for the binding of naphthyridinomycin and cyanocycline A to the hexanucleotide duplex d(ATGCAT)2, by using the molecular mechanics and dynamics modules of AMBER 3.0. It involved protonation on the oxazolidine-ring nitrogen, reduction of the quinone ring to a hydroquinone, formation of an iminium ion with loss of the C7 substituent, noncovalent binding in the minor groove with the hydroquinone ring in the 3'-direction from guanine, and covalent binding to the 2-amino group of this guanine with C7 adopting the R configuration. This model is consistent with the experimental evidence on the DNA binding of these drugs. An alternative binding mode based on opening of the oxazolidine ring and alkylation at C3a also was feasible according to molecular mechanics calculations. The geometry of naphthyridinomycin does not permit interstrand cross-linking involving both C3a and C7, but formation of a cross-link to protein appears possible. When the covalent naphthyridinomycin-d(ATGCAT)2 models were refined in the presence of water and counterions, the models with the most favorable net binding enthalpies were the same as those produced by simulation in vacuum. Qualitative estimates of the relative entropy changes resulting from adduct formation were based on the number of ordered (hydrogen bonded) water molecules released from d(ATGCAT)2 and from the drug. In all cases but one, d(ATGCAT)2 loses five water molecules. It loses six in the C3a covalent model with 5',S geometry. Naphthyridinomycin hydroquinone loses up to two water molecules, depending on the particular adduct. The 3',R model was again favored for the C7 covalent adduct. Among the C3a covalent models, the one with 5',R geometry lost the second most water molecules, but it had the best binding enthalpy.

Specific assignment of resonances in the 1H nuclear magnetic resonance spectrum of the polypeptide cardiac stimulant anthopleurin-A.

The specific assignment of resonances in the 300-MHz 1H nuclear magnetic resonance (NMR) spectrum of anthopleurin-A, a polypeptide cardiac stimulant from the sea anemone Anthopleura xanthogrammica, is described. Assignments have been made using two-dimensional NMR techniques, in particular the method of sequential assignments, where through-bond and through-space connectivities to the peptide backbone NH resonances are used to identify the spin systems of residues adjacent in the amino acid sequence. Complete assignments have been made of the resonances from 33 residues out of a total of 49, and partial assignments of a further 3. The resonances from several of the remaining residues have been identified but not yet specifically assigned. A complicating factor in making these assignments is the conformational heterogeneity exhibited by anthopleurin-A in solution. The resonances from a number of amino acid residues in the minor conformer have also been assigned. These assignments contribute towards identification of the origin of this heterogeneity, and permit some preliminary conclusions to be drawn regarding the secondary structure of the polypeptide.

Location of an alpha-helix in fragment 96-133 from bovine somatotropin by 1H NMR spectroscopy.

By use of two-dimensional NMR techniques most of the proton resonances (greater than 90%) are assigned for the tryptic digest fragment 96-133 of bovine somatotropin in 30% 2,2,2-trifluoroethanol-d3/70% H2O. Qualitative analysis of the nuclear Overhauser enhancement (NOE) data indicates that a region of alpha-helix spans residues 106-128, while the N- and C-terminal regions assume nonregular structures. Amide-exchange rates and comparison of two-dimensional NOE spectra indicate that the most stable piece of helix spans residues 120-125 and that this piece of helix is stable in water at 25 degrees C. Evidence is given to support the fact that intermolecular association of the helical segments stabilizes the helix.

Comparison of amide proton exchange in reduced and oxidized Rhodobacter capsulatus cytochrome c2: a 1H-15N NMR study.

The hydrogen-deuterium exchange rates of the reduced and oxidized forms of Rhodobacter capsulatus cytochrome c2 were studied by 1H-15N homonuclear multiple quantum correlation spectroscopy. Minimal differences were observed for the N- and C-terminal helices on changing redox state suggesting that although these helices are structurally important they do not affect the relative stability of the two redox states and hence may not be important in determining the redox potential differences observed amongst the class I c-type cytochromes. However, significant differences were observed for other regions of the protein. For example, all slow exchanging protons of the helix spanning Phe82 to Asp87 are similarly affected on reduction indicating that the unfolding equilibrium of this helix is altered between the two redox states. Other regions are not as simple to interpret; however, the difference in NH exchange rates between the redox states for a number of residues including His17, Leu37, Arg43, Ala45, Gly46, Ile57, Val58, Leu60, Gly61 and Leu100 suggest that interactions affecting the causes of these differences may be important factors in determining redox potential.

Conformation of MeAla6-cyclosporin A by NMR. Relationship of sidechain orientation of the MeBmt-1, MeLeu-9, and MeLeu-10 residues to immunosuppressive activity.

MeAla6-cyclosporin A (MeAla6-CsA) is a unique CsA analog that shows weak immunosuppressive activity and yet binds strongly to the proposed cytosolic protein receptor, cyclophilin (CyP). Preliminary 1H NMR data showed significant chemical shift differences between spectra of MeAla6-CsA and CsA, suggesting different preferred conformations. A more detailed study, however, revealed that the backbone conformations of the two molecules are essentially identical, and that the differences can be accounted for, principally, by the sidechain motions of the MeBmt-1, MeLeu-9, and -10 residues. ROE and coupling constant data show that in MeAla6-CsA, the preferred chi 1 rotamers for MeLeu-9 and -10 are + 180 degrees (T), whereas in CsA there is a more even distribution of rotamer populations for MeLeu-10, and a preferred -60 degrees (G-) chi 1 rotamer for MeLeu-9. Similar data argue that the sidechain of MeBmt-1 is more restricted in its motion in MeAla-CsA than in CsA. Temperature studies suggest that these preferred rotamers for MeAla6-CsA may increase the stability of the hydrogen bond between NH(7) and CO(11), but prevent particular residues, especially the essential MeBmt-1 sidechain, from adopting orientations required to elicit immunosuppressive activity. The significant changes observed in the preferred orientations for the sidechains of the MeBmt-1, MeLeu-9, and MeLeu-10 residues in MeAla6-CsA argue that the particular orientations which they assume in CsA are not essential for cyclophilin binding.

A spectroscopic analysis of the Pro35----Ala mutant of Rhodobacter capsulatus cytochrome c2. The strictly conserved Pro35 is not structurally essential.

Visible, near-ultraviolet circular dichroic, near-infrared and nuclear magnetic resonance spectroscopies show that the secondary and tertiary structures of the mutant Pro35----Ala Rhodobacter capsulatus ferrocytochrome c2 are similar to the wild-type protein. The near-infrared spectrum shows that the methionine-S--Fe-heme bond is intact; however, a small red shift in the heme M transition of the near-ultraviolet circular dichroic spectrum of the mutant indicates that the heme environment may differ slightly between the two proteins. This difference may be a consequence of changes in the ligand and hydrogen bonds of His17 [Gooley, P. R. & MacKenzie, N. E. (1990) FEBS Lett. 260, 225-228]. 1H and 15N chemical shift differences suggest that the microenvironment of pyrrole rings III and IV of the heme prosthetic group differs between the two proteins. As the rings of the Phe51 and Tyr53 flip faster in the mutant protein than the wild type, these chemical shift differences may reflect changes in the time-average ring-current effects and not structural alterations.

Mutations Pro----Ala-35 and Tyr----Phe-75 of Rhodobacter capsulatus ferrocytochrome c2 affect protein backbone dynamics: measurements of individual amide proton exchange rate constants by 1H-15N HMQC spectroscopy.

Comparisons of hydrogen-deuterium solvent exchange rate constants for the NH protons of wild-type Pro----Ala-35 (P35A) and Tyr----Phe-75 (Y75F) Rhodobacter capsulatus ferrocytochromes c2 were made by 1H-15N heteronuclear multiple-quantum correlation spectroscopy. Exchange rate constants increased for the NH protons of residues 45-46, 54, 57-58, 60-61, 82-87, 98, and 100 with Y75F and 16-18, 20, 34, 37, 43, 45-46, and 58 with P35A. The increases in exchange rate constants are consistent with changes in unfolding equilibria and protein dynamics. In Y75F the exchange rate constants of the observable NH protons of the helix spanning Pro-79-Asp-89, namely Phe-82-Leu-87, increase to a similar degree, suggesting that this helix is a single cooperative unfolding unit compatible with the local unfolding model. As the oxidation-reduction potential of Y75F is 59 mV lower than wild-type cytochrome c2 (367 mV), the dynamic changes in this mutant, compared to wild-type, are proposed to be important determinants of the oxidation-reduction potential. Several differences between wild-type and Y75F are in common with P35A, a mutation which does not affect the oxidation-reduction potential, implying that not all observed dynamic changes are functionally important.

Assignment of aromatic resonances in the 1H nuclear magnetic resonance spectra of cardioactive polypeptides from sea anemones.

High-resolution 1H NMR spectroscopy at 300 MHz has been used to investigate the aromatic residues of a series of homologous polypeptides from sea anemones: anthopleurin-A from Anthopleura xanthogrammica and toxins I and II from Anemonia sulcata. Using two-dimensional NMR techniques, specific assignments to individual protons have been made for all aromatic resonances in the spectra of these molecules. In all three polypeptides the resonances from the two conserved Trp residues, 23 and 33, are shifted significantly from their random coil values, and the indole NH resonance of Trp-23 is not observed. These shift perturbations are due in part to a mutual interaction of the two indole rings, which is also indicated by the observation of nuclear Overhauser enhancements between protons of the two rings. Several other nonpolar side chains also interact with these two Trp residues, forming a hydrophobic region, the overall structure of which is conserved throughout the series. The other aromatic residues in these polypeptides appear not to participate in this structural region.

NMR structure of human apolipoprotein C-II in the presence of sodium dodecyl sulfate.

The structure and protein-detergent interactions of apolipoprotein C-II (apoC-II) in the presence of SDS micelles have been investigated using circular dichroism and heteronuclear NMR techniques applied to (15)N-labeled protein. Micellar SDS, a commonly used mimetic of the lipoprotein surface, inhibits the aggregation of apoC-II and induces a stable structure containing approximately 60% alpha-helix as determined by circular dichroism. NMR reveals the first 12 residues of apoC-II to be structurally heterogeneous and largely disordered, with the rest of the protein forming a predominantly helical structure. Three regions of helical conformation, residues 16-36, 50-56, and 63-77, are well-defined by NMR-derived constraints, with the intervening regions showing more loosely defined helical conformation. The structure of apoC-II is compared to that determined for other apolipoproteins in a similar environment. Our results shed light on the lipid interactions of apoC-II and its mechanism of lipoprotein lipase activation.

The interaction of human apolipoprotein C-I with sub-micellar phospholipid.

Mature human apolipoprotein C-I (apoC-I), comprising 57 amino acids, is the smallest member of the plasma apolipoprotein family. Amphipathic helical regions within apoC-I, common to this class of proteins, are mediators of lipid binding, a process that underlies the functional properties of apoC-I, including the capacity to activate the plasma enzyme LCAT, to disrupt apoE mediated receptor interactions and to inhibit cholesterol ester transfer protein. To examine apoC-I/phospholipid interactions, we have developed an expression system in Escherichia coli to obtain purified apoC-I with yields of approximately 4-5 mg per L of culture. The purified product has properties similar to plasma-derived apoC-I including self-association in the lipid-free state and induced alpha-helical content in the presence of egg-yolk phosphatidylcholine and dimyristoylglycerophosphocholine vesicles. We chose the short-chain phospholipid, dihexanoylglycerophosphocholine (Hex2Gro-PCho), to examine the interaction of apoC-I with submicellar phospholipid. Circular dichroism spectroscopy and cross-linking experiments show that apoC-I acquires helical content and remains self-associated at submicellar concentrations of Hex2Gro-PCho (4 mM). Sedimentation equilibrium studies of apoC-I at submicellar levels of Hex2Gro-PCho and analysis of the effects of apoC-I on the 1H NMR spectrum of Hex2Gro-PCho indicate micelle induction by apoC-I, and establish the capacity of apoC-I to assemble individual phospholipid molecules.

Preferred conformational state of the N-terminus section of a bovine growth hormone fragment (residues 96-133) in water is an omega loop.

The solution structure of a 38-amino-acid-residue, biologically active fragment of bovine growth hormone (bGH96-133) was investigated with a combined nuclear magnetic resonance (NMR) and computer modeling approach. With the distance geometry program DISGEO and distance constraints derived from nuclear Overhauser enhancement (NOE) experiments, it was found that residues Ser-100 to Tyr-110 circumscribe and omega-loop, a recently categorized feature of nonregular secondary protein structure.

Assignment of the 1H and 15N NMR spectra of Rhodobacter capsulatus ferrocytochrome c2.

The peptide resonances of the 1H and 15N nuclear magnetic resonance spectra of ferrocytochrome c2 from Rhodobacter capsulatus are sequentially assigned by a combination of 2D 1H-1H and 1H-15N spectroscopy, the latter performed on 15N-enriched protein. Short-range nuclear Overhauser effect (NOE) data show alpha-helices from residues 3-17, 55-65, 69-88, and 103-115. Within the latter two alpha-helices, there are three single 3(10) turns, 70-72, 76-78, and 107-109. In addition alpha H-NHi+1 and alpha H-NHi+2 NOEs indicate that the N-terminal helix (3-17) is distorted. Compared to horse or tuna cytochrome c and cytochrome c2 of Rhodospirillium rubrum, there is a 6-residue insertion at residues 23-29 in R. capsulatus cytochrome c2. The NOE data show that this insertion forms a loop, probably an omega loop. 1H-15N heteronuclear multiple quantum correlation experiments are used to follow NH exchange over a period of 40 h. As the 2D spectra are acquired in short time periods (30 min), rates for intermediate exchanging protons can be measured. Comparison of the NH exchange data for the N-terminal helix of cytochrome c2 of R. capsulatus with the highly homologous horse heart cytochrome c [Wand, A. J., Roder, H., & Englander, S. W. (1986) Biochemistry 25, 1107-1114] shows that this helix is less stable in cytochrome c2.

Determination of local conformational stability in fragment 96-133 of bovine growth hormone by high-resolution H NMR spectroscopy.

The specific assignment of resonances in the 400-MHz nuclear magnetic resonance (NMR) spectrum of fragment 96-133 (AII) of bovine growth hormone (bSt) is described. Assignments have been made with homonuclear two-dimensional techniques, in particular that of sequential resonance assignment. Complete assignments were possible for the spin systems of 16 residues out of a total of 38 and partial assignments for another 5. Assignment of resonances to either residue type or a class of residue was possible for a number of other spin systems. Analysis of the type of nuclear Overhauser effect (NOE) indicates that segments 96-110 and 130-133 are nonregular stable structures and that the segment 111-127, which putatively spans the alpha-helix, is not sufficiently stable to generate NOEs.

Identification and comparison of the urinary metabolites of [1,2,3-13C3]acrylic acid and [1,2,3-13C3]propionic acid in the rat by homonuclear 13C nuclear magnetic resonance spectroscopy.

Acrylic acid (AA) and its esters are used extensively for the production of a variety of polymers. Despite their ubiquitous nature, little has been reported on the metabolism of the parent acid. The metabolites of AA may be volatile, unstable, polar, and thus difficult to isolate. Therefore, 13C NMR was used to help identify and compare directly the urinary metabolites of both 99% 13C-enriched AA and propionic acid (PA). Male Sprague-Dawley rats received [1,2,3-13C]AA (400 mg/kg in water p.o.) or an equimolar dose of [1,2,3-13C]propionate together with a radioactive tracer, [2,3-14C]AA, or [1-14C]propionate, respectively, and excreta were collected for 72 hr. For both acids, expiration of 14CO2 was the major route of elimination of radiolabel (approximately 80%). Approximately 6% of the dose was excreted in the urine. Urine was analyzed directly using proton-decoupled 13C and two-dimensional 13C homonuclear correlated NMR spectroscopy. The urine from AA-treated rats revealed major signals, the intensity of which was time-dependent, from at least five 13C-enriched metabolites of AA. Signals have been assigned to 3-hydroxypropionic acid, N-acetyl-S-(2-carboxyethyl)cysteine, and N-acetyl-S-(2-carboxyethyl)cysteine-S-oxide by comparison with spectra of authentic standards. No unchanged AA was detected. In contrast, the spectra of urine from a propionate-treated rat revealed only a few minor 13C-enriched signals that were assigned to methylmalonic acid. No unchanged PA was detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of active-site residues in diadenosine tetraphosphate hydrolase from Lupinus angustifolius.

Site-directed mutagenesis has been used to characterize the functions of key amino acid residues in the catalytic site of the 'nudix' hydrolase, (asymmetrical) diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) hydrolase (EC 3.6.1.17) from Lupinus angustifolius, the three-dimensional solution structure of which has recently been solved. Residues within the nudix motif, Gly-(Xaa)5-Glu-(Xaa)7-Arg-Glu-Uaa-Xaa-(Glu)2-Xaa-Gly (where Xaa represents unspecified amino acids and Uaa represents the bulky aliphatic amino acids Ile, Leu or Val) conserved in 'nudix enzymes', and residues important for catalysis from elsewhere in the molecule, were mutated and the expressed proteins characterized. The results reveal a high degree of functional conservation between lupin asymmetric Ap4A hydrolase and the 8-oxo-dGTP hydrolase from Escherichia coli. Charged residues in positions equivalent to those that ligate an enzyme-bound metal ion in the E. coli 8-oxo-dGTP hydrolase [Harris, Wu, Massiah and Mildvan (2000) Biochemistry 39, 1655-1674] were shown to contribute to catalysis to similar extents in the lupin enzyme. Mutations E55Q, E59Q and E125Q all reduced kcat markedly, whereas mutations R54Q, E58Q and E122Q had smaller effects. None of the mutations produced a substantial change in the Km)for Ap4A, but several extensively modified the pH-dependence and fluoride-sensitivities of the hydrolase. It was concluded that the precisely positioned glutamate residues Glu-55, Glu-59 and Glu-125 are conserved as functionally significant components of the hydrolytic mechanism in both of these members of the nudix family of hydrolases.