Interactions of hydrated metal ions with nucleotides: the crystal structure of barium adenosine 5'-monophosphate heptahydrate

The crystal and molecular structure of barium adenosine 5'-monophosphate heptahydrate was determined from x-ray diffraction data. Crystals of barium adenosine 5'-monophosphate heptahydrate are monoclinic, space group C2, with a = 32.559(3), b = 6.969(3), c = 9.597(1) A, and beta = 100.31(1) degrees. Intensity data were collected with an automated diffractometer. The structure was solved by the heavy-atom method and refined by least-squares to R = 0.034. This structure provides an example of an outer-sphere metal-nucleotide complex, in which a completely hydrated metal ion interacts with the nucleotide only through water bridges. The barium ion is coordinated to eight water molecules, which form a slightly distorted square antiprism. Seven of the eight water molecules from the barium hydration shell are hydrogen bonded to phosphate groups; three of these water molecules are also hydrogen bonded to other suitable acceptor sites on the base and ribose moieties. The conformation about the glycosidic bond is anti, with chiCN = 69 degrees, and, as in most nucleotide structures, the conformation about the C(4')-C(5') bond is gauche-gauche. However, the ribose displays an unusual conformation (best described as C(4')-exo) not previously observed in crystal structures of nucleosides or nucleotides, other than 3',5'-cyclic nucleotides. It is possible that this unusual conformation is a consequence of the metal-water-nucleotide bridging interactions.     

The family experiences of narcotic addicts and their subsequent parenting practices

The molecular structure of satellite tobacco mosaic virus (STMV) has been refined to 1.8 A resolution using X-ray diffraction data collected from crystals grown in microgravity. The final R value was 0.179 and Rfree was 0.184 for 219,086 independent reflections. The final model of the asymmetric unit contained amino acid residues 13 to 159 of a coat protein monomer, 21 nucleotides, a sulfate ion, and 168 water molecules. The nucleotides were visualized as 30 helical segments of nine base-pairs with an additional base stacked at each 3' end, plus a "free" nucleotide, not belonging to the helical segments, but firmly bound by the protein. Sulfate ions are located exactly on 5-fold axes and each is coordinated by ten asparagine side-chains. Of the 10,080 structural waters, 168 per asymmetric unit, about 20% serve to bridge the macromolecular components at protein-protein and protein-nucleic acid interfaces. Binding of RNA to the protein involves some salt linkages, particularly to the phosphate of the free nucleotide, but the major contribution is from an intricate network of hydrogen bonds. There are numerous water molecules in the RNA-protein interface, many serving as intermediate hydrogen bond bridges. The sugar-phosphate backbone contributes most of the donors and acceptors for the RNA. The helical RNA conformation is nearest that of A form DNA. The central region of a helical segment is most extensively involved in contacts with protein, and exhibits low thermal parameters which increase dramatically toward the ends. The visible RNA represents approximately 59% of the total nucleic acid in the virion and is derived from the single-stranded genome, which has folded upon itself to form helical segments. Linking of the helices and the free nucleotides in a contiguous and efficient manner severely restricts the disposition of the remaining, unseen nucleic acid. Using the remaining nucleotides it is possible to fold the RNA according to motifs that provide a periodic distribution of RNA structural elements compatible with the icosahedrally symmetrical arrangement seen in the crystallographic structure. The intimate relationship between protein and nucleic acid in STMV suggests an assembly pathway based on the cooperative and coordinated co-condensation of RNA with capsid protein dimers.     

Visualizing and quantifying molecular goodness-of-fit: small-probe contact dots with explicit hydrogen atoms

The technique of small-probe contact dot surfaces is described as a method for calculating and displaying the detailed atomic contacts inside or between molecules. It allows one both to measure and to visualize directly the goodness-of-fit of packing interactions. It requires both highly accurate structures and also the explicit inclusion of all hydrogen atoms and their van der Waals interactions. A reference dataset of 100 protein structures was chosen on the basis of resolution (1.7 A or better), crystallographic R-value, non-homology, and the absence of any unusual problems. Hydrogen atoms were added in standard geometry and, where needed, with rotational optimization of OH, SH, and NH+3 positions. Side-chain amide orientations were corrected where required by NH van der Waals clashes, as described in the accompanying paper. It was determined that, in general, methyl groups pack well in the default staggered conformation, except for the terminal methyl groups of methionine residues, which required rotational optimization. The distribution of serious clashes (i.e. non-H-bond overlap of >/=0.4 A) was studied as a function of resolution, alternate conformations, and temperature factor (B), leading to the decision that packing and other structural features would not be analyzed for residues in 'b' alternate conformations or with B-factors of 40 or above. At the level of the fine details analyzed here, structural accuracy improves quite significantly over the range from 1.7 to 1.0 A resolution. These high-resolution structures show impressively well-fitted packing interactions, with some regions thoroughly interdigitated and other regions somewhat sparser. Lower-resolution structures or model structures could undoubtedly be improved in accuracy by the incorporation of this additional information: for example, nucleic acid structures in non-canonical conformations are often very accurate for the bases and much less reliable for the backbone, whose conformation could be specified better by including explicit H atom geometry and contacts. The contact dots are an extremely sensitive method of finding problem areas, and often they can suggest how to make improvements. They can also provide explanations for structural features that have been described only as empirical regularities, which is illustrated by showing that the commonest rotamer of methionine (a left-handed spiral, with all chi values near -60 degrees) is preferred because it provides up to five good H atom van der Waals contacts. This methodology is thus applicable in two different ways: (1) for finding and correcting errors in structure models (either experimental or theoretical); and (2) for analyzing interaction patterns in the molecules themselves.     

The conformation of NADH bound to inosine 5'-monophosphate dehydrogenase determined by transferred nuclear Overhauser effect spectroscopy

Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes the oxidation of inosine 5-monophosphate (IMP) to xanthosine 5'-monophosphate (XMP). The reaction proceeds with concomitant conversion of NAD+ to NADH and is the rate-limiting step in the de novo biosynthesis of guanosine nucleotides. IMPDH is a target for numerous chemotherapeutic agents. The conformations of enzyme-bound substrates, enzyme-bound products and enzyme-bound ligands in general, are of interest for the understanding of the catalytic mechanism of the enzyme and the design of new inhibitors. Although several of the chemotherapeutic inhibitors of IMPDH are NAD+ or NADH analogues, no structural data for IMPDH-bound NAD+ (or NADH) are available. In the present work, we have used transferred nuclear Overhauser effect spectroscopy (TRNOESY) to determine the conformation of NADH bound to the active site of human type II IMPDH (IMPDH-h2). The inter-proton distances determined from TRNOESY data indicate that NADH binds to the enzyme active site in an overall extended conformation. The adenosine moiety and the nicotinamide riboside moiety are both in the anti conformation about the glycosidic bond, and both ribose rings are in approximately C4'-exo conformations. The nicotinamide amide group was found to be in a cis conformation. The anti conformation of the nicotinamide riboside moiety is in accord with the preferred conformations of several potent and selective dinucleotide inhibitors and is consistent with that implied by the stereospecificity of hydride transfer in the enzymatic reaction. The implications of this conformation for the catalytic mechanism of IMPDH-h2 are discussed.     

Kinetic properties of a single nucleotide binding site on chloroplast coupling factor 1 (CF1)

The kinetics of nucleotide binding to spinach chloroplast coupling factor CF1 in a fully inhibited state were investigated by stopped-flow experiments using the fluorescent trinitrophenyl analogue (NO2)3Ph-ADP. The CF1 was in a state in which two of the three binding sites on the beta subunits were irreversibly blocked with ADP, Mg2+ and fluoroaluminate, while the three binding sites on the alpha subunits were occupied by nucleotides [Garin, J., Vincon, M., Gagnon, J. & Vignais, P. V. (1994) Biochemistry 33, 3772-3777)]. Thus, it was possible to characterise a single nucleotide-binding site without superimposed nucleotide exchange or binding to an additional site. (NO2)3Ph-ADP binding to the remaining site on the third beta subunit was characterised by a high dissociation rate of 15 s(-1), leading to a very low affinity (dissociation constant higher than 150 microM). Subsequent to isolation, CF1 preparations contained two endogenously bound nucleotides. Pre-loading with ATP yielded CF1 with five tightly bound nucleotides and one free nucleotide-binding site on a beta subunit. Pre-loading with ADP, however, resulted in a CF1 preparation containing four tightly bound nucleotides and two free nucleotide binding sites. One of the two free binding sites was located on a beta subunit, while the other was probably located on an alpha subunit.     

Role of hydrophobicity and solvent-mediated charge-charge interactions in stabilizing alpha-helices

A theoretical study to identify the conformational preferences of lysine-based oligopeptides has been carried out. The solvation free energy and free energy of ionization of the oligopeptides have been calculated by using a fast multigrid boundary element method that considers the coupling between the conformation of the molecule and the ionization equilibria explicitly, at a given pH value. It has been found experimentally that isolated alanine and lysine residues have somewhat small intrinsic helix-forming tendencies; however, results from these simulations indicate that conformations containing right-handed alpha-helical turns are energetically favorable at low values of pH for lysine-based oligopeptides. Also, unusual patterns of interactions among lysine side chains with large hydrophobic contacts and close proximity (5-6 A) between charged NH3+ groups are observed. Similar arrangements of charged groups have been seen for lysine and arginine residues in experimentally determined structures of proteins available from the Protein Data Bank. The lowest-free-energy conformation of the sequence Ac-(LYS)6-NMe from these simulations showed large pKalpha shifts for some of the NH3+ groups of the lysine residues. Such large effects are not observed in the lowest-energy conformations of oligopeptide sequences with two, three, or four lysine residues. Calculations on the sequence Ac-LYS-(ALA)4-LYS-NMe also reveal low-energy alpha-helical conformations with interactions of one of the LYS side chains with the helix backbone in an arrangement quite similar to the one described recently by (Proc. Natl. Acad. Sci. U.S.A. 93:4025-4029). The results of this study provide a sound basis with which to discuss the nature of the interactions, such as hydrophobicity, charge-charge interaction, and solvent polarization effects, that stabilize right-handed alpha-helical conformations.     

Search for low-energy conformations of a neurotoxic protein by means of predictive rules, tests for hard-sphere overlaps, and energy minimization

A method to obtain models for the three-dimensional structure of the neurotoxin alpha from Naja nigricollis from its amino acid sequence is explored here. Empirical predictive rules were used to estimate the positions of helices, extended structures and bends; advantage was taken of the availability of 14 homologous sequences for the neurotoxins in an attempt to increase the reliability of these predictions. Unassigned residues were allowed to take up several possible conformations determined from the frequencies of occurrence of each type of conformation of that residue in x-ray structures of many proteins. The conformational space of the molecule was explored initially by testing for hard-sphere overlaps and approximate closure of disulfide loops with the aid of a computer; this procedure yielded a limited number of conformations, whose conformational energies were then determined and minimized by optimizing the backbone and side-chain dihedral angles of each residue. Five compact conformations with low energy were found for this neurotoxin. The procedure used here provides an illustration as to how empirical protein algorithms may be used to limit the conformational space, in which energy minimization has to be carried out.     

Ventral subiculum regulates hypothalamo-pituitary-adrenocortical and behavioural responses to cognitive stressors

The high resolution crystal structure of Saccharomyces cerevisiae phosphoglycerate mutase has been determined. This structure shows important differences from the lower resolution structure deposited in 1982. The crystal used to determine the new structure was of a different form, having spacegroup P2(1). The model was refined to a crystallographic R-factor of 18.9% and a free R-factor of 28.4% using all data between 25 and 2.3 A and employing a bulk solvent correction. The enzyme is a tetramer of identical, 246 amino acid subunits, whose structure is revealed to be a dimer of dimers, with four independent active sites located well away from the subunit contacts. Each subunit contains two domains, the larger with a typical nucleotide binding fold, although phosphoglycerate mutase has no physiological requirement to bind nucleotides. The catalytic-site histidine residues are no longer in a "clapping-hands" conformation, but more resemble the conformation seen in the distantly related enzymes prostatic acid phosphatase and fructose-2,6-bisphosphatase. However, the catalytic histidine residues in the mutase are found to be much closer to each other than in the phosphatase structures, perhaps due to the absence of bound ligands in the mutase crystal. An intricate web of H-bonds is found around the catalytic histidine residues, high-lighting residues probably important for maintaining their correct orientation and charge. The positions of certain other residues, including some found near the catalytic site and some lining the catalytic-site cleft, have been changed by the correction of registration errors between sequence and electron density in the original structure. Electron density was apparent for a portion of the functionally important C-terminal tail, which was absent from the earlier structure, showing it to adopt a mainly helical conformation.     

Circadian rhythm of intraocular pressure in the rat

In order to investigate the relationship between the bioactive conformation of a peptide and its set of thermodynamically accessible structures in solution, the conformational profile of the tetrapeptide Ac-Pro-Ala-Pro-Tyr-OH was characterized by computational methods. Search of the conformational space was performed within the molecular mechanics frame-work using the AMBER4.0 force field with an effective dielectric constant of 80. Unique structures of the peptide were compared with its bioactive conformation for the protein Streptomyces griseus Protease A, as taken from the crystal structure of the enzyme-peptide complex. The results show that the bound conformation is close to one of the unique conformations characterized in the conformational search of the isolated peptide. Moreover, the lowest energy minimum characterized in the conformational search exhibits large deviations when compared to the bound conformation of the crystal structure.     

Predicted ligand interactions of 3'5'-cyclic nucleotide-gated channel binding sites: comparison of retina and olfactory binding site models

Cyclic nucleotide-gated channels (CNGC) open in response to the binding of 3'5'-cyclic nucleotides. Members of the CNGC family vary as much as 100-fold in their ability to respond to cAMP and cGMP. Molecular models of the nucleotide binding domains of the bovine retina and catfish and rat olfactory CNGCs were built from the crystal structure of cAMP bound to catabolite gene activator protein (CAP) with AMMP, a program for molecular mechanics and dynamics. The nucleotide conformation can be predicted from the number of strong and weak interactions between the purine ring and the binding site. The amino acids predicted to be important for determining the nucleotide affinity and specificity are residues 61, 83 (mediated through a water molecule), 119 and 127 (CAP sequence numbers) which interact with the purine ring. These residues also dictate the conformation of the ligand in the binding pocket. cGMP is preferentially bound in the syn conformation in bovine retina, bovine olfactory and rat olfactory CNGCs due to Thr83, while either conformation can bind in catfish olfactory CNGC. cAMP is predicted to bind either in syn or anti conformation, depending on the interaction with residue 119: the anti conformation is preferentially bound in olfactory CNGCs.     

Preference for syn conformation: crystal structures of free acid and ammonium salt of adenosine 2'-monophosphate: an inhibitor of RNase T1

This paper reports the crystal structures of free acid and ammonium salt of adenosine 2'-monophosphate (2'-AMP). 2'-AMP crystallizes in the hexagonal space group P6(5)22 with a = 9.530(3) A, c = 73.422(2) A, and Z= 12. 2'-AMP.NH4 crystallizes in the trigonal space group P3(1) with a = 9.003(2) A, c = 34.743(2) A and Z= 6. Both the structures were solved by direct methods and refined by full matrix least- squares method to final R factors of 0.080 and 0.038 for 2'-AMP and 2'-AMP.NH4 respectively. The adenine bases of both the structures are in syn conformation contrasting with the anti geometry in 3'-AMP, 5'-AMP and the enzyme bound state. Ribose moiety of 2'-AMP is in C2' -endo conformation. However, the ribose moieties of both the nucleotide molecules display C2'-endo-C3'-exo twist conformation in 2'- AMP.NH4 structure. Both structures demonstrate g+ conformation about C4' -C5' bond. 2'-AMP and one of the nucleotide molecules of 2'-AMP.NH4 are protonated at N1 and the ammonium ion is involved in a bifurcated hydrogen bond with O3' B and O3A atoms. A characteristic feature of both the structures is the intramolecular O5' -N3 hydrogen bond. Our crystallographic results on 2'-AMP corroborates the earlier conclusion that the enzyme-bound state is not the lowest energy state of this nucleotide. 2' -AMP displays base-ribose 04' stacking not seen in the 2'-AMP.NH4 structure. Theoretical and experimental studies on 2'-, 3'- and 5'-AMP structures have been discussed.     

Structure of a DNA duplex containing a single 2'-O-methyl-beta-D-araT: combined use of NMR, restrained molecular dynamics, and full relaxation matrix refinement

Two-dimensional 1H NMR spectroscopy was used to determine the solution structure of the double-stranded DNA oligonucleotide d(5'-CGCATATAGCC-3'): d(5'-GGCTAXATGCG-3'), where X is 1-(2-O-methyl-beta-D-arabinofuranosyl)thymine. The structure determination was based on a total relaxation matrix analysis of NOESY cross-peak intensities using the MARDIGRAS program. The improved RANDMARDI procedure was used during the calculations to include the experimental "noise" in the NOESY spectra. The NOE-derived distance restraints were applied in restrained molecular dynamics calculations. Twenty final structures each were generated for the modified DNA duplex from both A-form and B-form DNA starting structures. The root-mean-square deviation of the coordinates for the 40 structures was 0.82 A. The duplex adopts a normal B-DNA-type helix, and the spectra as well as the structure show that the modified nucleotide X adopts a C2'-endo (S) sugar conformation. There are no significant changes in the helix originating from the modified nucleotide. The CH3O group on X is directed toward the major groove, and there seems to be free space for further modifications at this position.     

Cyclopropane-derived peptidomimetics. Design, synthesis, evaluation, and structure of novel HIV-1 protease inhibitors

Toward establishing the general efficacy of using trisubstituted cyclopropanes as peptide mimics to stabilize extended peptide structures, the cyclopropanes 20a-d were incorporated as replacements into 9-13, which are analogues of the known HIV-1 protease inhibitors 14 and 15. The syntheses of 20a-d commenced with the Rh2[5(S)-MEPY]4-catalyzed cyclization of the allylic diazoesters 16a-d to give the cyclopropyl lactones 17a-d in high enantiomeric excess. Opening of the lactone moiety using the Weinreb protocol and straightforward refunctionalization of the intermediate amides 18a-d gave 20a-d. A similar sequence of reactions was used to prepare the N-methyl-2-pyridyl analogue 28. Coupling of 20a-d and 28 with the known diamino diol 22 delivered 9-13. Pseudopeptides 9-12 were found to be competitive inhibitors of wild-type HIV-1 protease in biological assays having Kis of 0.31-0.35 nM for 9, 0.16-0.21 nM for 10, 0.47 nM for 11, and 0.17 nM for 12; these inhibitors were thus approximately equipotent to the known inhibitor 14(IC50 = 0.22 nM) from which they were derived. On the other hand 13 (Ki = 80 nM) was a weaker inhibitor than its analogue 15 (Ki = 0.11 nM). The solution structures of 9 and 10 were analyzed by NMR spectroscopy and simulated annealing procedures that included restraints derived from homo- and heteronuclear coupling constants and NOEs; because of the molecular symmetry of9 and 10, a special protocol to treat the NOE data was used. The final structure was checked by restrained and free molecular dynamic calculations using an explicit DMSO solvent box. The preferred solution conformations of 9 and 10 are extended structures that closely resemble the three-dimensional structure of 10 bound to HIV-1 protease as determined by X-ray crystallographic analysis of the complex. This work convincingly demonstrates that extended structures of peptides may be stabilized by the presence of substituted cyclopropanes that serve as peptide replacements. Moreover, the linear structure enforced in solution by the two cyclopropane rings in the pseudopeptides 9-12 appears to correspond closely to the biologically active conformation of the more flexible inhibitors 14 and 15. The present work, which is a combination of medicinal, structural, and quantum chemistry, thus clearly establishes that cyclopropanes may be used as structural constraints to reduce the flexibility of linear pseudopeptides and to help enforce the biologically active conformation of such ligands in solution.     

Perioperative pharmacodynamics of acetaminophen analgesia in children

The nitrogenase iron (Fe) protein binds two molecules of MgATP or MgADP, which results in protein conformational changes that are important for subsequent steps of the nitrogenase reaction mechanism. In the present work, isothermal titration calorimetry has been used to deconvolute the apparent binding constants (K'a1 and K'a2) and the thermodynamic terms (delta H' degree and delta S' degree) for each of the two binding events of MgATP or MgADP to either the reduced or oxidized states of the Fe protein from Azotobacter vinelandii. The Fe protein was found to bind two nucleotides with positive cooperativity and the oxidation state of the [4Fe-4S] cluster of the Fe protein was found to influence the affinity for binding nucleotides, with the oxidized ([4Fe-4S]2+) state having up to a 15-fold higher affinity for nucleotides when compared to the reduced ([4Fe-4S]1+) state. The first nucleotide binding reaction was found to be driven by a large favorable entropy change (delta S' degree = 10-21 cal mol-1 K-1), with a less favorable or unfavorable enthalpy change (delta H' degree = +1.5 to -3.3 kcal mol-1). In contrast, the second nucleotide binding reaction was found to be driven by a favorable change in enthalpy (delta H' degree = -3.1 to -13.0 kcal mol-1), with generally less favorable entropy changes. A plot of the associated enthalpy (-delta H' degree) and entropy terms (-T delta S' degree) for each nucleotide and protein binding reaction revealed a linear relationship with a slope of 1.12, consistent with strong enthalpy-entropy compensation. These results indicate that the binding of the first nucleotide to the nitrogenase Fe protein results in structural changes accompanied by the reorganization of bound water molecules, whereas the second nucleotide binding reaction appears to result in much smaller structural changes and is probably largely driven by bonding interactions. Evidence is presented that the total free energy change (delta G' degree) derived from the binding of two nucleotides to the Fe protein accounts for the total change in the midpoint potential of the [4Fe-4S] cluster.     

Recent developments in the long-term preservation of red blood cells

The complete nucleotide sequence of one of three linear DNA plasmids (pRS64-2) from the plant pathogenic fungus Rhizoctonia solani was determined. The pRS64-2 DNA consisted of 2877 nucleotides. The nucleotide sequences of the middle 2.2-kb regions of the other two plasmids (pRS64-1 and pRS64-3) were also determined. Comparison of the nucleotide sequences among the three plasmid DNAs indicated the presence of four regions with more than 86% sequence homology, suggesting the development of three plasmid DNAs from a common ancestor. A computer-based study of the pRS64-2 DNA-folding at both termini predicted hairpin loop structures. The hairpin loops consisted of the left- and right-hand terminal 113 and 105 nucleotides, respectively, and had no sequence homology. They had not undergone flip-flop inversion. The hairpins form cruciform base-paired structures.     

Absolute requirement of cyclic nucleotide in the activation of the G141Q mutant cAMP receptor protein from Escherichia coli

cAMP receptor protein (CRP), when interacting with cAMP, controls the expression of a network of catabolite-sensitive genes in Escherichia coli. To understand the molecular events that lead to the activation of CRP, a combined approach of site-directed mutagenesis and thermodynamic analysis was employed to study a member of a specific class of CRP mutant, CRP, which activates the in vivo expression of CRP-dependent operons in cya- strains in the absence of exogenous cAMP. Results from in vitro studies show that the CRP mutant G141Q absolutely requires cAMP for interacting with specific DNA. A quantitative comparison of the thermodynamic parameters governing ligand binding and DNA-protein complex formation in the presence of different cyclic nucleotides leads to the conclusion that this CRP mutant is activated only in the presence of cyclic nucleotides. The specificity toward cyclic nucleotides exhibited by wild-type CRP is lost in this mutant. Furthermore, the binding affinity of the ligand for the first binding site of the mutant is essentially the same as that of wild-type CRP regardless of the identity of the cyclic nucleotide. Hence, the observed in vivo activation of CRP-dependent operons by G141Q in the absence of exogenous cAMP is most likely the consequence of the replacement of cAMP by other cyclic nucleotides to activate the mutant. It is also possible that trace levels of cAMP present in the cya- strain could account for the in vivo activation of the mutant. Furthermore, these results indicate that this CRP mutant does not assume the activated conformation in the absence of cyclic nucleotides, in contrast to the current model derived from results of in vivo studies.     

Do active site conformations of small ligands correspond to low free-energy solution structures?

We compare the low free energy structures of ten small, polar ligands in solution to their conformations in their respective receptor active sites. The solution conformations are generated by a systematic search and the free energies of representative structures are computed with a continuum solvation model. Based on the values of torsion angles, we find little similarity between low energy solution structures of small ligands and their active site conformations. However, in nine out of ten cases, the positions of 'anchor points' (key atoms responsible for tight binding) in the lowest energy solution structures are very similar to the positions of these atoms in the active site conformations. A metric that more closely captures the essentials of binding supports the basic premise underlying pharmacophore mapping, namely that active site conformations of small flexible ligands correspond to their low energy structures in solution. This work supports the efforts of building pharmacophore models based on the information present in solution structures of small isolated ligands.