Water-inserted alpha-helical segments implicate reverse turns as folding intermediates.

Information relevant to the folding and unfolding of alpha helices has been extracted from an analysis of protein structures. The alpha helices in protein crystal structures have been found to be hydrated, either externally by a water molecule hydrogen bonding to the backbone carbonyl oxygen atom, or internally by inserting into the helix hydrogen bond and forming a hydrogen-bonded bridge between the backbone carbonyl oxygen and the amide nitrogen atoms. The water-inserted alpha-helical segments display a variety of reverse-turn conformations, such as type III, type II, type I, and opened out, that can be considered as folding intermediates that are trapped in the folding-unfolding process of alpha helices. Since the alpha helix, most turns, and the extended beta strand occupy contiguous regions in the conformational space of phi, psi dihedral angles, a plausible pathway can be proposed for the folding-unfolding process of alpha helices in aqueous solution.

Crystal structure of a naturally occurring dinucleoside monophosphate: uridylyl (3',5') adenosine hemihydrate.

The crystal structure of uridylyl (3',5') adenosine hemihydrate has been analyzed by x-ray diffraction. The two independent molecules found in the asymmetric unit exhibit conformations that differ significantly from those found in double-helical RNA. The conformational information obtained from this analysis provides considerable insight into the possible conformations of nonhelical "loop" regions of transfer RNA, as well as single-stranded regions of nucleic acids in general.

Molecular conformation of dihydrouridine: puckered base nucleoside of transfer RNA.

The crystal structure of dihydrouridine hemihydrate has been determined by x-ray diffraction. Crystals of dihydrouridine contain two independent molecules in the asymmetric unit and a molecule of water. The x-ray structure determination has shown that the conformations of both molecules differ in important respects in the saturated base and the ribose. The molecular conformation of dihydrouridine has, for the first time, provided structural evidence that the rare nucleoside can proinote "loop" formation in the sugar-phosphate chain.

X-ray diffraction patterns of transfer RNA consistent with the presence of short parallel helices in the molecule.

X-ray diffraction data of yeast formylmethionine transfer RNA, Escherichia coli phenylalanine transfer RNA, and Escherichia coli arginine transfer RNA single crystals are compared with the Fourier transform of a helix. The results are consistent with the presence of short parallel double helical segments in the transfer RNA molecules.

Molecular structure of troponin C from chicken skeletal muscle at 3-angstrom resolution.

The x-ray structure of chicken skeletal muscle troponin C (TnC), the Ca2+-binding subunit of the troponin complex, shows that the protein is about 70 angstroms long with an unusual dumbbell shape. The carboxyl and amino domains are separated by a single long alpha helix of about nine turns. Only the two high-affinity Ca2+-Mg2+ sites of the COOH-domain are occupied by metal ions resulting in conformational differences between the COOH- and NH2-domains. These differences are probably important in the triggering of muscle contraction by TnC. Also the structure of TnC is relevant in understanding the function of other calcium-regulated proteins, in particular that of calmodulin because of its strong similarity in amino acid sequence.

New crystal structures of nucleic acids and their complexes.

In the past year, X-ray crystallographic studies of representatives of all nucleic acid structural types have been reported. Among the most interesting structures are the parallel DNA tetraplex formed by d(TGGGGT), the four-stranded structure formed by d(CCCT) and a double drug bound side by side in an antiparallel orientation to the minor groove of a B-DNA. Certainly, the structure that has received most attention is that of the first complex of a ribozyme with an inhibitor DNA.

Crystal structure of a DNA.RNA hybrid duplex with a polypurine RNA r(gaagaagag) and a complementary polypyrimidine DNA d(CTCTTCTTC).

DNA.RNA hybrid duplexes are substrates of RNase H and reverse transcriptase. The crystal structure of a hybrid duplex, d(5'-CTCTTCTTC-3').r(5'-gaagaagag-3') (the uppercase letters indicate DNA and lowercase letters RNA), with a polypurine RNA strand and a complementary DNA strand has been determined at 1.8 A resolution. The structure was refined first at 1.9 A by XPLOR and subsequently by CNS at 1.8 A. The hybrid is found in a standard A-form conformation with all the sugars in the C3'-endo puckering. The 5'-terminal base dC of the DNA strand was clearly visible in the electron density map of the present structure, in contrast to the previously reported structure d(TTCTTBr(5)CTTC).r(gaagaagaa) where the 5'-terminal base dT was not visible, leaving the terminal rA unpaired. Thus, the comparison of the terminal base pairs, C.g versus T.a, in the two hybrid crystal structures provides information on the stability of these base pairs in hydrogen bonding (three versus two) and base stacking interactions. The differences in the terminal base pairs produce different kinks in the two structures. Minor groove widening is observed in the present structure at a distinctive kink in the lower half of the duplex, in contrast to the small widening of the minor groove and a very slight bend in the upper half of the T.a structure.

Synthesis and crystal structure of an octamer RNA r(guguuuac)/r(guaggcac) with G.G/U.U tandem wobble base pairs: comparison with other tandem G.U pairs.

We have determined the crystal structure of the RNA octamer duplex r(guguuuac)/r(guaggcac) with a tandem wobble pair, G.G/U.U (motif III), to compare it with U.G/G.U (motif I) and G.U/U.G (motif II) and to better understand their relative stabilities. The crystal belongs to the rhombohedral space group R3. The hexagonal unit cell dimensions are a = b = 41.92 A, c = 56.41 A, and gamma = 120 degrees, with one duplex in the asymmetric unit. The structure was solved by the molecular replacement method at 1.9 A resolution and refined to a final R: factor of 19.9% and R(free) of 23.3% for 2862 reflections in the resolution range 10.0-1.9 A with F >/= 2sigma(F). The final model contains 335 atoms for the RNA duplex and 30 water molecules. The A-RNA stacks in the familiar head-to-tail fashion forming a pseudo-continuous helix. The uridine bases of the tandem U.G pairs have slipped towards the minor groove relative to the guanine bases and the uridine O2 atoms form bifurcated hydrogen bonds with the N1 and N2 of guanines. The N2 of guanine and O2 of uridine do not bridge the 'locked' water molecule in the minor groove, as in motifs I and II, but are bridged by water molecules in the major groove. A comparison of base stacking stabilities of motif III with motifs I and II confirms the result of thermodynamic studies, motif I > motif III > motif II.

B-form to A-form conversion by a 3'-terminal ribose: crystal structure of the chimera d(CCACTAGTG)r(G).

The crystal structure of the chimerical decamer d(CCACTAGTG)r(G), bearing a 3'-terminal ribo-guanidine, has been solved and refined at 1.8 A resolution (R-factor 16.6%; free R-factor 22.8%). The decamer crystallizes in the orthorhombic space group P2(1)2(1)2(1) with unit cell constants a = 23.90 A, b = 45.76 A and c = 49.27 A. The structure was solved by molecular replacement using the coordinates of the isomorphous chimera r(GCG)d(TATACGC). The final model contains one duplex and 77 water molecules per asymmetric unit. Surprisingly, all residues adopt a conformation typical for A-form nucleic acids (C3'-endo type sugar pucker) although the all-DNA analog, d(CCACTAGTGG), has been crystallized in the B-form. Comparing circular dichroism spectra of the chimera and the corresponding all-DNA sequence reveals a similar trend of the former molecule to adopt an A-like conformation in solution. The results suggest that the preference of ribonucleotides for the A-form is communicated into the 5'-direction of an oligonucleotide strand, although direct interactions of the 2'-hydroxyl group can only be discerned with nucleotides in the 3'-direction of a C3'-endo puckered ribose. These observations imply that forces like water-mediated contacts, the concerted motions of backbone torsion angles, and stacking preferences, are responsible for such long-range influences. This bi-directional structural communication originating from a ribonucleotide can be expected to contribute to the stability of the A-form within all-RNA duplexes.

Crystal structure and conformation of a DNA-RNA hybrid duplex with a polypurine RNA strand: d(TTCTTBr5CTTC)-r(GAAGAAGAA).

BACKGROUND: . DNA-RNA hybrids are substrates for RNase H. This enzyme catalyzes the hydrolysis of the RNA strand in the hybrid form. The polypurine tract (PPT) in human immunodeficiency virus 1 (HIV-1) is a short stretch of purines ( approximately 15 bases) located at the 3'-end of the U3 region of the RNA genome. The PPT has the unique ability to resist digestion by RNase H and serves as a primer for plus-strand DNA synthesis. RESULTS: . The crystal structure of a DNA-RNA hybrid duplex containing a polypurine RNA strand, d(TTCTTBr5CTTC)-r(GAAGAAGAA), has been determined at 1.8 A resolution. The structure was solved by molecular replacement methods and refined to a final R factor of 20.1% (R free 23.7%). The hybrid duplex adopts a standard A-form conformation. All of the sugar rings and glycosidic torsion angles are found in the standard C3'-endo/anti conformation, as seen in A-RNA or A-DNA. The crystal packing is dominated by the DNA strand, where the terminal base pairs of the hybrid abut the neighboring A-DNA sugar-phosphate backbone on the minor groove side. CONCLUSIONS: . The present DNA-RNA hybrid duplex containing a polypurine RNA strand exhibits standard A-form geometry. This observation might suggest that the RNA PPT resists the RNase H activity of HIV reverse transcriptase as a result of its A-form conformation. In addition, there appears to be a correlation between the percentage purine content of the RNA and the DNA backbone conformation.

Conformational analysis of arabinonucleosides and nucleotides. A comparison with the ribonucleosides and nucleotides.

Conformations of arabino nucleosides and nucleotides have been analyzed by semiempirical energy calculations. It is found that the change in the configuration of the O(2')-hydroxyl group in arabinoses compared to riboses exerts significant influence on the conformational priorities of the glycosyl and the exocyclic C(4')-C(5') bond torsions. While the anti conformations for the bases are preferred, the anti in equilibrium or formed from syn interconversion is considerably hampered compared to ribosides due to large energy barrier. Further the preferred anti glycosyl torsions are shifted to higher values for C(3')-endo puckers and in ribosides. While the gauche+ conformation around the C(4')-C(5') bond is favored for C(3')-endo arabinosides, it is strongly stabilized for C(2')-endo arabinosides only in the presence of the intrasugar hydrogen bond O(2')-H ... O(5'). The net attractive electrostatic interactions between the phosphate and the base stabilizes the preferred conformations of 5'-arabinonucleotides also.

X-ray diffraction study of the zinc(II) binding sites in yeast phenylalanine transfer RNA. Preferential binding of zinc to guanines in purine-purine sequences.

An X-ray diffraction study of a zinc(II) complex of tRNAPhe from yeast reveals the present of five zinc-binding sites on the tRNA molecule. Two of the cooperatively bound Mg2+ in the native tRNA structure are replaced by Zn2+. The remaining sites involve direct coordination of zinc to the N7 position of tRNA guanine bases, G15, G43 and HG45. Thus, zinc displays a high specificity for binding to guanine bases in purine-purine sequences.

Crystal and molecular structure of the antibiotic blasticidin S hydrochloride pentahydrate.

The three-dimensional structure of blasticidin S hydrochloride pentahydrate, a member of the cytosine amino nucleoside antibiotics, has been solved using diffractometer data and refined to an R value of 0.115. The crystal data are a = 13.500(5), b = 20.387(7), c = 4.824 A, beta = 98.66(3) degrees, Z = 2, Dc = 1.389 g .cm-3, space group P21. The nucleoside base conformation is anti(chi = 86 degrees) and the 2',3'-unsaturated pyranosyl sugar exhibits a half-chair (degree H5) conformation. The amide plane is twisted from the trans position by about 10 degrees. The guanidium group and the amino group of the amino acid chain are positively charged, while the carboxyl group of the sugar is ionized. The chloride ion is surrounded by water molecules only, in a trigonal prismatic arrangement. The molecule has an extended conformation and there is an intramolecular hydrogen bond between the ammonium group and the carboxyl group. A striking feature of blasticidin is that all the hydrophilic groups lie on one side of the molecule and the hydrophobic groups on the other. Amicetin also shows a similar feature and this might be linked to the commonality of their antibiotic functions. Hydrogen bonds link the hydrophilic sides of adjacent molecules forming double chains parallel to the b-axis. The hydrophobic sides of adjacent double chains are separated by a water layer.

High resolution crystal structure of the A-DNA decamer d(CCCGGCCGGG). Novel intermolecular base-paired G*(G.C) triplets.

The DNA decamer d(CCCGGCCGGG) crystallizes in the orthorhombic space group P2(1)2(1)2(1) with a = 24.88, b = 44.60 and c = 46.97 A containing a duplex in the asymmetric unit. The structure was solved by molecular replacement and refined to an R factor of 18.5% using 6033 reflections at 1.65 A resolution. The decamer duplex adopts an A-DNA conformation. The abrupt dislocation of the duplex at the fourth base-pair G(4).C(17) by an abutting symmetry related molecule results in distortion of the backbone bonds of the fifth residue G(5), P-O(5')(alpha) and C(4')-C(5')(gamma), to the trans conformations from their favored gauche- and gauche+ conformations, respectively. In this close encounter the terminal G(10).C(11) base-pair of the symmetry related molecule hydrogen bonds to the G(4).C(17) base-pair forming a novel base-paired G(4)*(G10).C(11)) triplet, where G(4) is hydrogen bonded to both G(10) and C(11). To facilitate this hydrogen bonding the G(4).C(17) base-pair slides into the minor groove, causing a toll on the backbone conformation of the adjacent residue G(5). A similar triplet base-pairing interaction with somewhat weaker hydrogen bonds occurs at the pseudo dyad related C(7).G(14) base-pair with G(20) of another symmetry related duplex. This pseudo triplet interaction (C(7).G(14))*G(20), does not perturb the backgone alpha and gamma torsions of G(15). Both the novel base triplets are non-planar. The abrupt dislocation/bend at the G(4).C(17) base-pair jolts the global helical base-pair parameters, inclination, tilt, roll, tip, etc. quite markedly. Therefore a better description of the helix parameters is obtained by splitting the duplex and calculating the local helix axis for the top half consisting of the first three base-pairs, and the lower half consisting of the last six base-pairs, omitting the fourth base-pair. The two half duplexes are bent by only 10 degrees. This structure further demonstrates that crystal packing interactions, which can also be governed by base sequence, play a dominant role in determining DNA conformation.

Crystal structure of r(GUGUGUA)dC with tandem G x U/U x G wobble pairs with strand slippage.

To better understand the frequent occurrence of adjacent wobble pairs in ribosomal RNAs we have determined the crystal structure of the RNA duplex, r(GUGUGUA)dC with the 3'-terminal deoxy C residue. Two different crystal forms of the duplex were obtained and both belong to the rhombohedral space group, R3. Crystal form I has hexagonal unit cell dimensions, a = b = 40.82 A and c = 66.09 A and diffracts to 1.58 A resolution, while crystal form II has a = b = 47.11 A and c = 59.86 A, diffracting only to 2.50 A resolution. Both structures were solved by the molecular replacement method using different starting models. In spite of the large differences in the cell dimensions the overall structures in both crystals are similar. Instead of the expected blunt-end duplex with four consecutive G x U pairs, the slippage of the strands resulted in two different tandem G x U/U x G wobble pairs involving two of the central and two of the 5' overhang bases, still yielding a total of four wobble pairs. These tandem wobble pairs are flanked by two Watson-Crick pairs. The A-type duplexes stack in the familiar head-to-tail fashion forming a pseudocontinuous helix. The wobble pairs of the present motif II (G x U/U x G) structure stack with a low twist angle of 25.3 degrees in contrast to that of motif I (U x G/G x U), 38.1 degrees. The four wobble pairs are characteristically heavily hydrated in both the grooves accounting for their stability.

The potentially Z-DNA-forming sequence d(GTGTACAC) crystallizes as A-DNA.

(GT)n/(CA)n sequences have stimulated much interest because of their frequent occurrence in eukaryotic DNA and their potential for forming the left-handed Z-DNA structure. We here report the X-ray crystal structure of a self-complementary octadeoxynucleotide, d(GTGTACAC), at 2.5 A resolution. The molecule adopts a right-handed double-helical conformation belonging to the A-DNA family. In this alternating purine-pyrimidine DNA minihelix the roll and twist angles show alternations qualitatively consistent with Calladine's rules. The average tilt angle of 9.3 degrees is between the values found in A-DNA (19 degrees) and B-DNA (-6 degrees) fibers. It is envisaged that such intermediate conformations may render diversity to genomic DNA. The base-pair tilt angles and the base-pair displacements from the helix axis are found to be correlated for the known A-DNA double-helical fragments.

Crystal structures of the side-by-side binding of distamycin to AT-containing DNA octamers d(ICITACIC) and d(ICATATIC).

To understand the recognition interactions between AT-containing alternating DNA and minor groove binding drugs, the crystal structures of the side-by-side binding of two distamycin molecules to the DNA octamers d(ICITACIC)2 and d(ICATATIC)2, referred to here as TA and ATAT, respectively, have been determined at 1.6 A and 2.2 A, respectively. Compared to the previous 2:1 all-IC d(ICICICIC)2-distamycin complex, the substitutions of the I x C base-pairs by the A x T base-pairs enable the interactions of the drug with its natural target to be studied. Both complexes assume side-by-side drug binding, isomorphous to the all IC counterpart in the tetragonal space group P4(1)22 (a = b = 28.03 A, c = 58.04 A and a = b = 27.86 A, c = 58.62 A, respectively). The ATAT complex also crystallized in a new polymorphic monoclinic space group C2 (a = 33.38 A, b = 25.33 A, c = 28.11 A and beta = 120.45 degrees) and was solved at 1.9 A resolution. The structures of the three double drug x DNA complexes are very similar, characterized by systematic hydrogen bonding and van der Waals interactions. Each drug hydrogen bonds with the bases of the proximal DNA strand only and stacks with the sugar moiety, while the side-by-side drugs themselves exhibit pyrrole ring-peptide stacking. The pyrrole-peptide interaction is crucial for the side-by-side binding mode of the distamycin/netropsin family of drugs. The purine-pyrimidine alternation is probably responsible for the striking alternation in the helical and backbone conformations. The structures are conserved between the pure IC complex and the AT substituted complexes but further details of the side-by-side binding to DNA are provided by the 1.6 A resolution structure of TA.

Nine polymorphic crystal structures of d(CCGGGCCCGG), d(CCGGGCCm5CGG), d(Cm5CGGGCCm5CGG) and d(CCGGGCC(Br)5CGG) in three different conformations: effects of spermine binding and methylation on the bending and condensation of A-DNA.

The A-DNA decamer d(CCGGGCCm5CGG) crystallizes in the presence of spermine in three polymorphic forms and with one duplex in the asymmetric unit: hexagonal (P6(1)), unit cell of 55.0 A x 55.0 A x 45.9 A; orthorhombic (P2(1)2(1)2(1)), unit cell of 24.8 A x 44.6 A x 48.0 A, and a second orthorhombic (P2(1)2(1)2(1)), unit cell of 23.6 A x 40.8 A x 43.4 A. The reduction in cell volume among the three different forms is accompanied by a large reduction in solvent content (67% versus 40% versus 24%) and a significant reduction in volume per base-pair (2005 A(3) versus 1325 A(3) versus 1048 A(3)). There is also a concomitant increase in the number of bound spermine molecules per duplex (0 versus 1 versus 2) as well as an increase in DNA bending (10 degrees versus 16 degrees versus 31 degrees), which correspond to major groove widths of 8.0 A versus 4.5 A versus 1.3 A, respectively. The P6(1) crystal form, which represents a new space group for A-DNA decamers, supports one of the most hydrated and extended DNA duplexes to date, while the second orthorhombic form supports one of the least-hydrated and most-condensed non-Z-DNA duplexes. The unmethylated analogue d(CCGGGCCCGG), the double-methyl derivative d(Cm5CGGGCCm5CGG) and the bromine derivative d(CCGGGCC(Br)5CGG) also crystallize in at least two of the aforementioned conformations, and all nine crystal structures were determined. We report, in detail, on the three crystal structures of d(CCGGGCCm5CGG) and the effects of methylation and spermine binding on A-DNA conformation.

Crystal structure of an adenine bulge in the RNA chain of a DNA.RNA hybrid, d(CTCCTCTTC).r(gaagagagag).

Crystal structure of a DNA.RNA hybrid, d(CTCCTCTTC).r(gaagagagag), with an adenine bulge in the polypurine RNA strand was determined at 2.3 A resolution. The structure was solved by the molecular replacement method and refined to a final R-factor of 19.9% (Rfree 22.2%). The hybrid duplex crystallized in the space group I222 with unit cell dimensions, a = 46.66 A, b = 47.61 A and c = 54.05 A, and adopts the A-form conformation. All RNA and DNA sugars are in the C3'-endo conformation, the glycosyl angles in anti conformation and the majority of the C4'-C5' torsion angles in g+ except two trans angles, in conformity with the C3'-endo rigid nucleotide hypothesis. The adenine bulge is looped out and it is also in the anti C3'-endo conformation. The bulge is involved in a base-triple (C.g)*a interaction with the end base-pair (C9.g10) in the minor groove of a symmetry-related molecule. The 2' hydroxyl group of g15 is hydrogen bonded to O2P and O5' of g17, skipping the bulged adenine a16 and stabilizing the sugar-phosphate backbone of the hybrid. The hydrogen bonding and the backbone conformation at the bulged adenine site is very similar to that found in the crystal structure of a protein-RNA complex.

A single 2'-hydroxyl group converts B-DNA to A-DNA. Crystal structure of the DNA-RNA chimeric decamer duplex d(CCGGC)r(G)d(CCGG) with a novel intermolecular G-C base-paired quadruplet.

We have found that the introduction of a single 2'-hydroxyl group on the sugar-phosphate backbone of the B-DNA decamer d(CCGGCGCCGG) transforms it to A-DNA. Thus, for the first time the X-ray structures of the same sequence have been observed in both the A and B-DNA conformations, permitting a comparison. Crystals of the DNA-RNA chimeric decamer d(CCGGC)r(G)d(CCGG) belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions a = 25.63 A, b = 45.24 A and c = 47.99 A, and one decamer duplex in the asymmetric unit. The structure was solved by a rigid body search using the coordinates of the isomorphous structure d(CCCGGCCGGG) and refined to an R value of 0.136 using 2753 unique reflections at 1.9 A resolution. The final model contains 406 nucleotide atoms and 61 water molecules. The chimeric duplex exhibits typical A-DNA geometry, with all the sugars in the C(3')-endo puckering and the base-pairs inclined and displaced from the helix axis. The 2'-hydroxyl groups on rG6 and rG16 protrude into the minor groove surface and form different types of hydrogen bonds; that on strand 1 forms an intermolecular hydrogen bond with the furanose ring O(4') of a symmetry-related C1 residue, while that on strand 2 is involved in two water bridges. Crystal packing forces the G4-G17 base-pair in the top half of the duplex to slide significantly into the minor groove compared to the corresponding G7-G14 base-pair in the bottom half, resulting in these base-pairs exhibiting different base stacking and intermolecular interactions. The base G4 of the G4-G17 base-pair forms an unorthodox base "triple", G4*(G10-C11), hydrogen-bonding through its minor groove sites N(2) and N(3) to the minor groove atoms N(2) and O(2) of both bases of the G10-C11 base-pair of a symmetry-related molecule. The base G10 of this triple in turn forms a second similar unorthodox base triple, G10*(G3*C18), with the adjacent base-pair G3-C18 of the duplex, thus G10 is involved in a double triple. On the other hand, in the bottom half of the duplex, the C7-G14 base-pair is involved only in a single similar unorthodox base triple with G20, (C7-G14)*G20, while the adjacent base-pair rG6-C15 is involved in a novel quadruple with C1-G20, (rG6-C15) *(C1-G20), where the latter base-pairs are hydrogen-bonded to each other via the minor groove sites G(N(2))...C(O(2)).(ABSTRACT TRUNCATED AT 400 WORDS)