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PubMed2005

How sequence defines structure: a crystallographic map of DNA structure and conformation.

The fundamental question of how sequence defines conformation is explicitly answered if the structures of all possible sequences of a macromolecule are determined. We present here a crystallographic screen of all permutations of the inverted repeat DNA sequence d(CCnnnN6N7N8GG), where N6, N7, and N8 are any of the four naturally occurring nucleotides. At this point, 63 of the 64 possible permutations have been crystallized from a defined set of solutions. When combined with previous work, we have assembled a data set of 37 single-crystal structures from 29 of the sequences in this motif, representing three structural classes of DNA (B-DNA, A-DNA, and four-stranded Holliday junctions). This data set includes a unique set of amphimorphic sequence, those that crystallize in two different conformations and serve to bridge the three structural phases. We have thus constructed a map of DNA structures that can be walked through in single nucleotide steps. Finally, the resulting data set allows us to dissect in detail the stabilization of and conformational variations within structural classes and identify significant conformational deviations within a particular structural class that result from sequence rather than crystal or crystallization effects.

Base Sequence↗
PubMed2001

The crystal structure of the MJ0796 ATP-binding cassette. Implications for the structural consequences of ATP hydrolysis in the active site of an ABC transporter.

The crystal structure of the MJ0796 ATP-binding cassette, a member of the o228/LolD transporter family, has been determined at 2.7-A resolution with MgADP bound at its active site. Comparing this structure with that of the ATP-bound form of the HisP ATP-binding cassette (Hung, L. W., Wang, I. X., Nikaido, K., Liu, P. Q., Ames, G. F., and Kim, S. H. (1998) Nature 396, 703-707) shows a 5-A withdrawal of a phylogenetically invariant glutamine residue from contact with the gamma-phosphate of ATP in the active site. This glutamine is located in a protein segment that links the rigid F(1)-type ATP-binding core of the enzyme to an ABC transporter-specific alpha-helical subdomain that moves substantially away from the active site in the MgADP-bound structure of MJ0796 compared with the ATP-bound structure of HisP. A similar conformational effect is observed in the MgADP-bound structure of MJ1267 (Karpowich, N., et al. (2001) Structure, in press), establishing the withdrawal of the glutamine and the coupled outward rotation of the alpha-helical subdomain as consistent consequences of gamma-phosphate release from the active site of the transporter. Considering this subdomain movement in the context of a leading model for the physiological dimer of cassettes present in ABC transporters indicates that it produces a modest mechanical change that is likely to play a role in facilitating nucleotide exchange out of the ATPase active site. Finally, it is noteworthy that one of the intersubunit packing interactions in the MJ0796 crystal involves antiparallel beta-type hydrogen bonding interactions between the outermost beta-strands in the two core beta-sheets, leading to their fusion into a single extended beta-sheet, a type of structural interaction that has been proposed to play a role in mediating the aggregation of beta-sheet-containing proteins.

ATP-Binding Cassette Transporters↗
PubMed2004

Structure and assembly of the RNA binding domain of bluetongue virus non-structural protein 2.

Bluetongue virus non-structural protein 2 belongs to a class of highly conserved proteins found in orbiviruses of the Reoviridae family. Non-structural protein 2 forms large multimeric complexes and localizes to cytoplasmic inclusions in infected cells. It is able to bind single-stranded RNA non-specifically, and it has been suggested that the protein is involved in the selection and condensation of the Bluetongue virus RNA segments prior to genome encapsidation. We have determined the x-ray structure of the N-terminal domain (sufficient for the RNA binding ability of non-structural protein 2) to 2.4 A resolution using anomalous scattering methods. Crystals of this apparently insoluble domain were obtained by in situ proteolysis of a soluble construct. The asymmetric unit shows two monomers related by non-crystallographic symmetry, with each monomer folded as a beta sandwich with a unique topology. The crystal structure reveals extensive monomer-monomer interactions, which explain the ability of the protein to self-assemble into large homomultimeric complexes. Of the entire surface area of the monomer, one-third is used to create the interfaces of the curved multimeric assembly observed in the x-ray structure. The structure reported here shows how the N-terminal domain would be able to bind single-stranded RNA non-specifically protecting the bound regions in a heterogeneous multimeric but not polymeric complex.

Amino Acid Sequence↗
PubMed1993

Investigation of solution structure of d(GAATTTAAATTC)2 by 1H NMR, molecular dynamics, mechanics, refinement by back-calculation of the NOESY spectrum and analysis of this structure using X-ray data.

1H NMR spectroscopy, restrained molecular mechanics and dynamics and refinements after back-calculation of the NOESY spectrum have been performed to study the structure of the d(GAATTTAAATTC)2 duplex and to determine whether it is bent or not. It is found that the duplex adopts a B-type conformation; all sugar conformations belong to the C2' endo region and purines have a larger pseudorotation angle as compared to pyrimidines. The cross-strand AH2(n)-AH1' (m + 1) distance (where (n) and (m) are complementary residues), crucial for an anomalous A/T tract structure, is large on the TA step and gradually decreases at the 3' and 5' ends of the TTTAAA tract and follows the rules proposed previously [Chuprina, V.P., Lipanov, A.A., Fedoroff, O.Yu, Kim, S.G., Kintanar, A., and Reid, B.R., Proc. Natl. Acad. Sci. U.S.A. 88, 9087 (1991)]. The changes in this distance correlate with those in the T1 value for AH2 protons which we measured for several oligonucleotide sequences. A total number of about 250 interproton distance constraints were determined from NOESY spectra and were used for structure determination by molecular mechanics, dynamics and refinement by back-calculations. It is shown that these data are not enough to determine whether the duplex is bent or not. The whole family of B-type conformations including bent and straight structures fit well with the available NMR data. In principle, additional non-NMR data could be used in order to reduce the number of the allowable structures. The refinement of the structures with additional different non-NMR constraints (used as a driving force) on P-P or H1'-H1' minor groove width distances in the TA region shows a very good correlation between these distances and the angle of bending of the dodecamer. The more the minor groove width increases in the TA region the more the duplex is bent at the major groove of this region. On the other hand, there is also a very good correlation between P-P, H1'-H1' and AH2-H1' cross-strand distances as follows from analysis of X-ray B-type structures. These two correlations, together with the increased AH2-H1' cross-strand NMR distance in the TA region of the dodecamer indicate that the duplex should be characterized by a wider minor groove in the TA region and be bent in the major groove in this region.

Base Sequence↗
PubMed2004

Analysis of local convergence in NMR structure calculation for RNA by a classification system for nucleic acid structure (CSNA).

We are developing a program system, CSNA, to classify a set of structures into groups sharing similar structural characters. In the present study, CSNA was applied to the analysis of NMR structures obtained by the simulated annealing calculation to elucidate local convergences. A 34-mer RNA, U6-34, having a bulge-out region that is derived from the human U6 snRNA is used as a target molecule in the present study. Although the structure calculation was not converged with the conventional method, it was found by the CSNA analysis that the two stem regions in the molecule were converged well. Furthermore, one strand of the bulge-out region (A7-A11) was found to form a continuously stacked structure in two-thirds of calculated structures. In conclusion, CSNA can be a novel tool to elucidate the local convergence of the NMR structure calculations.

Base Sequence↗
PubMed2003

Application of the RNA structure classification system, CSNA, to NMR structure determination.

CSNA is a computer system which classifies a set of RNA structures based on their structural characters; hydrogen bond and base-base stacking. CSNA has been applied to the RNA structure determination by NMR and it was found that CSNA could provide well converged groups as the lowest energy structures. Here, we further applied CSNA to the structure determination of a 31mer RNA forming a psuedoknot structure. It was demonstrated that CSNA is a useful tool for the RNA structure determination by NMR.

Base Sequence↗
PubMed2002

Classification of RNA structures based on hydrogen bond and base-base stacking patterns: application for NMR structures.

A computational system, CSNA, for classifying RNA structures according to structural characters was developed. CSNA lists up all the hydrogen bonds and base-base stackings in the structures, and classifies the structures into sub-groups based on their patterns as the first step grouping. The frequency of each hydrogen bond or base-base stacking is calculated, the frequency score being defined as the sum of the frequency of existing hydrogen bonds or base-base stackings for each sub-group. Finally, the sub-groups are further classified into groups based on the frequency score defined in this study and the difference between the patterns. According to the frequency score, CSNA suggests a group that shares most frequently appearing hydrogen bonds and base-base stackings. CSNA was applied to the classification of the results of two individual simulated annealing calculations based on NMR information. It was found that CSNA could extract structures with lower energy without checking any energy term and could provide well converged groups as the lowest energy structures. Thus, CSNA could be a new tool for structural determination of nucleic acids.

Algorithms↗
PubMed2000

The structure and organization of lamprin genes: multiple-copy genes with alternative splicing and convergent evolution with insect structural proteins.

Lamprin is a unique structural protein which forms the extracellular matrix of several cartilaginous structures found in the lamprey. Lamprin is noncollagenous in nature but shows sequence similarities to elastins and to insect structural proteins. Here, we characterize the structure and organization of lamprin genes, demonstrating the presence of multiple similar but not identical copies of the lamprin gene in the genome of the lamprey. In at least one species of lamprey, Lampetra richardsoni, the multiple gene copies are arranged in tandem in the genome in a head-to-tail orientation. Lamprin genes from Petromyzon marinus contain either seven or eight exons, with exon 4 being alternatively spliced in all genes, resulting in a total of six different lamprin transcripts. All exon junctions are of class 1,1. An unusual feature of the lamprin gene structure is the distribution of the 3' untranslated region sequence among multiple exons. A TATA box and cap sequence have been identified in upstream sequences in close proximity to the transcription start site, but no CAAT box could be identified. Sequence and gene structure comparisons between lamprins, elastins, and insect structural proteins suggest that the regions of sequence similarity are the result of a process of convergent evolution.

Alternative Splicing↗
PubMed1973

The Structure of Plant Cell Walls: IV. A Structural Comparison of the Wall Hemicellulose of Cell Suspension Cultures of Sycamore (Acer PseudoPlatAnus) and of Red Kidney Bean (Phaseolus Vulgaris).

The molecular structure and chemical properties of the hemicellulose present in the isolated cell walls of suspension cultures of sycamore (Acer pseudoplatanus) cells has recently been described by Bauer et al. (Plant Physiol. 51: 174-187). The hemicellulose of the sycamore primary cell wall is a xyloglucan. This polymer functions as an important cross-link in the structure of the cell wall; the xyloglucan is hydrogen-bonded to cellulose and covalently attached to the pectic polymers.The present paper describes the structure of a xyloglucan present in the walls and in the extracellular medium of suspension-cultured Red Kidney bean (Phaseolus vulgaris) cells and compares the structure of the bean xyloglucan with the structure of the sycamore xyloglucan. Although some minor differences were found, the basic structure of the xyloglucans in the cell walls of these distantly related species is the same. The structure is based on a repeating heptasaccharide unit which consists of four residues of beta-1, 4-linked glucose and three residues of terminal xylose linked to the 6 position of three of the glucosyl residues.

Journal Article↗
PubMed2001

The joint probability distributions of structure-factor doublets in displacive incommensurately modulated structures and their applicability to direct methods.

In 1993, alternative normalized structure factors for incommensurately modulated structures were defined [Lam, Beurskens & van Smaalen (1993). Acta Cryst. A49, 709-721]. The probability distribution associated with the structure invariants E(-H)E(H')E(H - H') has approximately the same functional form as the Cochran distribution. It was shown, however, that triplet-phase relations are relatively less reliable when satellites are involved [de Gelder, Israël, Lam, Beurskens, van Smaalen, Fu & Fan (1996). Acta Cryst. A52, 947-954]. In the present paper, an alternative approach is presented: instead of studying the distribution of a three-phase invariant, the probability distribution of the phase sum of two first-order satellite reflections (h,k,l,1 and h',k',l',-1) has been derived under the assumption that the phase of the associated main reflection (h + h',k + k',l + l',0) can be calculated from the known main (or averaged) structure. Intensive tests with randomly generated artificial structures and one real structure show a significant improvement of direct-methods phase-sum statistics. Functional similarities with conventional direct methods, employing normalized structure factors and the Cochran distribution, are discussed.

Journal Article↗
PubMed2001

Structures of copper(II) and manganese(II) di(hydrogen malonate) dihydrate; effects of intensity profile truncation and background modelling on structure models.

The crystal structures of the title compounds were determined with net intensities I derived via the background-peak-background procedure. Least-squares optimizations reveal differences between the low-order (0 < s < 0.7 A(-1)) and high-order (0.7 < s < 1.0 A(-1)) structure models. The scale factors indicate discrepancies of up to 10% between the low-order and high-order reflection intensities. This observation is compound independent. It reflects the scan-angle-induced truncation error, because the applied scan angle (0.8 + 2.0 tan theta) degrees underestimates the wavelength dispersion in the monochromated X-ray beam. The observed crystal structures show pseudo-I-centred sublattices for three of its non-H atoms in the asymmetric unit. Our selection of observed intensities (I > 3 sigma) stresses that pseudo-symmetry. Model refinements on individual data sets with (h + k + l) = 2n and (h + k + l) = 2n + 1 illustrate the lack of model robustness caused by that pseudo-symmetry. To obtain a better balanced data set and thus a more robust structure we decided to exploit background modelling. We described the background intensities B(H-->) with an 11th degree polynomial in straight theta. This function predicts the local background b at each position H--> and defines the counting statistical distribution P(B), in which b serves as average and variance. The observation R defines P(R). This leads to P(I) = P(R)/P(B) and thus I = R - b and sigma(2)(I) = I so that the error sigma(I) is background independent. Within this framework we reanalysed the structure of the copper(II) derivative. Background modelling resulted in a structure model with an improved internal consistency. At the same time the unweighted R value based on all observations decreased from 10.6 to 8.4%. A redetermination of the structure at 120 K concluded the analysis.

Journal Article↗
PubMed2002

The structure of orange HgI2. I. Polytypic layer structure.

The metastable orange crystals of HgI(2) comprise three different crystal structures, all of which are built from corner-linked Hg(4)I(10) supertetrahedra. Two of them are end members with the maximum degree of order (MDO) of a polytypic layer structure; the third shows a three-dimensional linkage. This paper presents the determination from X-ray diffraction data of the tetragonal polytypic structures and their stacking disorder. Diffraction patterns show sharp Bragg reflections and rods of diffuse intensity with pronounced maxima. In a first step, the diffuse intensity was neglected and all maxima were treated as Bragg reflections. The crystal was supposed to be a conglomerate of the two MDO structures diffracting independently, and their parameters and volume ratio were refined against the single data set. The geometries and anisotropic displacement parameters of the layers in the two structures are shown to be nearly identical. Layer contacts in the two stacking modes are identical. The structures are fractal complications of the stable red form of HgI(2). In a second step, the stacking disorder has been quantitatively analyzed with a Markov chain model. Two probabilities describing next-nearest-layer interactions were visually adjusted to observed intensity profiles extracted from image-plate detector data. Results consistently show that the crystal comprises nearly equal volumes of MDO structures with an average domain thickness of about 5 layers or 30 A

Journal Article↗
PubMed2001

The X-ray structure of a recombinant major urinary protein at 1.75 A resolution. A comparative study of X-ray and NMR-derived structures.

Major urinary proteins belong to the lipocalin family and are present in the urine of rodents as an ensemble of isoforms with pheromonal activity. The crystal structure of a recombinant mouse MUP (rMUP) was solved by the molecular-replacement technique and refined to an R factor and R(free) of 20 and 26.5%, respectively, at 1.75 A resolution. The structure was compared with an NMR model and with a crystallographic structure of the wild-type form of the protein. The crystal structures determined in different space groups present significantly smaller conformational differences amongst themselves than in comparison with NMR models. Some, but not all, of the conformational differences between the crystal and solution structures can be explained by the influence of crystallographic contacts. Most of the differences between the NMR and X-ray structures were found in the N-terminus and loop regions. A number of side chains lining the hydrophobic pocket of the molecule are more tightly packed in the NMR structure than in the crystallographic model. Surprisingly, clear and continuous electron density for a ligand was observed inside the hydrophobic pocket of this recombinant protein. Conformation of the ligand modelled inside the density is coherent with the results of recent NMR experiments.

Amino Acid Sequence↗
PubMed2002

Structure-based enzyme inhibitor design: modeling studies and crystal structure analysis of Pneumocystis carinii dihydrofolate reductase ternary complex with PT653 and NADPH.

Structural data are reported for N-(2,4-diaminopteridin-6-yl)methyldibenz[b,f]azepine (PT653), an example of structure-based inhibitor design with 21-fold selectivity for Pneumocystis carinii dihydrofolate reductase (pcDHFR) relative to rat liver dihydrofolate reductase (rlDHFR). These data test the hypothesis that 2,4-diaminopteridines with a bulky N,N-diarylaminomethyl side chain at the 6-position could fit better into the larger active site of pcDHFR than into that of mammalian DHFR. The crystal structure of the ternary complex of NADPH, PT653 and pcDHFR, refined to 2.4 A resolution, reveals that PT653 binds in a different orientation than predicted from modeling studies reported previously [Rosowsky et al. (1999), J. Med. Chem. 42, 4853-4860]. These crystal data show that the pteridine-ring plane is tilted compared with that observed in the crystal structure of the pcDHFR methotrexate (MTX) NADPH ternary complex used as a template to model PT653 binding. Also, as a result of this tilt, the dibenzoazepine ring is bound deeper into the p-aminobenzoyl folate binding pocket of pcDHFR, thereby relieving close intermolecular contacts predicted from the modeling data. By far the most significant structural change, but more subtle in magnitude, is the ligand-induced conformational shift of 1.2 A away from the inhibitor of residues 61-66 in helix C. The other major effect is the unwinding of the short helical segment involving loop 47 which has a different conformation to that observed in other pcDHFR complexes [Cody et al. (1999), Biochemistry, 38, 4303-4312]. The favorable pcDHFR selectivity of PT653 could be a result of ligand-induced fit of the large hydrophobic dibenzazepine ring which occupies regions of the enzyme active site not probed by other antifolates and which take advantage of sequence and conformational differences between the structures of human and pcDHFR. These data suggest that such hydrophobic analogs could be used as lead compounds in the design of more pcDHFR-selective antifolates. Enzyme inhibition data also show that PT653 is 102-fold selective for Toxoplasma gondii (tg) DHFR relative to rlDHFR. Homology-modeling studies of the tgDHFR structure suggest that differences in ligand-binding orientation and enzyme sequence could influence the enhanced selectivity of PT653 for tgDHFR.

Crystallization↗
PubMed1994

Refined structure of concanavalin A complexed with methyl alpha-D-mannopyranoside at 2.0 A resolution and comparison with the saccharide-free structure.

The three-dimensional structure of the complex between methyl alpha-D-mannopyranoside and concanavalin A has been refined at 2.0 A resolution. Diffraction data were recorded from a single crystal (space group P2(1)2(1)2(1), a = 123.7, b = 128.6, c = 67.2 A) using synchrotron radiation at a wavelength of 1.488 A. The final model has good geometry and an R factor of 19.9% for 58 871 reflections (82% complete), within the resolution limits of 8 to 2 A, with F > 1.0sigma(F). The asymmetric unit contains four protein subunits arranged as a dimer of dimers with approximate 222 point symmetry. Each monomer binds one saccharide molecule. Each sugar is bound to the protein by hydrogen bonds and van der Waals contacts. Although the four subunits are not crystallographically equivalent, the protein-saccharide interactions are nearly identical in each of the four binding sites. The differences that do occur between the four sites are in the structure of the water network which surrounds each saccharide; these networks are involved in crystal packing. The structure of the complex is compared with a refined saccharide-free concanavalin A structure. The saccharide-free structure is composed of crystallographically identical subunits, again assembled as a dimer of dimers, but with exact 222 symmetry. In the saccharide complex the tetramer association is different in that the monomers tend to separate resulting in fewer intersubunit interactions. The average temperature factor of the mannoside complex is considerably higher than that of the saccharide-free protein. The binding site in the saccharide-free structure is occupied by three ordered water molecules and the side chain of Asp71 from a neighbouring molecule in the crystal. These occupy positions similar to those of the four saccharide hydroxyls which are hydrogen bonded to the site. Superposition of the saccharide-binding site from each structure shows that the major changes on binding involve expulsion of these ordered solvents and the reorientation of the side chain of Tyrl00. Overall the surface accessibility of the saccharide decreases from 370 to 100 A(2) when it binds to the protein. This work builds upon the earlier studies of Derewenda et al. [Derewenda, Yariv, Helliwell, Kalb (Gilboa), Dodson, Papiz, Wan & Campbell (1989). EMBO J. 8, 2198-2193] at 2.9 A resolution, which was the first detailed study of lectin-saccharide interactions.

Journal Article↗
PubMed1998

STEP--a trial-and-error procedure for crystal structure determination. II. The determination of two small protein structures.

This paper describes the difficulties in the process of using the trial-and-error SYSTEM90 program to determine ab initio the structures of two small proteins App [Woolfson & Yao (1990). Acta Cryst. A46, 409-413] and rubredoxin [Sheldrick et al. (1993). Acta Cryst. D49, 18-23] with high-resolution data. Some strategies for overcoming the difficulties are discussed and the upgraded SYSTEM95 program was used successfully to determine the two structures. The most characteristic feature of this structure-determination process is that the two proteins are treated as unknown structures with only their chemical compositions and high-resolution data sets known. A new figure of merit R(sc), replacing the old figure of merit, XDFOM, is quite effective in picking out a good set of phases in the multi-solution stage when the phases are overconsistent. Controlling the Fourier recycling technique and the residuals can separate the mixture of structures and the enantiomorph and finally give one absolute structure. The results are compared with known structures to verify their reliability.

Animals↗
PubMed2002

Rapid structural fluctuations of the free HIV protease flaps in solution: relationship to crystal structures and comparison with predictions of dynamics calculations.

Crystal structures have shown that the HIV-1 protease flaps, domains that control access to the active site, are closed when the active site is occupied by a ligand. Although flap structures ranging from closed to semi-open are observed in the free protease, crystal structures reveal that even the semi-open flaps block access to the active site, indicating that the flaps are mobile in solution. The goals of this paper are to characterize the secondary structure and fast (sub-ns) dynamics of the flaps of the free protease in solution, to relate these results to X-ray structures and to compare them with predictions of dynamics calculations. To this end we have obtained nearly complete backbone and many sidechain signal assignments of a fully active free-protease construct that is stabilized against autoproteolysis by three point mutations. The secondary structure of this protein was characterized using the chemical shift index, measurements of (3h)J(NC') couplings across hydrogen bonds, and NOESY connectivities. Analysis of these measurements indicates that the protease secondary structure becomes irregular near the flap tips, residues 49-53. Model-free analysis of (15)N relaxation parameters, T(1), T(2) (T(1rho)) and (15)N-[(1)H] NOE, shows that residues in the flap tips are flexible on the sub-ns time scale, in contrast with previous observations on the inhibitor-bound protease. These results are compared with theoretical predictions of flap dynamics and the possible biological significance of the sub-ns time scale dynamics of the flap tips is discussed.

Amino Acid Sequence↗
PubMed1994

Control of translation by mRNA secondary structure: the importance of the kinetics of structure formation.

RNA secondary structure is important in a wide variety of biological processes, but relatively little is known about the pathways and kinetics of RNA folding. When the IS10 transposase (tnp) gene is transcribed from a promoter outside the element, little increase in tnp expression is observed. This protection from outside transcription (pot) occurs at the translational level, presumably resulting from mRNA secondary structure proposed to sequester the tnp ribosome-binding site. Here, we confirm the pot RNA structure and show that it blocks 30S ribosomal subunit binding in vitro. Point mutations that abolish protection in vivo map to the pot structure. Surprisingly, these pot mutations do not severely alter the pot secondary structure or increase 30S subunit binding in vitro, except in one case. Using an oligonucleotide hybridization assay, we show that most of the pot mutations slow the kinetics of pot structure formation, with little or no effect on the inhibitory function of the final structure. Moreover, a suppressor mutation reverses this effect. We propose a pathway for pot mRNA folding that is consistent with the mutations and implicates the formation of important kinetic intermediates. The significance of these observations for the RNA folding problem in general is discussed.

Base Sequence↗