Biomedical subjects

D I Svergun

Publications and source records attributed to D I Svergun.

At least 19 recordsLinked to original sources

PubMed2003

Twists and turns: a tale of two shikimate-pathway enzymes.

We are studying two enzymes from the shikimate pathway, dehydroquinate synthase (DHQS) and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Both enzymes have been the subject of numerous studies to elucidate their reaction mechanisms. Crystal structures of DHQS and EPSPS in the presence and absence of substrates, cofactors and/or inhibitors are now available. These structures reveal movements of domains, rearrangements of loops and changes in side-chain positions necessary for the formation of a catalytically competent active site. The potential for using complementary small-angle X-ray scattering (SAXS) studies to confirm the presence of these structural differences in solution has also been explored. Comparative analysis of crystal structures, in the presence and absence of ligands, has revealed structural features critical for substrate-binding and catalysis. We have also analysed these structures by generating GRID energy maps to detect favourable binding sites. The combination of X-ray crystallography, SAXS and computational techniques provides an enhanced analysis of structural features important for the function of these complex enzymes.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗

PubMed2001

Solution structure of bacteriophage PRD1 vertex complex.

Bacteriophage PRD1 is a prototype of viruses with an internal membrane. The icosahedral capsid and major coat protein share structural similarity with the corresponding structures of adenovirus. The present study further explores similarities between these viruses, considering the 5-fold vertex assemblies. The vertex structure of bacteriophage PRD1 consists of proteins P2, P5, and P31. The vertex complex mediates host cell binding and controls double-stranded DNA delivery. Quaternary structures and interactions of purified spike proteins were studied by synchrotron radiation x-ray solution scattering. Low resolution models of the vertex proteins P5, P2, and P31 were reconstructed ab initio from the scattering data. Protein P5 is a long trimer that resembles the adenovirus spike protein pIV. The receptor-binding protein P2 is a 15.5-nm long, thin monomer and does not have an adenovirus counterpart. P31 forms a pentameric base with a maximum diameter of 8.5 nm, which is thinner than the adenovirus penton pIII. P5 further polymerize into a nonameric form ((P5(3))(3)). In the presence of P31, P5 associates into a P5(6):P31 complex. The constructed models of these assemblies provided support for a model of vertex assembly onto the virion. Although similar in overall architecture, clear differences between PRD1 and adenovirus spike assemblies have been revealed.

Bacteriophage PRD1↗

PubMed2001

Conformation of the Drosophila motor protein non-claret disjunctional in solution from X-ray and neutron scattering.

The quaternary structures of monomeric and dimeric Drosophila non-claret disjunctional (ncd) constructs were investigated using synchrotron x-ray and neutron solution scattering, and their low resolution shapes were restored ab initio from the scattering data. The experimental curves were further compared with those computed from crystallographic models of one monomeric and three available dimeric ncd structures in the microtubule-independent ADP-bound state. These comparisons indicate that accounting for the missing parts in the crystal structures for all these constructs is indispensable to obtain reasonable fits to the scattering patterns. A ncd construct (MC6) lacking the coiled-coil region is monomeric in solution, but the calculated scattering from the crystallographic monomer yields a poor fit to the data. A tentative configuration of the missing C-terminal residues in the form of an antiparallel beta-sheet was found that significantly improves the fit. The atomic model of a short dimeric ncd construct (MC5) without 2-fold symmetry is found to fit the data better than the symmetric models. Addition of the C-terminal residues to both head domains gives an excellent fit to the x-ray and neutron experimental data, although the orientation of the beta-sheet differs from that of the monomer. The solution structure of the long ncd construct (MC1) including complete N-terminal coiled-coil and motor domains is modeled by adding a straight coiled-coil section to the model of MC5.

Animals↗

PubMed2001

Structural Insights into the A1 ATPase from the archaeon, Methanosarcina mazei Gö1.

The low-resolution structure and overall dimensions of the A(3)B(3)CDF complex of the A(1) ATPase from Methanosarcina mazei Gö1 in solution is analyzed by synchrotron X-ray small-angle scattering. The radius of gyration and the maximum size of the complex are 5.03 +/- 0.1 and 18.0 +/- 0.1 nm, respectively. The low-resolution shape of the protein determined by two independent ab initio approaches has a knob-and-stalk-like feature. Its headpiece is approximately 9.4 nm long and 9.2 nm wide. The stalk, which is known to connect the headpiece to its membrane-bound A(O) part, is approximately 8.4 nm long. Limited tryptic digestion of the A(3)B(3)CDF complex was used to probe the topology of the smaller subunits (C-F). Trypsin was found to cleave subunit C most rapidly at three sites, Lys(20), Lys(21), and Arg(209), followed by subunit F. In the A(3)B(3)CDF complex, subunit D remained protected from proteolysis.

Adenosine Triphosphatases↗

PubMed2001

A novel quaternary structure of the dimeric alpha-crystallin domain with chaperone-like activity.

alphaB-crystallin, a member of the small heat-shock protein family and a major eye lens protein, is a high molecular mass assembly and can act as a molecular chaperone. We report a synchrotron radiation x-ray solution scattering study of a truncation mutant from the human alphaB-crystallin (alphaB57-157), a dimeric protein that comprises the alpha-crystallin domain of the alphaB-crystallin and retains a significant chaperone-like activity. According to the sequence analysis (more than 23% identity), the monomeric fold of the alpha-crystallin domain should be close to that of the small heat-shock protein from Methanococcus jannaschii (MjHSP16.5). The theoretical scattering pattern computed from the crystallographic model of the dimeric MjHSP16.5 deviates significantly from the experimental scattering by the alpha-crystallin domain, pointing to different quaternary structures of the two proteins. A rigid body modeling against the solution scattering data yields a model of the alpha-crystallin domain revealing a new dimerization interface. The latter consists of a strand-turn-strand motif contributed by each of the monomers, which form a four-stranded, antiparallel, intersubunit composite beta-sheet. This model agrees with the recent spin labeling results and suggests that the alphaB-crystallin is composed by flexible building units with an extended surface area. This flexibility may be important for biological activity and for the formation of alphaB-crystallin complexes of variable sizes and compositions.

Amino Acid Sequence↗

PubMed2001

Determination of domain structure of proteins from X-ray solution scattering.

An ab initio method for building structural models of proteins from x-ray solution scattering data is presented. Simulated annealing is employed to find a chain-compatible spatial distribution of dummy residues which fits the experimental scattering pattern up to a resolution of 0.5 nm. The efficiency of the method is illustrated by the ab initio reconstruction of models of several proteins, with known and unknown crystal structure, from experimental scattering data. The new method substantially improves the resolution and reliability of models derived from scattering data and makes solution scattering a useful technique in large-scale structural characterization of proteins.

Algorithms↗

PubMed2000

Structure of free Thermus flavus 5 S rRNA at 1.3 nm resolution from synchrotron X-ray solution scattering.

The shape of free Thermus flavus 5 S rRNA in solution at 1.3 nm resolution is restored from synchrotron x-ray scattering data using an ab initio simulated annealing algorithm. The free 5 S rRNA is a bent elongated molecule displaying a compact central region and two projecting arms, similar to those of the tRNA. The atomic models of the 5 S rRNA domains A-D-E and B-C in the form of elongated helices can be well accommodated within the shape, yielding a tentative model of the structure of the free 5 S rRNA in solution. Its comparison with the recent protein-RNA map in the ribosome (Svergun, D. I., and Nierhaus, K. H. (2000) J. Biol. Chem. 275, 14432-14439) indicates that the 5 S rRNA becomes essentially more compact upon complex formation with specific ribosomal proteins. A conceivable conformational change involves rotation of the B-C domain toward the A-D-E domain. The model of free 5 S rRNA displays no interactions between domains E and C, but such interactions are possible in the bound molecule.

Algorithms↗

PubMed2000

Evidence for major structural changes in the Manduca sexta midgut V1 ATPase due to redox modulation. A small angle X-ray scattering study.

The shape and overall dimensions of the oxidized and reduced form of the V(1) ATPase from Manduca sexta were investigated by synchrotron radiation x-ray solution scattering. The radius of gyration of the oxidized and reduced complex differ noticeably, with dimensions of 6. 20 +/- 0.06 and 5.84 +/- 0.06 nm, respectively, whereas the maximum dimensions remain constant at 22.0 +/- 0.1 nm. Comparison of the low resolution shapes of both forms, determined ab initio, indicates that the main structural alteration occurs in the head piece, where the major subunits A and B are located, and at the bottom of the stalk. In conjunction with the solution scattering data, decreased susceptibility to tryptic digestion and tryptophan fluorescence of the reduced V(1) molecule provide the first strong evidence for major structural changes in the V(1) ATPase because of redox modulation.

Animals↗

PubMed2000

Solution structure and conformational changes of the Streptomyces chitin-binding protein (CHB1).

The shape and overall dimensions of the recently discovered Streptomyces alpha-chitin-binding protein, CHB1, were investigated by synchrotron radiation X-ray solution scattering. The radius of gyration and the maximum size of CHB1 were determined to be 1.75 +/- 0.03 nm and 6.0 +/- 0.2 nm, respectively. Using two independent ab initio approaches the low-resolution shape of the protein was found to consist of two domains, an elongated main globule with a length of about 4 nm and a foot-like domain of about 2 nm width. The structural and functional properties of CHB1 depend strongly on the presence of disulfide bonds; upon their reduction, the protein loses its affinity to chitin.

Bacterial Proteins↗

PubMed2000

A map of protein-rRNA distribution in the 70 S Escherichia coli ribosome.

Neutron scattering exploits the enormous scattering difference between protons and deuterons. A set of 42 x-ray and neutron solution scattering curves from hybrid Escherichia coli ribosomes was obtained, where the proteins and rRNA moieties in the subunits were either protonated or deuterated in all possible combinations. This extensive data set is analyzed using a novel method. The volume defined by the cryoelectron microscopic model of Frank and co-workers (Frank, J., Zhu, J., Penczek, P., Li, Y. H., Srivastava, S., Verschoor, A., Radermacher, M., Grassucci, R., Lata, R. K., and Agrawal, R. K. (1995) Nature 376, 441-444) is divided into 7890 densely packed spheres of radius 0.5 nm. Simulated annealing is employed to assign each sphere to solvent, protein, or rRNA moieties to simultaneously fit all scattering curves. Twelve independent reconstructions starting from random approximations yielded reproducible results. The resulting model at a resolution of 3 nm represents the volumes occupied by rRNA and protein moieties at 95% probability threshold and displays 15 and 20 protein subvolumes in the 30 S and 50 S, respectively, connected by rRNA. 17 proteins with known atomic structure can be tentatively positioned into the protein subvolumes within the ribosome in agreement with the results from other methods. The protein-rRNA map enlarges the basis for the models of the rRNA folding and can further help to localize proteins in high-resolution crystallographic density maps.

Bacterial Proteins↗

PubMed2000

Solution structure of the E. coli 70S ribosome at 11.5 A resolution.

Over 73,000 projections of the E. coli ribosome bound with formyl-methionyl initiator tRNAf(Met) were used to obtain an 11.5 A cryo-electron microscopy map of the complex. This map allows identification of RNA helices, peripheral proteins, and intersubunit bridges. Comparison of double-stranded RNA regions and positions of proteins identified in both cryo-EM and X-ray maps indicates good overall agreement but points to rearrangements of ribosomal components required for the subunit association. Fitting of known components of the 50S stalk base region into the map defines the architecture of the GTPase-associated center and reveals a major change in the orientation of the alpha-sarcin-ricin loop. Analysis of the bridging connections between the subunits provides insight into the dynamic signaling mechanism between the ribosomal subunits.

Bacterial Proteins↗

PubMed2000

Low resolution structure of the sigma54 transcription factor revealed by X-ray solution scattering.

The sigma54 RNA polymerase holoenzyme functions in enhancer-dependent transcription. The structural organization of the sigma54 subunit of bacterial RNA polymerase in solution is analyzed by synchrotron x-ray scattering. Scattering patterns are collected from the full-length protein and from a large fragment able to bind the core RNA polymerase, and their low resolution shapes are restored using two ab initio shape determination techniques. The sigma54 subunit is a highly elongated particle, and the core binding fragment can be unambiguously positioned inside the full-length protein. The boomerang-like shape of the core binding fragment is similar to that of the atomic model of a fragment of the Escherichia coli sigma70 protein, indicating that, although the sigma54 and sigma70 factors are unrelated by primary sequence, they may share some structural similarity. Potential DNA binding surfaces of sigma54 are also predicted by comparison with the sigma54 core binding fragment.

Bacterial Proteins↗

PubMed2000

Crystal versus solution structures of thiamine diphosphate-dependent enzymes.

The quaternary structures of the thiamine diphosphate-dependent enzymes transketolase (EC 2.2.1.1; from Saccharomyces cerevisiae), pyruvate oxidase (EC 1.2.3.3; from Lactobacillus plantarum), and pyruvate decarboxylase (EC 4.1.1.1; from Zymomonas mobilis and brewers' yeast, the latter in the native and pyruvamide-activated forms) were examined by synchrotron x-ray solution scattering. The experimental scattering data were compared with the curves calculated from the crystallographic models of these multisubunit enzymes. For all enzymes noted above, except the very compact pyruvate decarboxylase from Z. mobilis, there were significant differences between the experimental and calculated profiles. The changes in relative positions of the subunits in solution were determined by rigid body refinement. For pyruvate oxidase and transketolase, which have tight intersubunit contacts in the crystal, relatively small modifications of the quaternary structure (root mean square displacements of 0.23 and 0.27 nm, respectively) sufficed to fit the experimental data. For the enzymes with looser contacts (the native and activated forms of yeast pyruvate decarboxylase), large modifications of the crystallographic models (root mean square displacements of 0.58 and 1.53 nm, respectively) were required. A clear correlation was observed between the magnitude of the distortions induced by the crystal environment and the interfacial area between subunits.

Artifacts↗

PubMed1999

Nonaggregating mutant of recombinant human hexokinase I exhibits wild-type kinetics and rod-like conformations in solution.

Hexokinase I governs the rate-limiting step of glycolysis in brain tissue, being inhibited by its product, glucose 6-phosphate, and allosterically relieved of product inhibition by phosphate. On the basis of small-angle X-ray scattering, the wild-type enzyme is a monomer in the presence of glucose and phosphate at protein concentrations up to 10 mg/mL, but in the presence of glucose 6-phosphate, is a dimer down to protein concentrations as low as 1 mg/mL. A mutant form of hexokinase I, specifically engineered by directed mutation to block dimerization, remains monomeric at high protein concentration under all conditions of ligation. This nondimerizing mutant exhibits wild-type activity, potent inhibition by glucose 6-phosphate, and phosphate reversal of product inhibition. Small-angle X-ray scattering data from the mutant hexokinase I in the presence of glucose/phosphate, glucose/glucose 6-phosphate, and glucose/ADP/Mg2+/AlF3 are consistent with a rodlike conformation for the monomer similar to that observed in crystal structures of the hexokinase I dimer. Hence, any mechanism for allosteric regulation of hexokinase I should maintain a global conformation of the polypeptide similar to that observed in crystallographic structures.

Brain↗

PubMed1999

Restoring low resolution structure of biological macromolecules from solution scattering using simulated annealing.

A method is proposed to restore ab initio low resolution shape and internal structure of chaotically oriented particles (e.g., biological macromolecules in solution) from isotropic scattering. A multiphase model of a particle built from densely packed dummy atoms is characterized by a configuration vector assigning the atom to a specific phase or to the solvent. Simulated annealing is employed to find a configuration that fits the data while minimizing the interfacial area. Application of the method is illustrated by the restoration of a ribosome-like model structure and more realistically by the determination of the shape of several proteins from experimental x-ray scattering data.

Biophysical Phenomena↗

PubMed1999

Biochemical and structural characterization of recombinant copper-metallothionein from Saccharomyces cerevisiae.

Methods were developed for large-scale purification of recombinant Cu-metallothionein (Cu-MT) for structural investigations and the determination of Cu-binding stoichiometry. Cu-MT of Saccharomyces cerevisiae overexpressed in Escherichia coli was purified using a procedure based on ion exchange and gel filtration chromatography followed by reversed-phase HPLC. The purified protein was fully characterized by electrophoresis, amino acid analysis, atomic absorption spectroscopy and elemental analysis, and was shown to contain 10 +/- 2 Cu(I) per molecule of protein. Small angle X-ray scattering measurements yielded a radius of gyration of 1.2 nm for the recombinant protein, indicating a more extended structure in solution than that derived from the recent NMR data [Peterson, C.W., Narula, S.S. & Armitage, I.A. (1996) FEBS Lett. 379, 85-93].

Carrier Proteins↗

PubMed1998

Quaternary structure of V1 and F1 ATPase: significance of structural homologies and diversities.

The V1 ATPase from the tobacco hornworm Manduca sexta and the Escherichia coli F1 ATPase were characterized by small-angle X-ray scattering (SAXS). The radii of gyration (Rg) of the complexes were 6.2 +/- 0.1 and 4.7 +/- 0.02 nm, respectively. The shape of the M. sexta V1 ATPase was determined ab initio from the scattering data showing six masses, presumed to be the A and B subunits, arranged in an alternating manner about a 3-fold axis. A seventh mass with a length of about 11.0 nm extends perpendicularly to the center of the hexameric unit. This central mass is presumed to be the stalk that connects V1 with the membrane domain (V(O)) in the intact V1V(O)-ATPase. In comparison, the shape of the F1 ATPase from E. coli possesses a quasi-3-fold symmetry over the major part of the enzyme. The overall asymmetry of the structure is given by a stem, assumed to include the central stalk subunits. The features of the V1 and F1 ATPase reveal structural homologies and diversities of the key components of the complexes.

Animals↗

PubMed1998

Evidence for a major structural change in Escherichia coli chorismate synthase induced by flavin and substrate binding.

Chorismate synthase (EC 4.6.1.4) catalyses the conversion of 5-enolpyruvylshikimate 3-phosphate (EPSP) into chorismate, and requires reduced FMN as a cofactor. The enzyme can bind first oxidized FMN and then EPSP to form a stable ternary complex which does not undergo turnover. This complex can be considered to be a model of the ternary complex between enzyme, EPSP and reduced FMN immediately before catalysis commences. It is shown that the binding of oxidized FMN and EPSP to chorismate synthase affects the properties and structure of the protein. Changes in small-angle X-ray scattering data, decreased susceptibility to tryptic digestion and altered Fourier-transform (FT)-IR spectra provide the first strong evidence for major structural changes in the protein. The tetrameric enzyme undergoes correlated screw movements leading to a more overall compact shape, with no change in oligomerization state. The changes in the FT-IR spectrum appear to reflect changes in the environment of the secondary-structural elements rather than alterations in their distribution, because the far-UV CD spectrum changes very little. Changes in the mobility of the protein during non-denaturing PAGE indicate that the ternary complex may exhibit less conformational flexibility than the apoprotein. Increased enzyme solubility and decreased tryptophan fluorescence are discussed in the light of the observed structural changes. The secondary structure of the enzyme was investigated using far-UV CD spectroscopy, and the tertiary structure was predicted to be an alpha-beta-barrel using discrete state-space modelling.

Circular Dichroism↗