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J Nyborg

Publications and source records attributed to J Nyborg.

At least 37 records · Page 2Linked to original sources
PubMed1998

EF-hands at atomic resolution: the structure of human psoriasin (S100A7) solved by MAD phasing.

BACKGROUND: The S100 family consists of small acidic proteins, belonging to the EF-hand class of calcium-binding proteins. They are primarily regulatory proteins, involved in cell growth, cell structure regulation and signal transduction. Psoriasin (S100A7) is an 11.7 kDa protein that is highly upregulated in the epidermis of patients suffering from the chronic skin disease psoriasis. Although its exact function is not known, psoriasin is believed to participate in the biochemical response which follows transient changes in the cellular Ca2+ concentration. RESULTS: The three-dimensional structure of holmium-substituted psoriasin has been determined by multiple anomalous wavelength dispersion (MAD) phasing and refined to atomic resolution (1.05 A). The structure represents the most accurately determined structure of a calcium-binding protein. Although the overall structure of psoriasin is similar to those of other S100 proteins, several important differences exist, mainly in the N-terminal EF-hand motif that contains a distorted loop and lacks a crucial calcium-binding residue. It is these minor differences that may account for the different specificities among members of this family. CONCLUSIONS: The structure of human psoriasin reveals that this protein, in contrast to other S100 proteins with known structure, is not likely to strongly bind more than one calcium ion per monomer. The present study contradicts the idea that calcium binding induces large changes in conformation, as suggested by previously determined structures of apo forms of S100 proteins. The substitution of Ca2+ ions in EF-hands by lanthanide ions may provide a general vehicle for structure determination of S100 proteins by means of MAD phasing.

Calcium↗
PubMed1998

Possible evolution of factors involved in protein biosynthesis.

The elongation factors of protein biosynthesis are well preserved through out evolution. They catalyze the elongation phase of protein biosynthesis, where on the ribosome amino acids are added one at a time to a growing peptide according to the genetic information transcribed into mRNA. Elongation factor Tu (EF-Tu) provides the binding of aminoacylated tRNA to the ribosome and protects the aminoester bond against hydrolysis until a correct match between the codon on mRNA and the anticodon on tRNA can be achieved. Elongation factor G (EF-G) supports the translocation of tRNAs and of mRNA on the ribosome so that a new codon can be exposed for decoding. Both these factors are GTP binding proteins, and as such exist in an active form with GTP and an inactive form with GDP bound to the nucleotide binding domain. Elongation factor Ts (EF-Ts) will catalyze the exchange of nucleotide on EF-Tu. This review describes structural work on EF-Tu performed in our laboratory over the last eight years. The structural results provide a rather complete picture of the major structural forms of EF-Tu, including the so called ternary complex of aa-tRNA:EF-Tu:GTP. The structural comparison of this ternary complex with the structure of EF-G:GDP displays an unexpected macromolecular mimicry, where three domains of EF-G mimick the shape of the tRNA in the ternary complex. This observation has initiated much speculation on the evolution of all factors involved in protein synthesis, as well as on the details of the ribosomal function in one part of elongation.

Amino Acid Sequence↗
PubMed1997

Crystallization and preliminary X-ray diffraction studies of psoriasin.

Crystals of psoriasin, a protein related to the skin disease psoriasis, have been grown in two different crystal forms. Form I represents the protein in the Ca(2+)-bound form, and form II represents the protein in the Zn(2+)- and Ca(2+)-bound form. The crystals of form I are orthorhombic belonging to the space group P2(1)2(1)2(1) with cell parameters a = 52.15, b = 56.67 and c = 76.38 A and diffract to 2.4 A. The crystals of form II are tetragonal and belong to the space group P4(1(3))2(1)2 with cell parameters a = b = 51.86, c = 115.93 A and diffract to 2.0 A.

Journal Article↗
PubMed1997

The ternary complex of EF-Tu and its role in protein biosynthesis.

The past year has seen a breakthrough in our structural understanding of how aminoacyl-tRNAs are selected and transported to the ribosomal A-site in order to decode genetic information contained in messenger RNA. All aminoacyl-tRNAs are recognized by the elongation factor EF-Tu in prokaryotes or EF-1alpha in eukaryotes. The recent determination of the structure of the ternary complex of aminoacyl-tRNA, EF-Tu and a GTP analogue shows how the CCA end of all aminoacyl-tRNA structures can be accommodated in a specific binding site on EF-Tu-GTP, and how part of the T-helix can be recognized by EF-Tu in a non-sequence-specific way. Furthermore, the structure of the ternary complex shows striking structural similarity to the structure of another prokaryotic elongation factor, EF-G, the tRNA translocase, in its GDP or empty form. This observation has led to the proposal of a general macromolecular mimicry of RNA and protein, which predicts elements of RNA-like structures will occur in other translation factors, such as initiation factors and release factors, that interact with similar sites on the ribosome.

Peptide Elongation Factor Tu↗
PubMed1997

Macromolecular mimicry in protein biosynthesis.

Elongation factor Tu (EF-Tu) is a G-protein which, in its active GTP conformation, protects and carries aminoacylated tRNAs (aa-tRNAs) to the ribosome during protein biosynthesis. EF-Tu consists of three structural domains of which the N-terminal domain consists of two special regions (switch I and switch II) which are structurally dependent on the type of the bound nucleotide. Structural studies of the complete functional cycle of EF-Tu reveal that it undergoes rather spectacular conformational changes when activated from the EF-Tu.GDP form to the EF-Tu.GTP form. In its active form, EF-Tu.GTP without much further structural change interacts with aa-tRNAs in the so-called ternary complex. The conformational changes of EF-Tu involve rearrangements of the secondary structures of both the switch I and switch II regions. As the switch II region forms part of the interface between domains 1 and 3, its structural rearrangement results in a very large change of the position of domain 1 relative to domains 2 and 3. The overall shape of the ternary complex is surprisingly similar to the overall shape of elongation factor G (EF-G). Thus, three domains of the protein EF-G seem to mimic the tRNA part of the ternary complex. This macromolecular mimicry has profound implications for the function of the elongation factors on the ribosome.

Amino Acid Sequence↗
PubMed1996

Isolation, crystallization and X-ray analysis of the quaternary complex of Phe-tRNA(Phe), EF-Tu, a GTP analog and kirromycin.

Kirromycin inhibits bacterial protein synthesis by acting on elongation factor Tu (EF-Tu). Complexes of the antibiotic, Phe-tRNA(Phe), the guanosine triphosphate analog GDPNP, and mesophilic (Escherichia coli), as well as thermophilic (Thermus thermophilus) EF-Tu were isolated. Crystallization was achieved at 4 degrees C, pH 6.4, using ammonium sulphate as precipitant. Crystallographic data were recorded at cryogenic temperature on crystals exposed to synchrotron radiation. Crystals of the thermophilic complex are based on a rhombohedral lattice with cell dimensions of 137.3 A, and angles of 54.0 degrees. Although related, these cell parameters are different from those found in the crystals of the recently solved structure of the ternary complex of Phe-tRNA(Phe), GDPNP, and Thermus aquaticus EF-Tu (Nissen, P., Kjeldgaard, M., Thirup, S., Polekhina, G., Reshetnikova, L., Clark, B.F. and Nyborg, J. (1995) Science 270, 1464-1472 [1]), possibly indicating some allosteric effect caused by kirromycin. Crystals of the mesophilic complex belong to the cubic space P432, with cell axis of 196.26 A. In both cases, the crystals contain one complex per asymmetric unit.

Guanosine Triphosphate↗
PubMed1996

Helix unwinding in the effector region of elongation factor EF-Tu-GDP.

BACKGROUND: Elongation factor Tu (EF-Tu) in its GTP conformation is a carrier of aminoacylated tRNAs (aa-tRNAs) to the ribosomal A site during protein biosynthesis. The ribosome triggers GTP hydrolysis, resulting in the dissociation of EF-Tu-GDP from the ribosome. The affinity of EF-Tu for other molecules involved in this process, some of which are unknown, is regulated by two regions (Switch I and Switch II) that have different conformations in the GTP and GDP forms. The structure of the GDP form of EF-Tu is known only as a trypsin-modified fragment, which lacks the Switch I, or effector, domain. The aim of this work was to establish the overall structure of intact EF-Tu-GDP, in particular the structure of the effector domain. RESULTS: The crystal structures of intact EF-Tu-GDP from Thermus aquaticus and Escherichia coli have been determined at resolutions of 2.7 A and 3.8 A, respectively. The structures confirm the domain orientation previously found in the structure of partially trypsin-digested EF-Tu-GDP. The structures of the effector region in T. aquaticus and E. coli EF-Tu-GDP are very similar. The C-terminal part of the effector region of EF-Tu-GDP is a beta hairpin; in EF-Tu-GTP, this region forms an alpha helix. This conformational change is not a consequence of crystal packing. CONCLUSIONS: EF-Tu undergoes major conformational changes upon GTP hydrolysis. Unlike other GTP-binding proteins, EF-Tu exhibits a dramatic conformational change in the effector region, involving an unwinding of a small helix and the formation of a beta hairpin structure. This change is presumably involved in triggering the release of tRNA, and EF-Tu, from the ribosome.

Bacterial Proteins↗
PubMed1996

Mapping Escherichia coli elongation factor Tu residues involved in binding of aminoacyl-tRNA.

Two residues of Escherichia coli elongation factor Tu involved in binding of aminoacyl-tRNA were identified and subjected to mutational analysis. Lys-89 and Asn-90 were each replaced by either Ala or Glu. The four single mutants were denoted K89A, K89E, N90A, and N90E, respectively. The mutants were characterized with respect to thermal and chemical stability, GTPase activity, tRNA affinity, and activity in an in vitro translation assay. Most conspicuously tRNA affinities were reduced for all mutants. The results verify our structural analysis of elongation factor Tu in complex with aminoacyl-tRNA, which suggested an important role of Lys-89 and Asn-90 in tRNA binding. Furthermore, our results indicate helix B to be an important target site for nucleotide exchange factor EF-Ts. Also the mutants His-66 to Ala and His-118 to either Ala or Glu were characterized in an in vitro translation assay. Their functional roles are discussed in relation to the structure of elongation factor Tu in complex with aminoacyl-tRNA.

Amino Acid Sequence↗
PubMed1996

The ternary complex of aminoacylated tRNA and EF-Tu-GTP. Recognition of a bond and a fold.

The refined crystal structure of the ternary complex of yeast Phe-tRNAPhe, Thermus aquaticus elongation factor EF-Tu and the non-hydrolyzable GTP analog, GDPNP, reveals many details of the EF-Tu recognition of aminoacylated tRNA (aa-tRNA). EF-Tu-GTP recognizes the aminoacyl bond and one side of the backbone fold of the acceptor helix and has a high affinity for all ordinary elongator aa-tRNAs by binding to this aa-tRNA motif. Yet, the binding of deacylated tRNA, initiator tRNA, and selenocysteine-specific tRNA (tRNASec) is effectively discriminated against. Subtle rearrangements of the binding pocket may occur to optimize the fit to any side chain of the aminoacyl group and interactions with EF-Tu stabilize the 3'-aminoacyl isomer of aa-tRNA. A general complementarity is observed in the location of the binding sites in tRNA for synthetases and for EF-Tu. The complex formation is highly specific for the GTP-bound conformation of EF-Tu, which can explain the effects of various mutants.

Aspartic Acid↗
PubMed1996

Elongation in bacterial protein biosynthesis.

The past year has brought some notable advances in our understanding of the structure and function of elongation factors (EFs) involved in protein biosynthesis. The structures of the ternary complex of aminoacylated tRNA with EF-Tu.GTP and of the complex EF-Tu.EF-Ts have been determined. Within the same period, new cryo-electron microscopy reconstructions of ribosome particles have been obtained.

Bacterial Proteins↗
PubMed1996

Identification of 13 new mutations in the vasopressin-neurophysin II gene in 17 kindreds with familial autosomal dominant neurohypophyseal diabetes insipidus.

Familial neurohypophyseal diabetes insipidus (FNDI) is an autosomal dominant disorder characterized by progressive postnatal deficiency of arginine vasopressin as a result of mutation in the gene that encodes the hormone. To determine the extent of mutations in the coding region that produce the phenotype, we studied members of 17 unrelated kindreds with the disorder. We sequenced all 3 exons of the gene by using a rapid, direct dye-terminator method and found the causative mutation in each kindred. In four kindreds, the mutations were each identical to mutations described in other affected families. In the other 13 kindreds each mutation was unique. There were two missense mutations that altered the cleavage region of the signal peptide, seven missense mutations in exon 2, which codes for the conserved portion of the protein, one nonsense mutation in exon 2, and three nonsense mutations in exon 3. These findings, together with the clinical features of FNDI, suggest that each of the mutations exerts an effect by directing the production of a pre-prohormone that cannot be folded, processed, or degraded properly and eventually destroys vasopressinergic neurons.

Arginine Vasopressin↗
PubMed1996

The GTP binding motif: variations on a theme.

GTP binding proteins (G-proteins) have wide-ranging functions in biology, being involved in cell proliferation, signal transduction, protein synthesis, and protein targeting. Common to their functioning is that they are active in the GTP-bound form and inactive in the GDP-bound form. The protein synthesis elongation factor EF-Tu was the first G-protein whose nucleotide binding domain was solved structurally by X-ray crystallography to yield a structural definition of the GDP-bound form, but a still increasing number of new structures of G-proteins are appearing in the literature, in both GDP and GTP bound forms. A common structural core for nucleotide binding is present in all these structures, and this core has long been known to include common consensus sequence elements involved in binding of the nucleotide. Nevertheless, subtle changes in the common sequences reflect functional differences. Therefore, it becomes increasingly important to focus on how these differences are reflected in the structures, and how these structural differences are related to function. The aim of this review is to describe to what extent this structural motif for GDP/GTP binding is common to other known structures of this class of proteins. We first describe the common structural core of the G-proteins. Next, examples are based on information available on the Ras protein superfamily, the targeting protein ARF, elongation factors EF-Tu and EF-G, and the heterotrimeric G-proteins. Finally, we discuss the important structures of complexes between GTP binding proteins and their substrates that have appeared in the literature recently.

Adenylosuccinate Synthase↗
PubMed1995

Crystal structure of the ternary complex of Phe-tRNAPhe, EF-Tu, and a GTP analog.

The structure of the ternary complex consisting of yeast phenylalanyl-transfer RNA (Phe-tRNAPhe), Thermus aquaticus elongation factor Tu (EF-Tu), and the guanosine triphosphate (GTP) analog GDPNP was determined by x-ray crystallography at 2.7 angstrom resolution. The ternary complex participates in placing the amino acids in their correct order when messenger RNA is translated into a protein sequence on the ribosome. The EF-Tu-GDPNP component binds to one side of the acceptor helix of Phe-tRNAPhe involving all three domains of EF-Tu. Binding sites for the phenylalanylated CCA end and the phosphorylated 5' end are located at domain interfaces, whereas the T stem interacts with the surface of the beta-barrel domain 3. The binding involves many conserved residues in EF-Tu. The overall shape of the ternary complex is similar to that of the translocation factor, EF-G-GDP, and this suggests a novel mechanism involving "molecular mimicry" in the translational apparatus.

Amino Acid Sequence↗
PubMed1995

Low resolution X-ray structure of human methylamine-treated alpha 2-macroglobulin.

The structure of methylamine-treated human alpha 2-macroglobulin (alpha 2M-Ma), a 720-kDa tetrameric inactivated proteinase inhibitor from plasma, has been determined to a resolution of 10 A. Data were collected with synchrotron radiation at 120 K, and phases were calculated by multiple isomorphous replacement and solvent flattening. A novel feature of the structure of alpha 2-M is present in its proteinase-binding cavity, dividing it into two compartments. The potential sites for proteinase entrapment in these compartments are sterically restricted. The positions of the thiol groups appearing from the functional important thiol esters upon their cleavage have been determined. They are found at the walls of the compartments at the center of the structure. The overall structure of alpha 2M-MA is much more sphere-like than previously inferred from electron microscopy studies. However, several aspects of the structure are well described by recent three-dimensional reconstructions. Possible models for the monomer, the disulfide bridged dimer, and native alpha 2M are discussed.

Crystallography, X-Ray↗
PubMed1995

Crystallisation and preliminary X-ray analysis of the receptor-binding domain of human and bovine alpha 2-macroglobulin.

The receptor-binding domains (RBDs) of human and bovine alpha 2-macroglobulin (alpha 2M) have been isolated after limited proteolysis of methylamine-treated alpha 2M with papain. Single crystals of the RBDs have been grown by vapour diffusion. Crystals of human RBD are very thin plates unsuited for data collection. However, crystals of RBD from bovine alpha 2M give diffraction patterns suitable for X-ray analysis, and a complete dataset with a maximum resolution of 2.3 A has been collected with synchrotron radiation at cryogenic temperature. The crystals belong to spacegroup P3(1)21 or P3(2)21 with cell parameters a = b = 106.8 A, c = 72.2 A.

Amino Acid Sequence↗
PubMed1995

Analysis and crystallization of a 25 kDa C-terminal fragment of cloned elongation factor Ts from Escherichia coli.

A 25 kDa C-terminal tryptic fragment of elongation factor Ts has been purified to homogeneity. Experimental evidence suggests that the 25 kDa C-terminal and the 5.3 kDa N-terminal fragments are structurally independent domains. The N-terminal fragment is shown to be essential for the nucleotide exchange activity. Crystals of the C-terminal fragment belong to space group P2 or P2(1). The diffraction pattern shows a pronounced pseudo-C2 symmetry at low resolution. This pseudo symmetry increases when the crystals are irradiated with X-rays for a few hours.

Amino Acid Sequence↗
PubMed1995

Nucleotide sequence encoding the snake venom fibrinolytic enzyme atroxase obtained from a Crotalus atrox venom gland cDNA library.

Atroxase, isolated from the venom of Crotalus atrox (western diamondback rattlesnake), is a nonhemorrhagic protease which has fibrinolytic activity in vivo. The primary structure of atroxase was deduced from the cDNA encoding the atroxase protein. The venom glands of Crotalus atrox were used to prepare a cDNA library. Degenerate oligonucleotides were synthesized based on the partial amino acid sequence of atroxase and were used as primers in the polymerase chain reaction to amplify overlapping cDNA fragments from the C. atrox cDNA library. The resulting cDNA fragments were subcloned, sequenced, and translated. The final nucleotide sequence shows high homology to previously described primary structures of nonhemorrhagic fibrinolytic proteases isolated from snake venom.

Amino Acid Sequence↗