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S Thirup

Publications and source records attributed to S Thirup.

14 recordsLinked to original sources
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↗
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

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↗
PubMed1994

Purification and crystallization of the ternary complex of elongation factor Tu:GTP and Phe-tRNA(Phe).

Elongation factor Tu (EF-Tu) is the most abundant protein in prokaryotic cells. Its general function in protein biosynthesis is well established. It is a member of the large family of G-proteins, all of which bind guanosine phosphates (GDP or GTP) as cofactors. In its active GTP bound state EF-Tu binds aminoacylated tRNA (aa-tRNA) forming the ternary complex EF-Tu:GTP:aa-tRNA. The ternary complex interacts with the ribosome where the anticodon on tRNA recognises a codon on mRNA, GTPase activity is induced and inactive EF-Tu:GDP is released. Here we report the successful crystallization of a ternary complex of Thermus aquaticus EF-Tu:GDPNP and yeast Phe-tRNA(Phe) after its purification by HPLC.

Chromatography, Gel↗
PubMed1993

The crystal structure of elongation factor EF-Tu from Thermus aquaticus in the GTP conformation.

BACKGROUND: Elongation factor Tu (EF-Tu) is a GTP-binding protein that is crucial for protein biosynthesis. In the GTP form of the molecule, EF-Tu binds tightly to aminoacyl-tRNA, forming a ternary complex that interacts with the ribosomal acceptor site. During this interaction, GTP is hydrolyzed, and EF-Tu.GDP is ejected. RESULTS: The crystal structure of EF-Tu from Thermus aquaticus, complexed to the GTP analogue GDPNP, has been determined at 2.5 A resolution and compared to the structure of Escherichia coli EF-Tu.GDP. During the transition from the GDP (inactive) to the GTP (active) form, domain 1, containing the GTP-binding site, undergoes internal conformational changes similar to those observed in ras-p21. In addition, a dramatic rearrangement of domains is observed, corresponding to a rotation of 90.8 degrees of domain 1 relative to domains 2 and 3. Residues that are affected in the binding of aminoacyl-tRNA are found in or near the cleft formed by the domain interface. CONCLUSION: GTP binding by EF-Tu leads to dramatic conformational changes which expose the tRNA binding site. It appears that tRNA binding to EF-Tu induces a further conformational change, which may affect the GTPase activity.

Amino Acid Sequence↗
PubMed1991

Crystallization and preliminary X-ray analysis of methylamine-treated alpha 2-macroglobulin and 3 alpha 2-macroglobulin-proteinase complexes.

Crystals of methylamine-treated alpha 2-macroglobulin (alpha 2M-MA), alpha 2-macroglobulin in complex with two molecules of trypsin, alpha 2M-T2, one molecule of plasmin, alpha 2M-PL, and one molecule of plasmin followed by methylamine-treatment, alpha 2M-PL(MA), have reproducibly been obtained using ammonium sulfate or magnesium sulfate as precipitants. The crystals are fragile tetragonal bipyramids of up to 1.5 mm in length. Crystals of alpha 2M-MA diffracted to at least 9 A resolution, crystals of alpha 2M-T2 diffracted to 10 A resolution and crystals of alpha 2M-PL and alpha 2M-PL(MA) diffracted to 11 A resolution. For alpha 2M-MA the cell parameters were determined as: a=b=257 A, c=555 A; and for alpha 2M-T2 as: a=b=247 A, c=559 A. For both preparations the space group was I4(1)22. As estimated from density measurements, the crystals of alpha 2M-MA and alpha 2M-T2 contain one 360 kDa alpha 2M dimer per asymmetric unit. The volume of the asymmetric unit/molecular weight, Vm, was estimated at 5.6 A3/Da. The crystal parameters of alpha 2M-PL and alpha 2M-PL(MA) were not determined.

Crystallization↗
PubMed1990

Structural determination of the functional sites of E. coli elongation factor Tu.

Recently, we have made significant progress in solving the structure of a nicked form of elongation factor (EF)-Tu complexed with GDP. The structure has been refined to an R factor of 19.2% at 2.6 A resolution, so that most of the structure is clearly visible in the electron density map. Here we describe what is known about functional sites of EF-Tu in terms of the structure, which still lacks amino acids 40-60.

Anti-Bacterial Agents↗
PubMed1990

ALMA, an editor for large sequence alignments.

A dedicated sequence editor, ALMA, was developed for aligning many sequences of proteins or RNA molecules or longer DNA fragments. Like previously published editors, ALMA is menu directed, screen oriented, and offers similarity and consensus display. ALMA has the additional features of collective movement of sequences, acceptance of input from many sources including structure files and databases, secondary structure display, and easy merging of alignments. In order to maintain sequence integrity and save disk space, gaps and sequences are stored separately. Automatic recovery of a session is possible. Finally, the program allows interaction between manual and automatic alignment.

Amino Acid Sequence↗
PubMed1986

Using known substructures in protein model building and crystallography.

Retinol binding protein can be constructed from a small number of large substructures taken from three unrelated proteins. The known structures are treated as a knowledge base from which one extracts information to be used in molecular modelling when lacking true atomic resolution. This includes the interpretation of electron density maps and modelling homologous proteins. Models can be built into maps more accurately and more quickly. This requires the use of a skeleton representation for the electron density which improves the determination of the initial chain tracing. Fragment-matching can be used to bridge gaps for inserted residues when modelling homologous proteins.

Models, Molecular↗
PubMed1985

Structural details of the binding of guanosine diphosphate to elongation factor Tu from E. coli as studied by X-ray crystallography.

Structural details of the guanosine diphosphate binding to a modified form of elongation factor Tu from Escherichia coli, resulting from X-ray crystallographic studies, are reported. The protein elements that take part in the nucleotide binding are located in four loops connecting beta-strands with alpha-helices. These loops correspond to regions in primary sequences which show a high degree of homology when compared with other prokaryotic and eukaryotic elongation factors and initiation factor 2.

Escherichia coli↗