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Biomedical subjects

A Liljas

Publications and source records attributed to A Liljas.

At least 19 recordsLinked to original sources
PubMed2001

Crystal structure of a superantigen bound to MHC class II displays zinc and peptide dependence.

The three-dimensional structure of a bacterial superantigen, Staphylococcus aureus enterotoxin H (SEH), bound to human major histocompatibility complex (MHC) class II (HLA-DR1) has been determined by X-ray crystallography to 2.6 A resolution (1HXY). The superantigen binds on top of HLA-DR1 in a completely different way from earlier co-crystallized superantigens from S.aureus. SEH interacts with high affinity through a zinc ion with the beta1 chain of HLA-DR1 and also with the peptide presented by HLA-DR1. The structure suggests that all superantigens interacting with MHC class II in a zinc-dependent manner present the superantigen in a common way. This suggests a new model for ternary complex formation with the T-cell receptor (TCR), in which a contact between the TCR and the MHC class II is unlikely.

Amino Acid Sequence↗
PubMed2001

Structure of ribosomal protein TL5 complexed with RNA provides new insights into the CTC family of stress proteins.

The crystal structure of Thermus thermophilus ribosomal protein TL5 in complex with a fragment of Escherichia coli 5S rRNA has been determined at 2.3 A resolution. The protein consists of two domains. The structure of the N-terminal domain is close to the structure of E. coli ribosomal protein L25, but the C-terminal domain represents a new fold composed of seven beta-strands connected by long loops. TL5 binds to the RNA through its N-terminal domain, whereas the C-terminal domain is not included in this interaction. Cd(2+) ions, the presence of which improved the crystal quality significantly, bind only to the protein component of the complex and stabilize the protein molecule itself and the interactions between the two molecules in the asymmetric unit of the crystal. The TL5 sequence reveals homology to the so-called general stress protein CTC. The hydrophobic cores which stabilize both TL5 domains are highly conserved in CTC proteins. Thus, all CTC proteins may fold with a topology close to that of TL5.

Amino Acid Sequence↗
PubMed2001

Mutations in the G-domain of elongation factor G from Thermus thermophilus affect both its interaction with GTP and fusidic acid.

Two hypersensitive and two resistant variants of elongation factor-G (EF-G) toward fusidic acid are studied in comparison with the wild type factor. All mutated proteins are active in a cell-free translation system and ribosome-dependent GTP hydrolysis. The EF-G variants with the Thr-84-->Ala or Asp-109-->Lys mutations bring about a strong resistance of EF-G to the antibiotic, whereas the EF-Gs with substitutions Gly-16-->Val or Glu-119-->Lys are the first examples of fusidic acid-hypersensitive factors. A correlation between fusidic acid resistance of EF-G mutants and their affinity to GTP are revealed in this study, although their interactions with GDP are not changed. Thus, fusidic acid-hypersensitive mutants have the high affinity to an uncleavable GTP analog, but the association of resistant mutants with GTP is decreased. The effects of either fusidic acid-sensitive or resistant mutations can be explained by the conformational changes in the EF-G molecule, which influence its GTP-binding center. The results presented in this paper indicate that fusidic acid-sensitive mutant factors have a conformation favorable for GTP binding and subsequent interaction with the ribosomes.

Alanine↗
PubMed2000

Structure of a mutant EF-G reveals domain III and possibly the fusidic acid binding site.

The crystal structure of Thermus thermophilus elongation factor G (EF-G) carrying the point mutation His573Ala was determined at a resolution of 2.8 A. The mutant has a more closed structure than that previously reported for wild-type EF-G. This is obtained by a 10 degrees rigid rotation of domains III, IV and V with regard to domains I and II. This rotation results in a displacement of the tip of domain IV by approximately 9 A. The structure of domain III is now fully visible and reveals the double split beta-alpha-beta motif also observed for EF-G domain V and for several ribosomal proteins. A large number of fusidic acid resistant mutations found in domain III have now been possible to locate. Possible locations for the effector loop and a possible binding site for fusidic acid are discussed in relation to some of the fusidic acid resistant mutations.

Amino Acid Motifs↗
PubMed2000

Archaeal ribosomal protein L1: the structure provides new insights into RNA binding of the L1 protein family.

BACKGROUND: L1 is an important primary rRNA-binding protein, as well as a translational repressor that binds mRNA. It was shown that L1 proteins from some bacteria and archaea are functionally interchangeable within the ribosome and in the repression of translation. The crystal structure of bacterial L1 from Thermus thermophilus (TthL1) has previously been determined. RESULTS: We report here the first structure of a ribosomal protein from archaea, L1 from Methanococcus jannaschii (MjaL1). The overall shape of the two-domain molecule differs dramatically from that of its bacterial counterpart (TthL1) because of the different relative orientations of the domains. Two strictly conserved regions of the amino acid sequence, each belonging to one of the domains and positioned close to each other in the interdomain cavity of TthL1, are separated by about 25 A in MjaL1 owing to a significant opening of the structure. These regions are structurally highly conserved and are proposed to be the specific RNA-binding sites. CONCLUSIONS: The unusually high RNA-binding affinity of MjaL1 might be explained by the exposure of its highly conserved regions. The open conformation of MjaL1 is strongly stabilized by nonconserved interdomain interactions and suggests that the closed conformations of L1 (as in TthL1) open upon RNA binding. Comparison of the two L1 protein structures reveals a high conformational variability of this ribosomal protein. Determination of the MjaL1 structure offers an additional variant for fitting the L1 protein into electron-density maps of the 50S ribosomal subunit.

Amino Acid Sequence↗
PubMed2000

Extremely thermostable elongation factor G from Aquifex aeolicus: cloning, expression, purification, and characterization in a heterologous translation system.

The fus gene of the translation factor G (EF-G) from the hyperthermophilic bacterium Aquifex aeolicus was cloned under control of a phage promoter and overexpressed in Escherichia coli with the T7 RNA polymerase system. A heat denaturation step at 95 degrees C was used to purify the protein from the cell extract. This approach simplified the chromatographic procedures and decreased the protein loss since most of Escherichia coli proteins were denatured and precipitated. Ten milligrams of the highly purified protein was isolated from 4 liters of induced culture. The overproduced EF-G was active in ribosome-dependent GTP hydrolysis and a poly(U)-directed polyphenylalanine translation system with E. coli 70S ribosomes. The method presented here might facilitate functional and structural studies of important components of the protein biosynthesis system.

Bacterial Proteins↗
PubMed2000

A decade of progress in understanding the structural basis of protein synthesis.

The key reaction of protein synthesis, peptidyl transfer, is catalysed in all living organisms by the ribosome - an advanced and highly efficient molecular machine. During the last decade extensive X-ray crystallographic and NMR studies of the three-dimensional structure of ribosomal proteins, ribosomal RNA components and their complexes with ribosomal proteins, and of several translation factors in different functional states have taken us to a new level of understanding of the mechanism of function of the protein synthesis machinery. Among the new remarkable features revealed by structural studies, is the mimicry of the tRNA molecule by elongation factor G, ribosomal recycling factor and the eukaryotic release factor 1. Several other translation factors, for which three-dimensional structures are not yet known, are also expected to show some form of tRNA mimicry. The efforts of several crystallographic and biochemical groups have resulted in the determination by X-ray crystallography of the structures of the 30S and 50S subunits at moderate resolution, and of the structure of the 70S subunit both by X-ray crystallography and cryo-electron microscopy (EM). In addition, low resolution cryo-EM models of the ribosome with different translation factors and tRNA have been obtained. The new ribosomal models allowed for the first time a clear identification of the functional centres of the ribosome and of the binding sites for tRNA and ribosomal proteins with known three-dimensional structure. The new structural data have opened a way for the design of new experiments aimed at deeper understanding at an atomic level of the dynamics of the system.

Amino Acid Sequence↗
PubMed2000

The end of the beginning: structural studies of ribosomal proteins.

Work on the structural biology of ribosomes has progressed rapidly over the past few years. It has come to a stage at which the structures of the individual components are no longer of interest, except for those that still present ambiguous information about their structure because of conformational dynamics, as well as for those that show very little homology with their counterparts from other species or other kingdoms. The recently solved structure of protein L7/L12 and its proposed modes of dimerization have helped to understand the structural flexibility of this protein, which occurs as two dimers in the ribosome. The structure provides a missing link for many previous biochemical and functional studies.

Crystallography, X-Ray↗
PubMed1999

Crystal structure of Thermotoga maritima ribosome recycling factor: a tRNA mimic.

Ribosome recycling factor (RRF), together with elongation factor G (EF-G), catalyzes recycling of ribosomes after one round of protein synthesis. The crystal structure of RRF was determined at 2.55 angstrom resolution. The protein has an unusual fold where domain I is a long three-helix bundle and domain II is a three-layer beta/alpha/beta sandwich. The molecule superimposes almost perfectly with a transfer RNA (tRNA) except that the amino acid-binding 3' end is missing. The mimicry suggests that RRF interacts with the posttermination ribosomal complex in a similar manner to a tRNA, leading to disassembly of the complex. The structural arrangement of this mimicry is entirely different from that of other cases of less pronounced mimicry of tRNA so far described.

Amino Acid Sequence↗
PubMed1999

N-terminal domain, residues 1-91, of ribosomal protein TL5 from Thermus thermophilus binds specifically and strongly to the region of 5S rRNA containing loop E.

In this work we show for the first time that the overproduced N-terminal fragment (residues 1-91) of ribosomal protein TL5 binds specifically to 5S rRNA and that the region of this fragment containing residues 80-91 is a necessity for its RNA-binding activity. The fragment of Escherichia coli 5S rRNA protected by TL5 against RNase A hydrolysis was isolated and sequenced. This 39 nucleotides fragment contains loop E and helices IV and V of 5S rRNA. The isolated RNA fragment forms stable complexes with TL5 and its N-terminal domain. Crystals of TL5 in complex with the RNA fragment diffracting to 2.75 A resolution were obtained.

Amino Acid Sequence↗
PubMed1999

Crystallization and preliminary X-ray analysis of Thermotoga maritima ribosome recycling factor.

Thermotoga maritima ribosome recycling factor (RRF) is one of the proteins catalyzing the fourth step in prokaryotic protein synthesis, ribosome recycling. The RRF protein was crystallized with ammonium sulfate. Native diffraction data to 2.55 A resolution were obtained at the MAX II synchrotron from a flash-frozen crystal at 100 K. The crystals belong to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 47, c = 298 A, and probably contain one monomer per asymmetric unit.

Bacterial Proteins↗
PubMed1998

The crystal structure of ribosomal protein L22 from Thermus thermophilus: insights into the mechanism of erythromycin resistance.

BACKGROUND: . The ribosomal protein L22 is one of five proteins necessary for the formation of an early folding intermediate of the 23S rRNA. L22 has been found on the cytoplasmic side of the 50S ribosomal subunit. It can also be labeled by an erythromycin derivative bound close to the peptidyl-transfer center at the interface side of the 50S subunit, and the amino acid sequence of an erythromycin-resistant mutant is known. Knowing the structure of the protein may resolve this apparent conflict regarding the location of L22 on the ribosome. RESULTS: . The structure of Thermus thermophilus L22 was solved using X-ray crystallography. L22 consists of a small alpha+beta domain and a protruding beta hairpin that is 30 A long. A large part of the surface area of the protein has the potential to be involved in interactions with rRNA. A structural similarity to other RNA-binding proteins is found, possibly indicating a common evolutionary origin. CONCLUSIONS: . The extensive surface area of L22 has the characteristics of an RNA-binding protein, consistent with its role in the folding of the 23S rRNA. The erythromycin-resistance conferring mutation is located in the protruding beta hairpin that is postulated to be important in L22-rRNA interactions. This region of the protein might be at the erythromycin-binding site close to the peptidyl transferase center, whereas the opposite end may be exposed to the cytoplasm.

Amino Acid Sequence↗
PubMed1998

An intact conformation at the tip of elongation factor G domain IV is functionally important.

Three variants of Thermus thermophilus EF-G with mutations in the loop at the distal end of its domain IV were obtained. The replacement of His-573 by Ala and double mutation H573A/D576A did not influence the functional activity of EF-G. On the other hand, the insertion of six amino acids into the loop between residues Asp-576 and Ser-577 reduced the translocational activity of EF-G markedly, while its GTPase activity was not affected. It is concluded that the native conformation of the loop is important for the factor-promoted translocation in the ribosome. The functional importance of the entire EF-G domain IV is discussed.

GTP Phosphohydrolase-Linked Elongation Factors↗
PubMed1998

Protein biosynthesis: structural studies of the elongation cycle.

The elongation cycle of protein synthesis on ribosomes is catalyzed by the elongation factors EF-Tu and EF-G. A thorough crystallographic analysis of the structures of the different functional states of EF-Tu has been made. Furthermore, the structure of EF-G:GDP is the form of EF-G that dissociates from the ribosome. Since it mimics the structure of the ternary complex of EF-Tu:GTP with aminoacyl-tRNA, which subsequently binds to the ribosome, EF-G:GDP leaves an imprint on the ribosome for the ternary complex. In addition, electron cryomicroscopy studies of ribosomes with tRNA as well as the ternary complex bound are beginning to give a solid structural basis for the functional description of elongation.

Peptide Chain Elongation, Translational↗
PubMed1998

Crystal structure of ribosomal protein S8 from Thermus thermophilus reveals a high degree of structural conservation of a specific RNA binding site.

S8 is one of the core ribosomal proteins. It binds to 16 S RNA with high affinity and independently of other ribosomal proteins. It also acts as a translational repressor in Escherichia coli by binding to its own mRNA. The structure of Thermus thermophilus S8 has been determined by the method of multiple isomorphous replacement at 2.9 A resolution and refined to a crystallographic R-factor of 16.2% (Rfree 27.5%). The two domains of the structure have an alpha/beta fold and are connected by a long protruding loop. The two molecules in the asymmetric unit of the crystal interact through an extensive hydrophobic core and form a tightly associated dimer, while symmetry-related molecules form a joint beta-sheet of mixed type. This type of protein-protein interaction could be realized within the ribosomal assembly. A comparison of the structures of T. thermophilus and Bacillus stearothermophilus S8 shows that the interdomain loop is eight residues longer in the former and reveals high structural conservation of an extensive region, located in the C-terminal domain. From mutational studies this region was proposed earlier to be involved in specific interaction with RNA. On the basis of these data and on the comparison of the two structures of S8, it is proposed that the three-dimensional structure of specific RNA binding sites in ribosomal proteins is highly conserved among different species.

Amino Acid Sequence↗
PubMed1998

Increased functional activity of elongation factor G with G16V mutation in the GTP-binding domain.

Oligonucleotide-directed mutagenesis was used to obtain elongation factor G from Thermus thermophilus with the G16V mutation in its GTP-binding domain. Functional studies of the mutated protein and elongation factor G from E. coli were carried out. The data revealed that the G16V mutant retains high thermostability, has an increased ribosome-dependent GTPase activity, and its translation activity in cell-free translation system is equal to that of the factor G from E. coli. The mutated protein with an uncleavable GTP analog also has an increased affinity to the ribosomes.

Base Sequence↗