Eleven down and nine to go.
A series of new crystal structures of aminoacyl-tRNA synthetases sheds light on the evolution of specificity in this ancient family of enzymes.
Amino Acyl-tRNA Synthetases↗
Biomedical subjects
S Cusack
Publications and source records attributed to S Cusack.
A new additive for protein crystallization.
The potential usefulness of the new zwitterionic solubilizing agent, dimethyl ethylammonium propane sulfonate (NDSB195), in protein crystallization was shown using hen egg-white lysozyme. In the presence of this agent, highly diffracting crystals were obtained using ammonium sulphate as a precipitant, whereas in its absence only amorphous precipitates were obtained. The crystals possess a triclinic unit cell not previously described and diffract to a resolution of 2 A. To ascertain that the new reagent had not produced significant changes in the protein fold the structure was determined to a resolution of 2.6 A. Only minor differences were observed (notably in regions of crystal contacts) with the known tetragonal lysozyme structure (Brookhaven Protein Data Bank entry 1HEL).
Crystallization↗
Structure of influenza virus RNP. I. Influenza virus nucleoprotein melts secondary structure in panhandle RNA and exposes the bases to the solvent.
The influenza virus genome consists of eight segments of negative-sense RNA, i.e. the viral (v) RNA forms the template for the mRNA. Each segment is encapsidated by the viral nucleoprotein to form a ribonucleoprotein (RNP) particle and each RNP carries its own polymerase complex. We studied the interaction of purified nucleoprotein with RNA in vitro, by using a variety of enzymatic and chemical probes for RNA conformation. Our results suggest that the nucleoprotein binds to the vRNA backbone without apparent sequence specificity, exposing the bases to the outside and melting all secondary structure. In this way, the viral polymerase may transcribe the RNA without the need for dissociating the nucleoprotein and without being stopped by RNA secondary structure, and the viral RNPs are ready to start transcription as soon as they enter the host cell.
Base Composition↗
The 2.9 A crystal structure of T. thermophilus seryl-tRNA synthetase complexed with tRNA(Ser).
The crystal structure of Thermus thermophilus seryl-transfer RNA synthetase, a class 2 aminoacyl-tRNA synthetase, complexed with a single tRNA(Ser) molecule was solved at 2.9 A resolution. The structure revealed how insertion of conserved base G20b from the D loop into the core of the tRNA determines the orientation of the long variable arm, which is a characteristic feature of most serine specific tRNAs. On tRNA binding, the antiparallel coiled-coil domain of one subunit of the synthetase makes contacts with the variable arm and T psi C loop of the tRNA and directs the acceptor stem of the tRNA into the active site of the other subunit. Specificity depends principally on recognition of the shape of tRNA(Ser) through backbone contacts and secondarily on sequence specific interactions.
Adenosine Triphosphate↗
A small percentage of influenza virus M1 protein contains zinc but zinc does not influence in vitro M1-RNA interaction.
A peptide containing the CCHH motif, the putative zinc-binding sequence of influenza virus M1 protein, was found to bind zinc in a one-to-one complex with the characteristics of a typical zinc-binding peptide. Intact influenza virus also contained zinc and we show that this zinc is bound to the M1 protein in the virus. However, only a small proportion of M1 contained zinc: 4% in virus and 6 to 9% in isolated protein. One strain, B/Yamagata/16/88, consistently contained more zinc: 15 to 20% both in virus and in isolated protein. We also determined the RNA binding and transcription inhibition activities of various M1 proteins and found that the zinc content of M1 had no influence on either activity. We suggest that the zinc in M1 has a structural role in the virion other than nucleic acid binding.
Amino Acid Sequence↗
Calcium is needed for the thermostability of influenza B virus neuraminidase.
The activity and stability of influenza virus neuraminidase is known to depend on the presence of calcium ions. The atomic structure of the tetrameric neuraminidase head shows two distinct Ca2+ binding sites, one with low affinity on the molecular fourfold symmetry axis and one with high affinity close to the active site in each of the monomers. Here we show that Ca is essential for the thermostability of the isolated neuraminidase tetramer. Inactivation of Ca-free neuraminidase at high temperatures is accompanied by changes in protein structure leading to protease sensitivity. More than one Ca ion per tetramer is involved in stabilization, suggesting a role for the high affinity Ca binding site and the cooperative stabilization of the subunits. Sites which are located close to the fourfold axis of the neuraminidase tetramer and which are able to bind a variety of different metal ions are also described.
Calcium↗
Refined crystal structure of the seryl-tRNA synthetase from Thermus thermophilus at 2.5 A resolution.
The three-dimensional structure of the seryl-tRNA synthetase from Thermus thermophilus has been determined and refined at 2.5 A resolution. The final model consists of a dimer of 421 residues each and 190 water molecules. The R-factor is 18.4% for all the data between 10 and 2.5 A resolution. The structure is very similar to that of the homologous enzyme from Escherichia coli, with an r.m.s. difference of 1.5 A for the 357 alpha-carbon atoms considered equivalent. The comparison of the two structures indicates increased hydrophobicity, reduced conformational entropy and reduced torsional strain as possible mechanisms by which thermostability is obtained in the enzyme from the thermophile.
Amino Acid Sequence↗
Influenza B virus neuraminidase can synthesize its own inhibitor.
BACKGROUND: Neuraminidase, one of the two surface glycoproteins of influenza virus, cleaves terminal sialic acid residues from glycolipids or glycoproteins. Its crystal structure is known at high resolution, but the mechanism of glycosyl hydrolysis remains unclear. RESULTS: We have determined the crystal structure at 1.8 A resolution of two complexes of influenza B/Beijing neuraminidase containing either the reaction product, sialic acid, or the transition state analogue inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA). The sialic acid is bound in a distorted 'boat' conformation closely resembling that of bound DANA, stabilized by a conserved tyrosine residue (Tyr408). This distortion also gives rise to a suicidal side reaction that converts sialic acid to DANA at a low rate. CONCLUSIONS: The mechanism of neuraminidase action is distinct from that of other known glycosyl hydrolases. Substrate distortion appears to be the driving force in glycosyl bond hydrolysis and the proton required for catalysis can probably be donated by water, rather than by residues in the active site, thus allowing the enzyme to operate at high pH. The side reaction converting sialic acid to DANA appears reasonably favourable, and it is unclear how this is minimized by the enzyme.
Crystallography, X-Ray↗
Crystallization of the seryl-tRNA synthetase:tRNAS(ser) complex of Escherichia coli.
Crystals of the complex between seryl-tRNA synthetase and tRNA(2ser) from Escherichia coli have been obtained from ammonium sulphate solutions. The crystals are of the 1:2 enzyme:tRNA complex, belong to the space group C222(1), have cell dimensions of a = 128.9 A, b = 164.9 A, c = 127.3 A and diffract anisotropically from 3.5 to 4.5 A. An X-ray diffraction data set to 4 A has been collected. The combination of molecular replacement using the refined structure of the catalytic domain of the native enzyme, data from a heavy atom derivative and solvent flattening was used to produce a map at 4 A resolution. This shows that a tRNA molecule binds across the dimer, the anticodon stem and loop do not contact the protein and the helical arm of the enzyme contacts the T psi C loop and the long extra arm of the tRNA.
Crystallization↗
Adenovirus type 40 virions contain two distinct fibers.
Human subgroup F adenoviruses, types 40 and 41, have two genes that could specify different fiber proteins, a long fiber with 21 or 22 pseudorepeat motifs in the shaft, and a short fiber with 12 motifs. We show that for adenovirus type 40, both proteins are expressed from separate late regions of the genome. They are incorporated into the virion with only one fiber per penton base, unlike avian adenoviruses which have two fibers extending from each penton. Comparison of known adenovirus fiber sequences suggests that the two fibers in adenovirus 40 and 41 arose through an external recombination event rather than having evolved from each other directly. Two alternative fibers on the virion may imply the existence of two distinct receptors.
Adenoviruses, Human↗
Comparison of structure and sequence of influenza B/Yamagata and B/Beijing neuraminidases shows a conserved "head" but much greater variability in the "stalk" and NB protein.
In 1988 the B/Yamagata/16/88 influenza virus appeared, which was antigenically quite different from the other B viruses circulating at that time. Neuraminidase heads of this strain were purified, crystallized, and analyzed by X-ray crystallography and compared with the structure of B/Beijing/1/87 neuraminidase. Only two point mutations could be detected in the "head" of the molecule leading to local rearrangement of amino acid side-chains. The nucleotide sequence, however, revealed significant differences in the stalk region and in the NB protein.
Amino Acid Sequence↗
Sequence, structure and evolutionary relationships between class 2 aminoacyl-tRNA synthetases: an update.
The seven class 2 aminoacyl-tRNA synthetases that are alpha 2 dimers have previously been divided by sequence homology into class 2a (seryl-, threonyl-, prolyl- and histidyl-) and class 2b (aspartyl-, asparaginyl- and lysyl-). It has been more difficult to classify the glycyl-, phenylalanyl- and alanyl-tRNA synthetases which have different subunit stoichiometries and which did not apparently contain all three canonical class 2 motifs. New sequence and structural information relating to the three problematic synthetases will be discussed permitting a step forward to be taken in the understanding of the evolutionary relationships between the class 2 synthetases.
Amino Acid Sequence↗
Survival from cardiac arrest in an accident and emergency department: the impact of out of hospital advisory defibrillation.
A prospective 1-year audit of cardiac arrests treated in the Accident and Emergency department of the Royal Infirmary, Edinburgh is presented. During the period January 1st, 1991 to December 31st, 1991, 325 patients with cardiac arrest were treated. Two-hundred ninety-seven of these were 'out-of-hospital' and 28 were 'in-department' arrests. Of patients with 'out-of-hospital' ventricular fibrillation/pulseless ventricular tachycardia 22.8% were discharged. Survival rates for patients with asystole or electromechanical dissociation were very poor. The impact of semiautomatic out-of-hospital defibrillation upon the survival and number of patients presenting to the department is discussed.
Aged↗
The introduction of automatic blood pressure monitoring to an accident & emergency resuscitation room.
Machines for automatic non-invasive blood pressure (BP) monitoring are increasingly available in British accident and emergency departments. Our department recently acquired two machines with this capability for use in the resuscitation room. This provided us with an opportunity to compare the speed and frequency of automatic BP recording with the previously used manual method. We found no significant difference in either the median time to the first recording of BP or in the median number of documented recordings in the first hour. However, the overall frequency of BP recording did show a statistically significant increase. We conclude that automation alone does not improve standard practice in this area greatly.
Automation↗
A new crystal form of the complex between seryl-tRNA synthetase and tRNA(Ser) from Thermus thermophilus that diffracts to 2.8 A resolution.
Two distinct complexes between seryl-tRNA synthetase and tRNA(Ser) from Thermus thermophilus have been crystallized using ammonium sulphate as a precipitant. Form III crystals grow from solutions containing a 1:2.5 stoichiometry of synthetase dimer to tRNA. They are of monoclinic space group C2 with unit cell dimensions a = 211.6 A, b = 126.8 A, c = 197.1 A, beta = 132.4 degrees and diffract to about 3.5 A. Preliminary crystallographic results show that the crystallographic asymmetric unit contains two synthetase dimers. Form IV crystals grow from solutions containing a 1:1.5 stoichiometry of synthetase dimer to tRNA. They are of orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions a = 124.5 A, b = 128.9 A, c = 121.2 A and diffract to 2.8 A resolution. Preliminary crystallographic results show that these crystals contain only one tRNA molecule bound to a synthetase dimer.
Crystallization↗
New triple-helical model for the shaft of the adenovirus fibre.
The adenovirus fibre is a trimeric protein with a globular head on a long thin shaft that projects from the twelve fivefold vertices of the virion. The shaft region of the fibre primary sequence has a unique pseudo-repeating motif of 15 residues. Using constraints derived from sequence analysis, the trimeric nature of the fibre, the experimental determination of the shaft length and general knowledge about protein structure, an atomic model of the fibre shaft has been constructed by computer modelling techniques. In the final model the three monomers form a left-handed triple-helical structure with threefold symmetry and with successive 15-residue repeats on the same chain related by an axial rise of 13.1 A and a left-handed azimuthal rotation of close to 300 degrees. Three threefold related beta-sheets with short strands are formed by inter-monomer main-chain hydrogen bonds giving rise to superhelical ribbons covering the surface of the shaft. The model satisfies criteria of extensive hydrogen bonding, reasonable backbone torsion angles, burial of most hydrophobic residues and good packing of the hydrophobic core. Furthermore, the model is consistent with the observed shaft length of about 290 A and its calculated X-ray fibre diffraction patterns shows the characteristic features found in the diffraction pattern of crystals of fibre, notably layer lines with a spacing of about 1/26 A-1 and strong meridional intensity at 1/4.4 A-1.
Adenoviruses, Human↗
Crystallization of the seryl-tRNA synthetase-tRNA(Ser) complex from Thermus thermophilus.
The complex between seryl-tRNA synthetase and its cognate tRNA from the extreme thermophile Thermus thermophilus has been crystallized from ammonium sulphate solutions. Two different tetragonal crystal forms have been characterized, both diffracting to about 6 A using synchrotron radiation. One form grows as large bipyramids and has cell dimensions a = b = 127 A, c = 467 A, and the second form occurs as long, thin square prisms with cell dimensions a = b = 101 A, c = 471 A. Analysis of washed and dissolved crystals demonstrates the presence of both protein and tRNA.
Crystallization↗
The 2.2 A resolution crystal structure of influenza B neuraminidase and its complex with sialic acid.
Influenza virus neuraminidase catalyses the cleavage of terminal sialic acid, the viral receptor, from carbohydrate chains on glycoproteins and glycolipids. We present the crystal structure of the enzymatically active head of influenza B virus neuraminidase from the strain B/Beijing/1/87. The native structure has been refined to a crystallographic R-factor of 14.8% at 2.2 A resolution and its complex with sialic acid refined at 2.8 A resolution. The overall fold of the molecule is very similar to the already known structure of neuraminidase from influenza A virus, with which there is amino acid sequence homology of approximately 30%. Two calcium binding sites have been identified. One of them, previously undescribed, is located between the active site and a large surface antigenic loop. The calcium ion is octahedrally co-ordinated by five oxygen atoms from the protein and one water molecule. Sequence comparisons suggest that this calcium site should occur in all influenza A and B virus neuraminidases. Soaking of sialic acid into the crystals has enabled the mode of binding of the reaction product in the putative active site pocket to be revealed. All the large side groups of the sialic acid are equatorial and are specifically recognized by nine fully conserved active site residues. These in turn are stabilized by a second shell of 10 highly conserved residues principally by an extensive network of hydrogen bonds.
Amino Acid Sequence↗