Biomedical subjects

S Cusack

Publications and source records attributed to S Cusack.

At least 19 recordsLinked to original sources

PubMed1995

The structural basis for seryl-adenylate and Ap4A synthesis by seryl-tRNA synthetase.

BACKGROUND: Seryl-tRNA synthetase is a homodimeric class II aminoacyl-tRNA synthetase that specifically charges cognate tRNAs with serine. In the first step of this two-step reaction, Mg.ATP and serine react to form the activated intermediate, seryl-adenylate. The serine is subsequently transferred to the 3'-end of the tRNA. In common with most other aminoacyl-tRNA synthetases, seryl-tRNA synthetase is capable of synthesizing diadenosine tetraphosphate (Ap4A) from the enzyme-bound adenylate intermediate and a second molecule of ATP. Understanding the structural basis for the substrate specificity and the catalytic mechanism of aminoacyl-tRNA synthetases is of considerable general interest because of the fundamental importance of these enzymes to protein biosynthesis in all living cells. RESULTS: Crystal structures of three complexes of seryl-tRNA synthetase from Thermus thermophilus are described. The first complex is of the enzyme with ATP and Mn2+. The ATP is found in an unusual bent conformation, stabilized by interactions with conserved arginines and three manganese ions. The second complex contains seryl-adenylate in the active site, enzymatically produced in the crystal after soaking with ATP, serine and Mn2+. The third complex is between the enzyme, Ap4A and Mn2+. All three structures exhibit a common Mn2+ site in which the cation is coordinated by two active-site residues in addition to the alpha-phosphate group from the bound ligands. CONCLUSIONS: Superposition of these structures allows a common reaction mechanism for seryl-adenylate and Ap4A formation to be proposed. The bent conformation of the ATP and the position of the serine are consistent with nucleophilic attack of the serine carboxyl group on the alpha-phosphate by an in-line displacement mechanism leading to the release of the inorganic pyrophosphate. A second ATP molecule can bind with its gamma-phosphate group in the same position as the beta-phosphate of the original ATP. This can attack the seryl-adenylate with the formation of Ap4A by an identical in-line mechanism in the reverse direction. The divalent cation is essential for both reactions and may be directly involved in stabilizing the transition state.

Adenosine Monophosphate↗

PubMed1995

Cocrystallization of lysyl-tRNA synthetase from Thermus thermophilus with its cognate tRNAlys and with Escherichia coli tRNAlys.

Lysyl-tRNA synthetase from Thermus thermophilus has been cocrystallized with either its cognate tRNAlys or Escherichia coli tRNAlys using ammonium sulfate as precipitant. The crystals grow from solutions containing a 1:2.5 stoichiometry of synthetase dimer to tRNA in 18-22% ammonium sulfate in 50 mM Tris-maleate buffer at pH 7.5. Both complexes form square prismatic, tetragonal crystals with very similar unit cell parameters (a = b = 233 A, c = 119 A) and diffract to at least 2.7 A resolution. However the homocomplex is of space group P42(1)2 and the heterocomplex of space group I422.

Crystallization↗

PubMed1995

Crystallization of Thermus thermophilus histidyl-tRNA synthetase and its complex with tRNAHis.

Histidyl-tRNA synthetase (HisRS) has been purified from the extreme thermophile Thermus thermophilus. The protein has been crystallized separately with histidine and with its cognate tRNAHis. Both crystals have been obtained using the vapor diffusion method with ammonium sulphate as precipitant. The crystals of HisRS with histidine belong to the spacegroup P2(1)2(1)2 with cell parameters a = 171.3 A, b = 214.7 A, c = 49.3 A, alpha = beta = gamma = 90 degrees. A complete data set to a resolution of 2.7A with an Rmerge on intensities of 4.1% has been collected on a single frozen crystal. A partial data set collected on a crystal of HisRS in complex with tRNAHis shows that the crystals are tetragonal with cell parameters a = b = 232 A, c = 559 A, alpha = beta = gamma = 90 degrees and diffract to about 4.5 A resolution.

Crystallography, X-Ray↗

PubMed1995

Adenovirus fiber.

Explore the source record for details and available documents.

Adenoviridae↗

PubMed1995

Structure, function and evolution of seryl-tRNA synthetases: implications for the evolution of aminoacyl-tRNA synthetases and the genetic code.

Two aspects of the evolution of aminoacyl-tRNA synthetases are discussed. Firstly, using recent crystal structure information on seryl-tRNA synthetase and its substrate complexes, the coevolution of the mode of recognition between seryl-tRNA synthetase and tRNA(ser) in different organisms is reviewed. Secondly, using sequence alignments and phylogenetic trees, the early evolution of class 2 aminoacyl-tRNA synthetases is traced. Arguments are presented to suggest that synthetases are not the oldest of protein enzymes, but survived as RNA enzymes during the early period of the evolution of protein catalysts. In this view, the relatedness of the current synthetases, as evidenced by the division into two classes with their associated subclasses, reflects the replacement of RNA synthetases by protein synthetases. This process would have been triggered by the acquisition of tRNA 3' end charging activity by early proteins capable of activating small molecules (e.g., amino acids) with ATP. If these arguments are correct, the genetic code was essentially frozen before the protein synthetases that we know today came into existence.

Acylation↗

PubMed1995

Evaluation of outcome following cardiac arrest in patients presenting to two Scottish emergency departments.

OBJECTIVES: To compare and contrast outcomes following cardiac arrest managed in two Accident and Emergency departments, and to identify factors which might account for such differences. DESIGN: Prospective 1-year evaluation of patients sustaining an out-of-hospital cardiac arrest. SETTING: The Accident and Emergency departments of the Edinburgh (ERI) and Glasgow (GRI) Royal Infirmaries which serve two large urban municipalities. PATIENTS: All patients sustaining a prehospital cardiac arrest and brought to ERI or GRI were included. Children (< 13 years), those declared dead on arrival at the scene, and events related to poisoning, near drowning, trauma and pregnancy were excluded. MEASUREMENTS AND MAIN RESULTS: There were 297 prehospital arrests from ERI, and 158 from GRI. Eighty-two (27.6%) were admitted as 'in-patients' to ERI and 23 (14.6%) to GRI (P < 0.01). Thirty-nine (13.1%) survived to hospital discharge from ERI; 13 (8.2%) survived to discharge from GRI (NS). The proportion of VF/VT:Asystole observed was significantly different between the two centres--162:98 from ERI, 54:73 from GRI (P < 0.001). Significantly more prehospital arrests were witnessed and received bystander CPR in those brought to ERI (P < 0.02). For the combined VF/VT/Asystole groups the ERI ambulance response times were significantly shorter (P < 0.01). However, there was no significant difference in the collapse to EMS arrival at the scene times between ERI and GRI. Two survivors from ERI had asystole as their initial observed rhythm. From GRI, one survivor had asystole, one had electromechanical dissociation and in another the initial rhythm was unknown. No survivor to discharge had severe neurological disability. CONCLUSIONS: Patients suffering out-of-hospital cardiac arrests in Edinburgh have a significantly better chance of being admitted to a ward. There is a trend favouring better survival to discharge in Edinburgh, but with the numbers investigated this does not achieve statistical significance. Amongst those factors which contribute to survival there are fewer witnessed arrests, less bystander CPR and slower ambulance response times in those brought to GRI. There is a need to investigate the environment in which patients collapse, to train the public in CPR, and to review the efficiency and resourcing of the ambulance service.

Adolescent↗

PubMed1995

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↗

PubMed1994

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↗

PubMed1994

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↗

PubMed1994

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↗

PubMed1994

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↗

PubMed1993

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↗

PubMed1992

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↗

PubMed1992

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↗

PubMed1992

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↗

PubMed1992

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↗

PubMed1992

Flying squad response to out-of-hospital cardiac arrest--a decade of experience.

The Flying Squad (MEDIC I) based at the Royal Infirmary, Edinburgh, commenced operation in 1980. The MEDIC I response to out of hospital non-traumatic cardiac arrest over the past decade is reported. On-scene resuscitation was attempted in 384 patients. A total of 149 (39%) patients were successfully resuscitated and transferred to hospital. Thirty-six (9.4%) patients survived to discharge from hospital. Patients receiving basic life support prior to the arrival of MEDIC I and in ventricular fibrillation had a survival rate of 14.5% (25/174). During 1988-89, 21 patients were initially attended by ambulance crews equipped with semi-automatic external defibrillators and eight (38%) of these patients survived. The response of a hospital-based flying squad to support trained ambulance crews, especially when equipped with a defibrillator may provide an economically and operationally feasible alternative to training all first responders in the full range of paramedic skills.

Adolescent↗

PubMed1992

Exposure of the hands to ionizing radiation in the resuscitation room of an accident & emergency department.

Exposure of the hands to ionizing radiation in the resuscitation room of an A&E department was measured in eight health care personnel over 3 consecutive months. The radiation levels did not exceed those limits currently recommended by the International Commission on Radiological Protection 1990. The level recorded in one individual did exceed the level set at the Lothian Health Boards investigation limit. In only five of 85 occasions were lead gloves worn by the doctor during cross table lateral cervical radiographs. Recommendations are made for reducing the radiation exposure to the hands.

Emergency Service, Hospital↗