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S Cusack

Publications and source records attributed to S Cusack.

At least 55 records · Page 3Linked to original sources
PubMed1996

Crystallization and preliminary X-ray analysis of the 9 kDa protein of the mouse signal recognition particle and the selenomethionyl-SRP9.

Two different crystal forms of the 9 kDa protein of the signal recognition particle (SRP9) have been prepared by the hanging drop vapor diffusion technique using 28% (w/v) PEG8000 or 28% saturated ammonium sulphate as precipitant. The crystals are hexagonal bipyramids with average dimensions of 0.2 X 0.1 X 0.1 mm(3) and they diffract to a resolution of 2.3 Angstroms. They belong to the space groups P6(2)22/P6(4)22 or P3(1)21/P3(2)21 with cell dimensions a = b = 63.0 Angstroms, and c = 111.5 Angstroms. Crystals have also been grown from the selenomethionyl protein and multiwavelength data sets have been collected.

Animals↗
PubMed1996

The structure of the Escherichia coli EF-Tu.EF-Ts complex at 2.5 A resolution.

The crystal structure of the EF-Tu.EF-Ts complex from Escherichia coli has been determined to a resolution of 2.5 A. The complex contains two subunits of each of the elongation factors. The two EF-Ts molecules form a tight dimer, but there is little contact between the two EF-Tu molecules. The interaction of EF-Ts with EF-Tu results principally in the disruption of the Mg2+ ion binding site, thereby reducing the affinity of EF-Tu for guanine nucleotides.

Amino Acid Sequence↗
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.

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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

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↗
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

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↗
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↗
PubMed1993

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↗