Biomedical subjects

Stephen Cusack

Publications and source records attributed to Stephen Cusack.

At least 19 recordsLinked to original sources

PubMed2006

Structure of dimeric SecA, the Escherichia coli preprotein translocase motor.

SecA is the preprotein translocase ATPase subunit and a superfamily 2 (SF2) RNA helicase. Here we present the 2 A crystal structures of the Escherichia coli SecA homodimer in the apo form and in complex with ATP, ADP and adenosine 5'-[beta,gamma-imido]triphosphate (AMP-PNP). Each monomer contains the SF2 ATPase core (DEAD motor) built of two domains (nucleotide binding domain, NBD and intramolecular regulator of ATPase 2, IRA2), the preprotein binding domain (PBD), which is inserted in NBD and a carboxy-terminal domain (C-domain) linked to IRA2. The structures of the nucleotide complexes of SecA identify an interfacial nucleotide-binding cleft located between the two DEAD motor domains and residues critical for ATP catalysis. The dimer comprises two virtually identical protomers associating in an antiparallel fashion. Dimerization is mediated solely through extensive contacts of the DEAD motor domains leaving the C-domain facing outwards from the dimerization core. This dimerization mode explains the effect of functionally important mutations and is completely different from the dimerization models proposed for other SecA structures. The repercussion of these findings on translocase assembly and catalysis is discussed.

Adenosine Diphosphate↗

PubMed2006

Solution structure of NOD1 CARD and mutational analysis of its interaction with the CARD of downstream kinase RICK.

NOD1 is a cytosolic signalling host pattern-recognition receptor composed of a caspase-activating and recruitment domain (CARD), a nucleotide-binding and oligomerization domain (NOD) and leucine-rich repeats. It plays a crucial role in innate immunity by activating the NF-kappaB pathway via its downstream effector the kinase RICK (RIP2) following the recognition of a specific bacterial ligand. RICK is recruited by NOD1 through interaction of their respective CARDs. Here we present the high resolution NMR structure of the NOD1 CARD. It is generally similar to other CARDs of known structure, consisting of six tightly packed helices, although the length and orientation of the last helix is unusual. Mutations in both the NOD1 and RICK CARD domains were assayed by immuno-precipitation of cell lysates and in vivo NF-kappaB activation in order to define residues important for CARD-CARD interaction and downstream signalling. The results show that the interaction is critically dependent on three acidic residues on NOD1 CARD and three basic residues on RICK CARD and thus is likely to have a strong electrostatic component, similar to other characterised CARD-CARD interactions.

Amino Acid Sequence↗

PubMed2006

Structural genomics of the Epstein-Barr virus.

Epstein-Barr virus is a herpesvirus that causes infectious mononucleosis, carcinomas and immunoproliferative disease. Its genome encodes 86 proteins, which provided targets for a structural genomics project. After updating the annotation of the genome, 23 open reading frames were chosen for expression in Escherichia coli, initially selecting for those with known enzyme activity and then supplementing this set based on a series of predicted properties, in particular secondary structure. The major obstacle turned out to be poor expression and low solubility. Surprisingly, this could not be overcome by modifications of the constructs, changes of expression temperature or strain or renaturation. Of the eight soluble proteins, five were crystallized using robotic nanolitre-drop crystallization trials, which led to four solved structures. Although these results depended on individual treatment rather than standardized protocols, a high-throughput miniaturized crystallization screening protocol was a key component of success with these difficult proteins.

Cloning, Molecular↗

PubMed2006

Crystal structure of the UPF2-interacting domain of nonsense-mediated mRNA decay factor UPF1.

UPF1 is an essential eukaryotic RNA helicase that plays a key role in various mRNA degradation pathways, notably nonsense-mediated mRNA decay (NMD). In combination with UPF2 and UPF3, it forms part of the surveillance complex that detects mRNAs containing premature stop codons and triggers their degradation in all organisms studied from yeast to human. We describe the 3 A resolution crystal structure of the highly conserved cysteine-histidine-rich domain of human UPF1 and show that it is a unique combination of three zinc-binding motifs arranged into two tandem modules related to the RING-box and U-box domains of ubiquitin ligases. This UPF1 domain interacts with UPF2, and we identified by mutational analysis residues in two distinct conserved surface regions of UPF1 that mediate this interaction. UPF1 residues we identify as important for the interaction with UPF2 are not conserved in UPF1 homologs from certain unicellular parasites that also appear to lack UPF2 in their genomes.

Amino Acid Sequence↗

PubMed2006

Structural and mutational analysis of human Ad37 and canine adenovirus 2 fiber heads in complex with the D1 domain of coxsackie and adenovirus receptor.

Adenovirus fibers from most serotypes bind the D1 domain of coxsackie and adenovirus receptor (CAR), although the binding residues are not strictly conserved. To understand this further, we determined the crystal structures of canine adenovirus serotype 2 (CAV-2) and the human adenovirus serotype 37 (HAd37) in complex with human CAR D1 at 2.3 and 1.5A resolution, respectively. Structure comparison with the HAd12 fiber head-CAR D1 complex showed that the overall topology of the interaction is conserved but that the interfaces differ in number and identity of interacting residues, shape complementarity, and degree of conformational adaptation. Using surface plasmon resonance, we characterized the binding affinity to CAR D1 of wild type and mutant CAV-2 and HAd37 fiber heads. We found that CAV-2 has the highest affinity but fewest direct interactions, with the reverse being true for HAd37. Moreover, we found that conserved interactions can have a minor contribution, whereas serotype-specific interactions can be essential. These results are discussed in the light of virus evolution and design of adenovirus vectors for gene transfer.

Adenoviridae↗

PubMed2006

Discovering new classes of Brugia malayi asparaginyl-tRNA synthetase inhibitors and relating specificity to conformational change.

SLIDE software, which models the flexibility of protein and ligand side chains while docking, was used to screen several large databases to identify inhibitors of Brugia malayi asparaginyl-tRNA synthetase (AsnRS), a target for anti-parasitic drug design. Seven classes of compounds identified by SLIDE were confirmed as micromolar inhibitors of the enzyme. Analogs of one of these classes of inhibitors, the long side-chain variolins, cannot bind to the adenosyl pocket of the closed conformation of AsnRS due to steric clashes, though the short side-chain variolins identified by SLIDE apparently bind isosterically with adenosine. We hypothesized that an open conformation of the motif 2 loop also permits the long side-chain variolins to bind in the adenosine pocket and that their selectivity for Brugia relative to human AsnRS can be explained by differences in the sequence and conformation of this loop. Loop flexibility sampling using Rigidity Optimized Conformational Kinetics (ROCK) confirms this possibility, while scoring of the relative affinities of the different ligands by SLIDE correlates well with the compounds' ranks in inhibition assays. Combining ROCK and SLIDE provides a promising approach for exploiting conformational flexibility in structure-based screening and design of species selective inhibitors.

Animals↗

PubMed2006

Structures of two bacterial prolyl-tRNA synthetases with and without a cis-editing domain.

Prolyl-tRNA synthetases (ProRSs) are unique among synthetases in that they have diverse architectures, notably the variable presence of a cis-editing domain homologous to the freestanding deacylase proteins YbaK and ProX. Here, we describe crystal structures of two bacterial ProRSs from the pathogen Enterococcus faecalis, which possesses an editing domain, and from Rhodopseudomonas palustris, which does not. We compare the overall structure and binding mode of ATP and prolyl-adenylate with those of the archael/eukaryote-type ProRS from Thermus thermophilus. Although structurally more homologous to YbaK, which preferentially hydrolyzes Cys-tRNA(Pro), the editing domain of E. faecalis ProRS possesses key elements similar to ProX, with which it shares the activity of hydrolyzing Ala-tRNA(Pro). The structures give insight into the complex evolution of ProRSs, the mechanism of editing, and structural differences between prokaryotic- and eukaryotic-type ProRSs that can be exploited for antibiotic design.

Adenosine Triphosphate↗

PubMed2006

The impact of a temporary ice-rink on an emergency department service.

BACKGROUND: A temporary ice-rink opened close to Cork city for 6 weeks from 30 November 2003. During this time, a number of patients presented to the local emergency departments with ice-skating-related injuries. We documented these injuries. METHODS: All patients presenting to emergency departments in Cork city with ice-skating-related complaints were included. Information on age and sex, mechanism of injury, diagnosis, follow-up/disposition and ambulance service utilization was recorded. RESULTS: One hundred and twenty-five ice-rink-related attendances were reported at Cork emergency departments, representing 1.25% of total attendances. One hundred and twenty-three patients presented with skating-related injuries and two with medical complaints occurring at the ice-rink: 70.8% were female patients and 29.2% were male patients. In the 4-14-year age group, however, 48.5% were girls and 51.5% were boys. Most injuries were directly due to falls; 5.6% were due to skate blades. The commonest site of injury was the upper limb. Fractures and dislocations accounted for 53.9% of injuries, with 20.5% of these requiring orthopaedic admission. Lacerations and digital injuries accounted for 7.1%, with 11% of these required admission for surgery. One minor head injury was reported. 38.1% had soft tissue injuries. Fifteen patients were transported by ambulance. These attendances represented a minimum overall cost of 77,510 euro to the local health service. CONCLUSIONS: A temporary ice-rink had a significant impact on local emergency departments. Currently, there is no specific legislation in Ireland relating to public health and safety in ice-rinks. We recommend consultation with local public bodies before opening such facilities, and appropriate regulation.

Adolescent↗

PubMed2006

Structural and biochemical characterization of a human adenovirus 2/12 penton base chimera.

The vertex of the adenoviral capsid is formed by the penton, a complex of two proteins, the pentameric penton base and the trimeric fiber protein. The penton contains all necessary components for viral attachment and entry into the host cell. After initial attachment via the head domain of the fiber protein, the penton base interacts with cellular integrins through an Arg-Gly-Asp (RGD) motif located in a hypervariable surface loop, triggering virus internalization. In order to investigate the structural and functional role of this region, we replaced the hypervariable loop of serotype 2 with the corresponding, but much shorter, loop of serotype 12 and compared it to the wild type. Here, we report the 3.6 A crystal structure of a human adenovirus 2/12 penton base chimera crystallized as a dodecamer. The structure is generally similar to human adenovirus 2 penton base, with the main differences localized to the fiber protein-binding site. Fluorescence anisotropy assays using a trimeric fiber protein mimetic called the minifiber and wild-type human adenovirus 2 and chimeric penton base demonstrate that fiber protein binding is independent of the hypervariable loop, with a K(d) for fiber binding estimated in the 1-2 microm range. Interestingly, competition assays using labeled and unlabeled minifiber demonstrated virtually irreversible binding to the penton base, which we ascribe to a conformational change, on the basis of comparisons of all available penton base structures.

Adenoviruses, Human↗

PubMed2005

Inhibition of mRNA deadenylation by the nuclear cap binding complex (CBC).

Poly(A)-specific ribonuclease (PARN) is a cap-interacting and poly(A)-specific 3'-exoribonuclease. Here we have investigated how the cap binding complex (CBC) affects human PARN activity. We showed that CBC, via its 80-kDa subunit (CBP80), inhibited PARN, suggesting that CBC can regulate mRNA deadenylation. The CBC-mediated inhibition of PARN was cap-independent, and in keeping with this, the CBP80 subunit alone inhibited PARN. Our data suggested a new function for CBC, identified CBC as a potential regulator of PARN, and emphasized the importance of communication between the two extreme ends of the mRNA as a key strategy to regulate mRNA degradation. Based on our data, we have proposed a model for CBC-mediated regulation of PARN, which relies on an interaction between CBP80 and PARN. Association of CBC with PARN might have importance in the regulated recruitment of PARN to the nonsense-mediated decay pathway during the pioneer round of translation.

Exoribonucleases↗

PubMed2005

The crystal structure of leucyl-tRNA synthetase complexed with tRNALeu in the post-transfer-editing conformation.

Leucyl-tRNA synthetase (LeuRS) has a specific post-transfer editing activity directed against mischarged isoleucine and similar noncognate amino acids. We describe the post-transfer-editing and product complexes of Thermus thermophilus LeuRS (LeuRSTT) with tRNA(Leu) at 2.9- to 3.3-A resolution. In the post-transfer-editing configuration, A76 binds in the editing active site exactly as previously found for the adenosine moiety of a small-molecule editing-substrate analog. The 60 C-terminal residues of LeuRSTT, unseen in previous structures, fold into a compact domain flexibly linked to the rest of the molecule and interacting with the G19-C56 tertiary base pair of tRNA(Leu). LeuRS recognition of tRNA(Leu) depends essentially on tRNA shape rather than base-specific interactions. The structures show that considerable domain rotations, notably of the editing domain, accompany the tRNA-3' end dynamics associated successively with aminoacylation, post-transfer editing and product release.

Amino Acid Sequence↗

PubMed2005

Specificity of recognition of mRNA 5' cap by human nuclear cap-binding complex.

The heterodimeric nuclear cap-binding complex (CBC) binds to the mono-methylated 5' cap of eukaryotic RNA polymerase II transcripts such as mRNA and U snRNA. The binding is important for nuclear maturation of mRNAs and possibly in the first round of translation and nonsense-mediated decay. It is also essential for nuclear export of U snRNAs in metazoans. We report characterization by fluorescence spectroscopy of the recognition of 5' capped RNA by human CBC. The association constants (K(as)) for 17 mono- and dinucleotide cap analogs as well as for the oligomer m7GpppA(m2') pU(m2')pA(m2') cover the range from 1.8 x 10(6) M(-1) to 2.3 x 10(8) M(-1). Higher affinity for CBC is observed for the dinucleotide compared with mononucleotide analogs, especially for those containing a purine nucleoside next to m7G. The mRNA tetramer associates with CBC as tightly as the dinucleotide analogs. Replacement of Tyr138 by alanine in the CBP20 subunit of CBC reduces the cap affinity except for the mononucleotide analogs, consistent with the crystallographic observation of the second base stacking on this residue. Our spectroscopic studies showed that contrary to the other known cap-binding proteins, the first two nucleotides of a capped-RNA are indispensable for its specific recognition by CBC. Differences in the cap binding of CBC compared with the eukaryotic translation initiation factor 4E (eIF4E) are analyzed and discussed regarding replacement of CBC by eIF4E.

Binding Sites↗

PubMed2005

A quasi-atomic model of human adenovirus type 5 capsid.

Adenoviruses infect a wide range of vertebrates including humans. Their icosahedral capsids are composed of three major proteins: the trimeric hexon forms the facets and the penton, a noncovalent complex of the pentameric penton base and trimeric fibre proteins, is located at the 12 capsid vertices. Several proteins (IIIa, VI, VIII and IX) stabilise the capsid. We have obtained a 10 A resolution map of the human adenovirus 5 by image analysis from cryo-electron micrographs (cryoEMs). This map, in combination with the X-ray structures of the penton base and hexon, was used to build a quasi-atomic model of the arrangement of the two major capsid components and to analyse the hexon-hexon and hexon-penton interactions. The secondary proteins, notably VIII, were located by comparing cryoEM maps of native and pIX deletion mutant virions. Minor proteins IX and IIIa are located on the outside of the capsid, whereas protein VIII is organised with a T=2 lattice on the inner face of the capsid. The capsid organisation is compared with the known X-ray structure of bacteriophage PRD1.

Adenoviruses, Human↗

PubMed2005

The structure of the human adenovirus 2 penton.

The adenovirus penton, a noncovalent complex of the pentameric penton base and trimeric fiber proteins, comprises the vertices of the adenovirus capsid and contains all necessary components for viral attachment and internalization. The 3.3 A resolution crystal structure of human adenovirus 2 (hAd2) penton base shows that the monomer has a basal jellyroll domain and a distal irregular domain formed by two long insertions, a similar topology to the adenovirus hexon. The Arg-Gly-Asp (RGD) motif, required for interactions with cellular integrins, occurs on a flexible surface loop. The complex of penton base with bound N-terminal fiber peptide, determined at 3.5 A resolution, shows that the universal fiber motif FNPVYPY binds at the interface of adjacent penton base monomers and results in a localized structural rearrangement in the insertion domain of the penton base. These results give insight into the structure and assembly of the adenovirus capsid and will be of use for gene-therapy applications.

Adenoviruses, Human↗

PubMed2005

Adenovirus complex structures.

Adenovirus has, for a long time, been a model system for understanding complex virus structure, assembly and interference in host cell processes. Recent structures of adenoviral capsid proteins critical for cell entry have given new insights into both interactions with host cell receptors and inter-capsid protein interactions, which determine the capsid architecture. Such studies are of importance in engineering adenovirus for use in various gene transfer applications. Remarkable and unexpected similarities have been revealed between the cell-attachment proteins and primary receptors of adenovirus and the unrelated reovirus, and between the capsid proteins and architecture of adenovirus, the enveloped bacteriophage PRD1 and other large DNA viruses.

Adenoviridae↗

PubMed2005

X-ray crystallographic and NMR studies of the third KH domain of hnRNP K in complex with single-stranded nucleic acids.

The heterogeneous nuclear ribonucleoprotein (hnRNP) K is implicated in multiple functions in the regulation of gene expression and acts as a hub at the intersection of signaling pathways and processes involving nucleic acids. Central to its function is its ability to bind both ssDNA and ssRNA via its KH (hnRNP K homology) domains. We determined crystal structures of hnRNP K KH3 domain complexed with 15-mer and 6-mer (CTC(4)) ssDNAs at 2.4 and 1.8 A resolution, respectively, and show that the KH3 domain binds specifically to both TCCC and CCCC sequences. In parallel, we used NMR to compare the binding affinity and mode of interaction of the KH3 domain with several ssRNA ligands and CTC(4) ssDNA. Based on a structure alignment of the KH3-CTC(4) complex with known structures of other KH domains in complex with ssRNA, we discuss recognition of tetranucleotide sequences by KH domains.

Amino Acid Sequence↗

PubMed2005

The monomeric dUTPase from Epstein-Barr virus mimics trimeric dUTPases.

Deoxyuridine 5'-triphosphate pyrophosphatases (dUTPases) are ubiquitous enzymes cleaving dUTP into dUMP and pyrophosphate. They occur as monomeric, dimeric, or trimeric molecules. The trimeric and monomeric enzymes both contain the same five characteristic sequence motifs but in a different order, whereas the dimeric enzymes are not homologous. Monomeric dUTPases only occur in herpesviruses, such as Epstein-Barr virus (EBV). Here, we describe the crystal structures of EBV dUTPase in complex with the product dUMP and a substrate analog alpha,beta-imino-dUTP. The molecule consists of three domains forming one active site that has a structure extremely similar to one of the three active sites of trimeric dUTPases. The three domains functionally correspond to the subunits of the trimeric form. Domains I and II have the dUTPase fold, but they differ considerably in the regions that are not involved in the formation of the unique active site, whereas domain III has only little secondary structure.

Amino Acid Sequence↗

PubMed2005

Novel way of capping mRNA trimer and studies of its interaction with human nuclear cap-binding complex.

Binding of mRNA 5' cap by the nuclear cap-binding complex (CBC) is crucial for a wide variety of mRNA metabolic events. The interaction involving the CBP20 subunit of CBC is mediated by numerous hydrogen bonds and by stacking of the tyrosine sidechains with two first bases of the capped mRNA. To examine a possible role of a longer mRNA chain in the CBC-cap recognition, we have synthesized an mRNA tetramer using a novel way of capping an RNA trimer and determined its affinity for CBC by fluorescence titration.

Dose-Response Relationship, Drug↗