Biomedical subjects

Stephen Cusack

Publications and source records attributed to Stephen Cusack.

32 records · Page 2Linked to original sources

PubMed2005

Crystal structure of enteric adenovirus serotype 41 short fiber head.

Human enteric adenoviruses of species F contain two fibers in the same virion, a long fiber which binds to coxsackievirus and adenovirus receptor (CAR) and a short fiber of unknown function. We have determined the high-resolution crystal structure of the short fiber head of human adenovirus serotype 41 (Ad41). The short fiber head has the characteristic fold of other known fiber heads but has three unusual features. First, it has much shorter loops between the beta-strands. Second, one of the usually well-ordered beta-strands on the distal face of the fiber head is highly disordered and this same region is sensitive to digestion with pepsin, an enzyme occurring naturally in the intestinal tract, the physiological environment of Ad41. Third, the AB loop has a deletion giving it a distinct conformation incompatible with CAR binding.

Adenoviruses, Human↗

PubMed2004

Conserved tertiary base pairing ensures proper RNA folding and efficient assembly of the signal recognition particle Alu domain.

Proper folding of the RNA is an essential step in the assembly of functional ribonucleoprotein complexes. We examined the role of conserved base pairs formed between two distant loops in the Alu portion of the mammalian signal recognition particle RNA (SRP RNA) in SRP assembly and functions. Mutations disrupting base pairing interfere with folding of the Alu portion of the SRP RNA as monitored by probing the RNA structure and the binding of the protein SRP9/14. Complementary mutations rescue the defect establishing a role of the tertiary loop-loop interaction in RNA folding. The same mutations in the Alu domain have no major effect on binding of proteins to the S domain suggesting that the S domain can fold independently. Once assembled into a complete SRP, even particles that contain mutant RNA are active in arresting nascent chain elongation and translocation into microsomes, and, therefore, tertiary base pairing does not appear to be essential for these activities. Our results suggest a model in which the loop-loop interaction and binding of the protein SRP9/14 play an important role in the early steps of SRP RNA folding and assembly.

Alu Elements↗

PubMed2004

Crystallization, microPIXE and preliminary crystallographic analysis of the complex between the third KH domain of hnRNP K and single-stranded DNA.

hnRNP K is one of the major proteins found in hnRNP particles which are ribonucleoprotein complexes containing proteins and pre-mRNA. hnRNP K contains hnRNP K homology (KH) domains which bind both CT-rich single-stranded DNA (ssDNA) and CU-rich ssRNA. Co-crystallization of the third KH domain of human hnRNP K with a 15-mer ssDNA gave rod-shaped crystals belonging to the trigonal space group P3(1)21 (unit-cell parameters a = 54.0, c = 149.7 A) and diffracting to 2.4 A resolution. MicroPIXE (proton-induced X-ray emission) experiments showed that the crystals contained the complex and that the phosphorus to sulfur atomic ratio was consistent with the asymmetric unit containing three KH3 domains per 15-mer ssDNA. This was confirmed by structure solution by molecular replacement.

Cloning, Molecular↗

PubMed2004

The structural basis for the interaction between nonsense-mediated mRNA decay factors UPF2 and UPF3.

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism by which eukaryotic cells detect and degrade transcripts containing premature termination codons. Three 'up-frameshift' proteins, UPF1, UPF2 and UPF3, are essential for this process in organisms ranging from yeast to human. We present a crystal structure at a resolution of 1.95 A of the complex between the interacting domains of human UPF2 and UPF3b, which are, respectively, a MIF4G (middle portion of eIF4G) domain and an RNP domain (ribonucleoprotein-type RNA-binding domain). The protein-protein interface is mediated by highly conserved charged residues in UPF2 and UPF3b and involves the beta-sheet surface of the UPF3b RNP domain, which is generally used by these domains to bind nucleic acids. We show that the UPF3b RNP does not bind RNA, whereas the UPF2 construct and the complex do. Our results advance understanding of the molecular mechanisms underlying the NMD quality control process.

Amino Acid Sequence↗

PubMed2004

Crystal structure of species D adenovirus fiber knobs and their sialic acid binding sites.

Adenovirus serotype 37 (Ad37) belongs to species D and can cause epidemic keratoconjunctivitis, whereas the closely related Ad19p does not. Primary cell attachment by adenoviruses is mediated through receptor binding of the knob domain of the fiber protein. The knobs of Ad37 and Ad19p differ at only two positions, Lys240Glu and Asn340Asp. We report the high-resolution crystal structures of the Ad37 and Ad19p knobs, both native and in complex with sialic acid, which has been proposed as a receptor for Ad37. Overall, the Ad37 and Ad19p knobs are very similar to previously reported knob structures, especially to that of Ad5, which binds the coxsackievirus-adenovirus receptor (CAR). Ad37 and Ad19p knobs are structurally identical with the exception of the changed side chains and are structurally most similar to CAR-binding knobs (e.g., that of Ad5) rather than non-CAR-binding knobs (e.g., that of Ad3). The two mutations in Ad19p result in a partial loss of the exceptionally high positive surface charge of the Ad37 knob but do not affect sialic acid binding. This site is located on the top of the trimer and binds both alpha(2,3) and alpha(2,6)-linked sialyl-lactose, although only the sialic acid residue makes direct contact. Amino acid alignment suggests that the sialic acid binding site is conserved in several species D serotypes. Our results show that the altered viral tropism and cell binding of Ad19p relative to those of Ad37 are not explained by a different binding ability toward sialyl-lactose.

Adenoviruses, Human↗

PubMed2003

Structural and mechanistic basis of pre- and posttransfer editing by leucyl-tRNA synthetase.

The aminoacyl-tRNA synthetases link tRNAs with their cognate amino acid. In some cases, their fidelity relies on hydrolytic editing that destroys incorrectly activated amino acids or mischarged tRNAs. We present structures of leucyl-tRNA synthetase complexed with analogs of the distinct pre- and posttransfer editing substrates. The editing active site binds the two different substrates using a single amino acid discriminatory pocket while preserving the same mode of adenine recognition. This suggests a similar mechanism of hydrolysis for both editing substrates that depends on a key, completely conserved aspartic acid, which interacts with the alpha-amino group of the noncognate amino acid and positions both substrates for hydrolysis. Our results demonstrate the economy by which a single active site accommodates two distinct substrates in a proofreading process critical to the fidelity of protein synthesis.

Adenosine↗

PubMed2003

Structural basis for orthogonal tRNA specificities of tyrosyl-tRNA synthetases for genetic code expansion.

The archaeal/eukaryotic tyrosyl-tRNA synthetase (TyrRS)-tRNA(Tyr) pairs do not cross-react with their bacterial counterparts. This 'orthogonal' condition is essential for using the archaeal pair to expand the bacterial genetic code. In this study, the structure of the Methanococcus jannaschii TyrRS-tRNA(Tyr)-L-tyrosine complex, solved at a resolution of 1.95 A, reveals that this archaeal TyrRS strictly recognizes the C1-G72 base pair, whereas the bacterial TyrRS recognizes the G1-C72 in a different manner using different residues. These diverse tRNA recognition modes form the basis for the orthogonality. The common tRNA(Tyr) identity determinants (the discriminator, A73 and the anticodon residues) are also recognized in manners different from those of the bacterial TyrRS. Based on this finding, we created a mutant TyrRS that aminoacylates the amber suppressor tRNA with C34 65 times more efficiently than does the wild-type enzyme.

Amino Acid Sequence↗

PubMed2002

Co-crystallization of the human nuclear cap-binding complex with a m7GpppG cap analogue using protein engineering.

The nuclear cap-binding complex (CBC) binds the 7-methyl-G(5')ppp(5')N cap structure at the 5' end of pre-messenger and uracil-rich small nuclear RNAs in the nucleus. It mediates interaction of these capped RNAs with various nuclear machineries involved in RNA maturation and is co-exported with them to the cytoplasm. The structure of human CBC, which comprises the subunits CBP20 and CBP80, has previously been determined in a mildly trypsinated form which can no longer bind the cap. Here, the engineering and crystallization of two variant CBCs with deletions in CBP80 which do not affect function are described. A complex with a small N-terminal deletion in CBP80 was crystallized in space group C2 with one complex per asymmetric unit. The crystals diffract to 2 A resolution and give the first structure of intact but cap-free CBC. An additional internal deletion in CBP80 of a prominent solvent-exposed coiled coil gives rise to a more compact complex. This was co-crystallized with the cap analogue m(7)GpppG in two different crystal forms which could grow in the same drop. Form 1 belongs to space group P3(1)21 with one complex per asymmetric unit and diffracts to 2.15 A resolution. Form 2 belongs to space group P2(1)2(1)2(1) with two complexes per asymmetric unit and diffracts to 2.3 A resolution. In both forms, strong extra electron density is observed for the cap analogue and for the N- and C-terminal extensions of CBP20 which was absent or disordered in all previous structures.

Base Sequence↗

PubMed2002

Large-scale induced fit recognition of an m(7)GpppG cap analogue by the human nuclear cap-binding complex.

The heterodimeric nuclear cap-binding complex (CBC) binds to the 5' cap structure of RNAs in the nucleus and plays a central role in their diverse maturation steps. We describe the crystal structure at 2.1 A resolution of human CBC bound to an m(7)GpppG cap analogue. Comparison with the structure of uncomplexed CBC shows that cap binding induces co-operative folding around the dinucleotide of some 50 residues from the N- and C-terminal extensions to the central RNP domain of the small subunit CBP20. The cap-bound conformation of CBP20 is stabilized by an intricate network of interactions both to the ligand and within the subunit, as well as new interactions of the CBP20 N-terminal tail with the large subunit CBP80. Although the structure is very different from that of other known cap-binding proteins, such as the cytoplasmic cap-binding protein eIF4E, specificity for the methylated guanosine again is achieved by sandwiching the base between two aromatic residues, in this case two conserved tyrosines. Implications for the transfer of capped mRNAs to eIF4E, required for translation initiation, are discussed.

Amino Acid Sequence↗

PubMed2002

Class I tyrosyl-tRNA synthetase has a class II mode of cognate tRNA recognition.

Bacterial tyrosyl-tRNA synthetases (TyrRS) possess a flexibly linked C-terminal domain of approximately 80 residues, which has hitherto been disordered in crystal structures of the enzyme. We have determined the structure of Thermus thermophilus TyrRS at 2.0 A resolution in a crystal form in which the C-terminal domain is ordered, and confirm that the fold is similar to part of the C-terminal domain of ribosomal protein S4. We have also determined the structure at 2.9 A resolution of the complex of T.thermophilus TyrRS with cognate tRNA(tyr)(G Psi A). In this structure, the C-terminal domain binds between the characteristic long variable arm of the tRNA and the anti-codon stem, thus recognizing the unique shape of the tRNA. The anticodon bases have a novel conformation with A-36 stacked on G-34, and both G-34 and Psi-35 are base-specifically recognized. The tRNA binds across the two subunits of the dimeric enzyme and, remarkably, the mode of recognition of the class I TyrRS for its cognate tRNA resembles that of a class II synthetase in being from the major groove side of the acceptor stem.

Amino Acid Sequence↗

PubMed2002

Crystallization and preliminary X-ray analysis of the Escherichia coli adaptor protein ClpS, free and in complex with the N-terminal domain of ClpA.

Protein degradation in Escherichia coli is accomplished by a handful of large oligomeric complexes. In most cases, these proteolytic machines are comprised of a chaperone (e.g. ClpA) that is required to prepare the substrate for degradation by the peptidase (e.g. ClpP). Recently, it was shown that the substrate recognition of the chaperone ClpA could be modified by the adaptor protein ClpS. To investigate the structural implications of this change in substrate specificity, ClpS was crystallized alone and in complex with the N-terminal domain of ClpA (ClpA(N)). Crystals of ClpS diffract to 2.9 A resolution and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 82.63, b = 145.67, c = 152.31 A. Two different crystal forms of the ClpA(N)-ClpS complex were characterized. Crystal form I (CFI) belongs to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 91.63, b = 112.47, c = 38.47 A; data to 1.92 A resolution were collected. Crystals of form II (CFII) belong to space group P4(1/3)2(1)2, with unit-cell parameters a = b = 93.57, c = 78.77 A, and diffract to 1.85 A resolution. Data sets collected from heavy-atom derivatives of CFI indicated the incorporation of Pt and Hg atoms. Structure solution using MIR and MAD methods is currently under way.

Adenosine Triphosphatases↗

PubMed2002

tRNA-like recognition of group I introns by a tyrosyl-tRNA synthetase.

The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) functions in splicing group I introns by promoting the formation of the catalytically active RNA structure. Previous work suggested that CYT-18 recognizes a conserved tRNA-like structure of the group I intron catalytic core. Here, directed hydroxyl-radical cleavage assays show that the nucleotide-binding fold and C-terminal domains of CYT-18 interact with the expected group I intron cognates of the aminoacyl-acceptor stem and D-anticodon arms, respectively. Further, three-dimensional graphic modeling, supported by biochemical data, shows that conserved regions of group I introns can be superimposed over interacting regions of the tRNA in a Thermus thermophilus TyrRS/tRNA(Tyr) cocrystal structure. Our results support the hypothesis that CYT-18 and other aminoacyl-tRNA synthetases interact with group I introns by recognizing conserved tRNA-like structural features of the intron RNAs.

Cloning, Molecular↗

PubMed2002

Towards the structure of the mammalian signal recognition particle.

The signal recognition particle (SRP) is a ubiquitous ribonucleoprotein particle involved in the co-translational targeting of proteins to membranes. Crystal structures are now available for three protein-RNA subcomplexes from the SRP, which give insights into fundamental aspects of protein-RNA recognition, the assembly of stable ribonucleoprotein particles and the mechanism of action of the SRP.

Alu Elements↗

PubMed2002

Structural analysis of the adaptor protein ClpS in complex with the N-terminal domain of ClpA.

In Escherichia coli, protein degradation is performed by several proteolytic machines, including ClpAP. Generally, the substrate specificity of these machines is determined by chaperone components, such as ClpA. In some cases, however, the specificity is modified by adaptor proteins, such as ClpS. Here we report the 2.5 A resolution crystal structure of ClpS in complex with the N-terminal domain of ClpA. Using mutagenesis, we demonstrate that two contact residues (Glu79 and Lys 84) are essential not only for ClpAS complex formation but also for ClpAPS-mediated substrate degradation. The corresponding residues are absent in the chaperone ClpB, providing a structural rationale for the unique specificity shown by ClpS despite the high overall similarity between ClpA and ClpB. To determine the location of ClpS within the ClpA hexamer, we modeled the N-terminal domain of ClpA onto a structurally defined, homologous AAA+ protein. From this model, we proposed a molecular mechanism to explain the ClpS-mediated switch in ClpA substrate specificity.

Adenosine Triphosphatases↗