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Instruments / Technical Area	Publication Details	Published
I04-Macromolecular Crystallography	A structurally conserved motif in γ-herpesvirus uracil-DNA glycosylases elicits duplex nucleotide-flipping Christopher Earl , Claire Bagneris , Kara Zeman , Ambrose Cole , Tracey Barrett , Renos Savva Nucleic Acids Research 332 DOI: 10.1093/nar/gky217 Diamond Proposal Number(s): [9204, 12305, 17201] Open Access Show Abstract ▼ Abstract: Efficient γ-herpesvirus lytic phase replication requires a virally encoded UNG-type uracil-DNA glycosylase as a structural element of the viral replisome. Uniquely, γ-herpesvirus UNGs carry a seven or eight residue insertion of variable sequence in the otherwise highly conserved minor-groove DNA binding loop. In Epstein–Barr Virus [HHV-4] UNG, this motif forms a disc-shaped loop structure of unclear significance. To ascertain the biological role of the loop insertion, we determined the crystal structure of Kaposi’s sarcoma-associated herpesvirus [HHV-8] UNG (kUNG) in its product complex with a uracil-containing dsDNA, as well as two structures of kUNG in its apo state. We find the disc-like conformation is conserved, but only when the kUNG DNA-binding cleft is occupied. Surprisingly, kUNG uses this structure to flip the orphaned partner base of the substrate deoxyuridine out of the DNA duplex while retaining canonical UNG deoxyuridine-flipping and catalysis. The orphan base is stably posed in the DNA major groove which, due to DNA backbone manipulation by kUNG, is more open than in other UNG–dsDNA structures. Mutagenesis suggests a model in which the kUNG loop is pinned outside the DNA-binding cleft until DNA docking promotes rigid structuring of the loop and duplex nucleotide flipping, a novel observation for UNGs.	Mar 2018
I04-Macromolecular Crystallography	KSHV SOX mediated host shutoff: the molecular mechanism underlying mRNA transcript processing Hyunah Lee , Anathe O. M. Patschull , Claire Bagneris , Hannah Ryan , Christopher M. Sanderson , Bahram Ebrahimi , Irene Nobeli , Tracey E. Barrett Nucleic Acids Research DOI: 10.1093/nar/gkw1340 Diamond Proposal Number(s): [9204] Open Access Show Abstract ▼ Abstract: Onset of the lytic phase in the KSHV life cycle is accompanied by the rapid, global degradation of host (and viral) mRNA transcripts in a process termed host shutoff. Key to this destruction is the virally encoded alkaline exonuclease SOX. While SOX has been shown to possess an intrinsic RNase activity and a potential consensus sequence for endonucleolytic cleavage identified, the structures of the RNA substrates targeted remained unclear. Based on an analysis of three reported target transcripts, we were able to identify common structures and confirm that these are indeed degraded by SOX in vitro as well as predict the presence of such elements in the KSHV pre-microRNA transcript K12-2. From these studies, we were able to determine the crystal structure of SOX productively bound to a 31 nucleotide K12-2 fragment. This complex not only reveals the structural determinants required for RNA recognition and degradation but, together with biochemical and biophysical studies, reveals distinct roles for residues implicated in host shutoff. Our results further confirm that SOX and the host exoribonuclease Xrn1 act in concert to elicit the rapid degradation of mRNA substrates observed in vivo, and that the activities of the two ribonucleases are co-ordinated.	Jan 2017
I03-Macromolecular Crystallography	Crystallographic and Cellular Characterisation of Two Mechanisms Stabilising the Native Fold of á1-Antitrypsin: Implications for Disease and Drug Design Bibek Gooptu , Elena Miranda , Irene Nobeli , Meera Mallya , Andrew Purkiss , Sarah C. Leigh Brown , Charlotte Summers , Russell L. Phillips , David A. Lomas , Tracey Barrett Journal Of Molecular Biology 387 (4), 857 - 868 DOI: 10.1016/j.jmb.2009.01.069 PMID: 19232354 Open Access Show Abstract ▼ Abstract: The common Z mutant (Glu342Lys) of ?1-antitrypsin results in the formation of polymers that are retained within hepatocytes. This causes liver disease whilst the plasma deficiency of an important proteinase inhibitor predisposes to emphysema. The Thr114Phe and Gly117Phe mutations border a surface cavity identified as a target for rational drug design. These mutations preserve inhibitory activity but reduce the polymerisation of wild-type native ?1-antitrypsin in vitro and increase secretion in a Xenopus oocyte model of disease. To understand these effects, we have crystallised both mutants and solved their structures. The 2.2 Å structure of Thr114Phe ?1-antitrypsin demonstrates that the effects of the mutation are mediated entirely by well-defined partial cavity blockade and allows in silico screening of fragments capable of mimicking the effects of the mutation. The Gly117Phe mutation operates differently, repacking aromatic side chains in the helix F–?-sheet A interface to induce a half-turn downward shift of the adjacent F helix. We have further characterised the effects of these two mutations in combination with the Z mutation in a eukaryotic cell model of disease. Both mutations increase the secretion of Z ?1-antitrypsin in the native conformation, but the double mutants remain more polymerogenic than the wild-type (M) protein. Taken together, these data support different mechanisms by which the Thr114Phe and Gly117Phe mutations stabilise the native fold of ?1-antitrypsin and increase secretion of monomeric protein in cell models of disease.	Apr 2009

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