I03-Macromolecular Crystallography
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Abstract: The intracellular subtilisin proteases (ISPs) are the only known members of the important and ubiquitous subtilisin family that function exclusively within the cell, constituting a major component of the degradome in many Gram-positive bacteria. The first ISP structure reported herein at a spacing of 1.56 A reveals features unique among subtilisins that has enabled potential functional and physiological roles to be assigned to sequence elements exclusive to the ISPs. Unlike all other subtilisins, ISP from B. clausii is dimeric, with residues from the C terminus making a major contribution to the dimer interface by crossing over to contact the partner subunit. A short N-terminal extension binds back across the active site to provide a potential novel regulatory mechanism of intrinsic proteolytic activity: a proline residue conserved throughout the ISPs introduces a kink in the polypeptide backbone that lifts the target peptide bond out of reach of the catalytic residues.
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Jun 2010
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I04-Macromolecular Crystallography
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Antonio
Ariza
,
Jens M.
Eklof
,
Oliver
Spadiut
,
Wendy A.
Offen
,
Shirley M.
Roberts
,
Werner
Besenmatter
,
Esben P.
Friis
,
Michael
Skjot
,
Keith S.
Wilson
,
Harry
Brumer
,
Gideon
Davies
Diamond Proposal Number(s):
[1221]
Open Access
Abstract: The enzymatic degradation of plant polysaccharides is emerging as one of the key environmental goals of the early 21st century, impacting on many processes in the textile and detergent industries as well as biomass conversion to biofuels. One of the well known problems with the use of nonstarch (nonfood)-based substrates such as the plant cell wall is that the cellulose fibers are embedded in a network of diverse polysaccharides, including xyloglucan, that renders access difficult. There is therefore increasing interest in the “accessory enzymes,” including xyloglucanases, that may aid biomass degradation through removal of “hemicellulose” polysaccharides. Here, we report the biochemical characterization of the endo-?-1,4-(xylo)glucan hydrolase from Paenibacillus polymyxa with polymeric, oligomeric, and defined chromogenic aryl-oligosaccharide substrates. The enzyme displays an unusual specificity on defined xyloglucan oligosaccharides, cleaving the XXXG-XXXG repeat into XXX and GXXXG. Kinetic analysis on defined oligosaccharides and on aryl-glycosides suggests that both the ?4 and +1 subsites show discrimination against xylose-appended glucosides. The three-dimensional structures of PpXG44 have been solved both in apo-form and as a series of ligand complexes that map the ?3 to ?1 and +1 to +5 subsites of the extended ligand binding cleft. Complex structures are consistent with partial intolerance of xylosides in the ?4? subsites. The atypical specificity of PpXG44 may thus find use in industrial processes involving xyloglucan degradation, such as biomass conversion, or in the emerging exciting applications of defined xyloglucans in food, pharmaceuticals, and cellulose fiber modification.
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Sep 2011
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[6386]
Abstract: Crimean-Congo hemorrhagic fever virus (CCHFV) is an emerging tick-borne virus of the Bunyaviridae family responsible for fatal human disease for which preventative or therapeutic measures do not exist. We solved the crystal structure of the Baghdad-12 strain CCHFV nucleocapsid protein (N), a potential therapeutic target, at a resolution of 2.1 angstroms.
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Oct 2012
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Antonio
Ariza
,
Sian
Tanner
,
Cheryl T.
Walter
,
Kyle C.
Dent
,
Dale A.
Shepherd
,
Weining
Wu
,
Susan V.
Matthews
,
Julian A.
Hiscox
,
Todd J.
Green
,
Ming
Luo
,
Richard M.
Elliott
,
Anthony R.
Fooks
,
Alison E.
Ashcroft
,
Nicola J.
Stonehouse
,
Neil A.
Ranson
,
John N.
Barr
,
Thomas A.
Edwards
Open Access
Abstract: All orthobunyaviruses possess three genome segments of single-stranded negative sense RNA that are encapsidated with the virus-encoded nucleocapsid (N) protein to form a ribonucleoprotein (RNP) complex, which is uncharacterized at high resolution. We report the crystal structure of both the Bunyamwera virus (BUNV) N–RNA complex and the unbound Schmallenberg virus (SBV) N protein, at resolutions of 3.20 and 2.75 Å, respectively. Both N proteins crystallized as ring-like tetramers and exhibit a high degree of structural similarity despite classification into different orthobunyavirus serogroups. The structures represent a new RNA-binding protein fold. BUNV N possesses a positively charged groove into which RNA is deeply sequestered, with the bases facing away from the solvent. This location is highly inaccessible, implying that RNA polymerization and other critical base pairing events in the virus life cycle require RNP disassembly. Mutational analysis of N protein supports a correlation between structure and function. Comparison between these crystal structures and electron microscopy images of both soluble tetramers and authentic RNPs suggests the N protein does not bind RNA as a repeating monomer; thus, it represents a newly described architecture for bunyavirus RNP assembly, with implications for many other segmented negative-strand RNA viruses.
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Apr 2013
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I04-Macromolecular Crystallography
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Antonio
Ariza
,
Olga
Moroz
,
Elena
Blagova
,
Johan
Turkenburg
,
Jitka
Waterman
,
Shirley
Roberts
,
Jesper
Vind
,
Carsten
Sjøholm
,
Søren F.
Lassen
,
Leonardo
De Maria
,
Vibe
Glitsoe
,
Lars K.
Skov
,
Keith
Wilson
Open Access
Abstract: Phytases hydrolyse phytate (myo-inositol hexakisphosphate), the principal form of phosphate stored in plant seeds to produce phosphate and lower phosphorylated myo-inositols. They are used extensively in the feed industry, and have been characterised biochemically and structurally with a number of structures in the PDB. They are divided into four distinct families: histidine acid phosphatases (HAP), β-propeller phytases, cysteine phosphatases and purple acid phosphatases and also split into three enzyme classes, the 3-, 5- and 6-phytases, depending on the position of the first phosphate in the inositol ring to be removed. We report identification, cloning, purification and 3D structures of 6-phytases from two bacteria, Hafnia alvei and Yersinia kristensenii, together with their pH optima, thermal stability, and degradation profiles for phytate. An important result is the structure of the H. alvei enzyme in complex with the substrate analogue myo-inositol hexakissulphate. In contrast to the only previous structure of a ligand-bound 6-phytase, where the 3-phosphate was unexpectedly in the catalytic site, in the H. alvei complex the expected scissile 6-phosphate (sulphate in the inhibitor) is placed in the catalytic site.
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May 2013
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I02-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Sian J.
Tanner
,
Antonio
Ariza
,
Charles-adrien
Richard
,
Hannah F.
Kyle
,
Rachel L.
Dods
,
Maire-lise
Blondot
,
Weining
Wu
,
J.
Trincao
,
Chi H.
Trinh
,
Julian A.
Hiscox
,
Miles W.
Carroll
,
Nigel J.
Silman
,
Jean-francois
Eleouet
,
Thomas A.
Edwards
,
John N.
Barr
Diamond Proposal Number(s):
[8367]
Abstract: The M2-1 protein of the important pathogen human respiratory
syncytial virus is a zinc-binding transcription antiterminator that
is essential for viral gene expression. We present the crystal
structure of full-length M2-1 protein in its native tetrameric form
at a resolution of 2.5 Å. The structure reveals that M2-1 forms
a disk-like assembly with tetramerization driven by a long helix
forming a four-helix bundle at its center, further stabilized by
contact between the zinc-binding domain and adjacent protomers.
The tetramerization helix is linked to a core domain responsible for
RNA binding activity by a flexible region onwhich lie two functionally
critical serine residues that are phosphorylated during infection. The
crystal structure of a phosphomimetic M2-1 variant revealed altered
charge density surrounding this flexible region although its position
was unaffected. Structure-guided mutagenesis identified residues
that contributed to RNA binding and antitermination activity, revealing
a strong correlation between these two activities, and further
defining the role of phosphorylation in M2-1 antitermination activity.
The data we present here identify surfaces critical for M2-1 function
that may be targeted by antiviral compounds.
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Jan 2014
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[9306]
Open Access
Abstract: Poly(ADP-ribosyl)ation is a common post-translational modification that mediates a wide variety of cellular processes including DNA damage repair, chromatin regulation, transcription, and apoptosis. The difficulty associated with accessing poly(ADP-ribose) (PAR) in a homogeneous form has been an impediment to understanding the interactions of PAR with poly(ADP-ribose) glycohydrolase (PARG) and other binding proteins. Here we describe the chemical synthesis of the ADP-ribose dimer, and we use this compound to obtain the first human PARG substrate-enzyme cocrystal structure. Chemical synthesis of PAR is an attractive alternative to traditional enzymatic synthesis and fractionation, allowing access to products such as dimeric ADP-ribose, which has been detected but never isolated from natural sources. Additionally, we describe the synthesis of an alkynylated dimer and demonstrate that this compound can be used to synthesize PAR probes including biotin and fluorophore-labeled compounds. The fluorescently labeled ADP-ribose dimer was then utilized in a general fluorescence polarization-based PAR-protein binding assay. Finally, we use intermediates of our synthesis to access various PAR fragments, and evaluation of these compounds as substrates for PARG reveals the minimal features for substrate recognition and enzymatic cleavage. Homogeneous PAR oligomers and unnatural variants produced from chemical synthesis will allow for further detailed structural and biochemical studies on the interaction of PAR with its many protein binding partners.
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Mar 2015
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Johannes Gregor Matthias
Rack
,
Rosa
Morra
,
Eva
Barkauskaite
,
Rolf
Kraehenbuehl
,
Antonio
Ariza
,
Yue
Qu
,
Mary
Ortmayer
,
Orsolya
Leidecker
,
David r
Cameron
,
Ivan
Matic
,
Anton y.
Peleg
,
David
Leys
,
Ana
Traven
,
Ivan
Ahel
Diamond Proposal Number(s):
[9306]
Open Access
Abstract: Sirtuins are an ancient family of NAD+-dependent deacylases connected with the regulation of fundamental cellular processes including metabolic homeostasis and genome integrity. We show the existence of a hitherto unrecognized class of sirtuins, found predominantly in microbial pathogens. In contrast to earlier described classes, these sirtuins exhibit robust protein ADP-ribosylation activity. In our model organisms, Staphylococcus aureus and Streptococcus pyogenes, the activity is dependent on prior lipoylation of the target protein and can be reversed by a sirtuin-associated macrodomain protein. Together, our data describe a sirtuin-dependent reversible protein ADP-ribosylation system and establish a crosstalk between lipoylation and mono- ADP-ribosylation. We propose that these posttranslational modifications modulate microbial virulence by regulating the response to host-derived reactive oxygen species.
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Jul 2015
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8367]
Open Access
Abstract: Hazara virus (HAZV) is a member of the Bunyaviridae family of segmented negative stranded RNA viruses, and shares the same serogroup as Crimean-Congo haemorrhagic fever virus (CCHFV). CCHFV is responsible for fatal human disease with a mortality rate approaching 30 %, which has an increased recent incidence within southern Europe. There are no preventative or therapeutic treatments for CCHFV-mediated disease, and thus CCHFVis classified as a hazard group 4 pathogen. In contrast HAZV is not associated with serious human disease, although infection of interferon receptor knockout mice with either CCHFV or HAZV results in similar disease progression. To characterise further similarities between HAZV and CCHFV, and support the use of HAZV as a model for CCHFV infection, we investigated the structure of the HAZV nucleocapsid protein (N) and compared it to CCHFV N. N performs an essential role in the viral life cycle by encapsidating the viral RNA genome, and thus, N represents a potential therapeutic target.
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Dec 2015
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I24-Microfocus Macromolecular Crystallography
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Jon
Agirre
,
Antonio
Ariza
,
Wendy
Offen
,
Johan
Turkenburg
,
Shirley
Roberts
,
Stuart
Mcnicholas
,
Paul V.
Harris
,
Brett
Mc Brayer
,
Jan
Dohnalek
,
Kevin
Cowtan
,
Gideon
Davies
,
Keith
Wilson
Diamond Proposal Number(s):
[1221]
Open Access
Abstract: The industrial conversion of cellulosic plant biomass into useful products such as biofuels is a major societal goal. These technologies harness diverse plant degrading enzymes, classical exo- and endo-acting cellulases and, increasingly, cellulose-active lytic polysaccharide monooxygenases, to deconstruct the recalcitrant β-D-linked polysaccharide. A major drawback with this process is that the exo-acting cellobiohydrolases suffer from severe inhibition from their cellobiose product. β-D-Glucosidases are therefore important for liberating glucose from cellobiose and thereby relieving limiting product inhibition. Here, the three-dimensional structures of two industrially important family GH3 β-D-glucosidases from Aspergillus fumigatus and A. oryzae, solved by molecular replacement and refined at 1.95 Å resolution, are reported. Both enzymes, which share 78% sequence identity, display a three-domain structure with the catalytic domain at the interface, as originally shown for barley β-D-glucan exohydrolase, the first three-dimensional structure solved from glycoside hydrolase family GH3. Both enzymes show extensive N-glycosylation, with only a few external sites being truncated to a single GlcNAc molecule. Those glycans N-linked to the core of the structure are identified purely as high-mannose trees, and establish multiple hydrogen bonds between their sugar components and adjacent protein side chains. The extensive glycans pose special problems for crystallographic refinement, and new techniques and protocols were developed especially for this work. These protocols ensured that all of the D-pyranosides in the glycosylation trees were modelled in the preferred minimum-energy 4C1 chair conformation and should be of general application to refinements of other crystal structures containing O- or N-glycosylation. The Aspergillus GH3 structures, in light of other recent three-dimensional structures, provide insight into fungal β-D-glucosidases and provide a platform on which to inform and inspire new generations of variant enzymes for industrial application.
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Feb 2016
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