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1.  Taking a molecular motor for a spin: helicase mechanism studied by spin labeling and PELDOR 
Nucleic Acids Research  2015;44(2):954-968.
The complex molecular motions central to the functions of helicases have long attracted attention. Protein crystallography has provided transformative insights into these dynamic conformational changes, however important questions about the true nature of helicase configurations during the catalytic cycle remain. Using pulsed EPR (PELDOR or DEER) to measure interdomain distances in solution, we have examined two representative helicases: PcrA from superfamily 1 and XPD from superfamily 2. The data show that PcrA is a dynamic structure with domain movements that correlate with particular functional states, confirming and extending the information gleaned from crystal structures and other techniques. XPD in contrast is shown to be a rigid protein with almost no conformational changes resulting from nucleotide or DNA binding, which is well described by static crystal structures. Our results highlight the complimentary nature of PELDOR to crystallography and the power of its precision in understanding the conformational changes relevant to helicase function.
PMCID: PMC4737156  PMID: 26657627
2.  The structural biology of patellamide biosynthesis 
The biosynthetic pathways for patellamide and related natural products have recently been studied by structural biology. These pathways produce molecules that have a complex framework and exhibit a diverse array of activity due to the variability of the amino acids that are found in them. As these molecules are difficult to synthesize chemically, exploitation of their properties has been modest. The patellamide pathway involves amino acid heterocyclization, peptide cleavage, peptide macrocyclization, heterocycle oxidation and epimerization; closely related products are also prenylated. Enzyme activities have been identified for all these transformations except epimerization, which may be spontaneous. This review highlights the recent structural and mechanistic work on amino acid heterocyclization, peptide cleavage and peptide macrocyclization. This work should help in using the enzymes to produce novel analogs of the natural products enabling an exploitation of their properties.
PMCID: PMC4268494  PMID: 25460274
3.  Structural analysis of leader peptide binding enables leader-free cyanobactin processing 
Nature chemical biology  2015;11(8):558-563.
Regioselective modification of amino acids within the context of a peptide is common to a number of biosynthetic pathways and many such products have potential as therapeutics. The ATP dependent enzyme LynD heterocyclizes multiple cysteine residues to thiazolines within a peptide substrate. The enzyme requires the substrate to have conserved N-terminal leader for full activity. Catalysis is almost insensitive to immediately flanking residues in the substrate suggesting recognition occurs distant from the active site. Nucleotide and peptide substrate co-complex structures of LynD reveal the substrate leader peptide binds to and extends the β-sheet of a conserved domain of LynD, whilst catalysis is accomplished in another conserved domain. The spatial segregation of catalysis from recognition combines seemingly contradictory properties of regioselectivity and promiscuity; it appears to be a conserved strategy in other peptide modifying enzymes. A variant of LynD that efficiently processes substrates without a leader peptide has been engineered.
PMCID: PMC4512242  PMID: 26098679
4.  Structural basis for the RING catalyzed synthesis of K63 linked ubiquitin chains 
The RING E3 ligase catalysed formation of lysine 63 linked ubiquitin chains by the Ube2V2–Ubc13 E2 complex is required for many important biological processes. Here we report the structure of the RING domain dimer of rat RNF4 in complex with a human Ubc13~Ub conjugate and Ube2V2. The structure has captured Ube2V2 bound to the acceptor (priming) ubiquitin with Lys63 in a position that could lead to attack on the linkage between the donor (second) ubiquitin and Ubc13 that is held in the active “folded back” conformation by the RING domain of RNF4. The interfaces identified in the structure were verified by in vitro ubiquitination assays of site directed mutants. This represents the first view of the synthesis of Lys63 linked ubiquitin chains in which both substrate ubiquitin and ubiquitin-loaded E2 are juxtaposed to allow E3 ligase mediated catalysis.
PMCID: PMC4529489  PMID: 26148049
5.  A coiled-coil domain acts as a molecular ruler in LPS chain length regulation 
Long-chain bacterial polysaccharides play important roles in pathogenicity. In Escherichia coli O9a, a model for ABC transporter dependent polysaccharide assembly, a large extracellular carbohydrate with a narrow distribution of size is polymerized from monosaccharides by a complex of two proteins, WbdA (polymerase) and WbdD (terminating protein). Such careful control of polymerization is recurring theme in biology. Combining crystallography and small angle X-ray scattering, we show that the C-terminal domain of WbdD contains an extended coiled-coil that physically separates WbdA from the catalytic domain of WbdD. The effects of insertions and deletions within the coiled-coil region were analyzed in vivo, revealing that polymer size is controlled by varying the length of the coiled-coil domain. Thus, the coiled-coil domain of WbdD functions as a molecular ruler that, along with WbdA:WbdD stoichiometry, controls the chain length of a model bacterial polysaccharide.
PMCID: PMC4650267  PMID: 25504321
6.  Unraveling the Specific Regulation of the Central Pathway for Anaerobic Degradation of 3-Methylbenzoate* 
The Journal of Biological Chemistry  2015;290(19):12165-12183.
Background: The specific transcriptional regulation of the mbd pathway for anaerobic 3-methylbenzoate degradation is unknown.
Results: The MbdR/3-methylbenzoyl-CoA couple controls the induction of the mbd genes.
Conclusion: MbdR is the regulator of the mbd pathway in Azoarcus sp. CIB.
Significance: This work highlights the importance of the regulatory systems in the evolution and adaptation of bacteria to the anaerobic degradation of aromatic compounds.
The mbd cluster encodes the anaerobic degradation of 3-methylbenzoate in the β-proteobacterium Azoarcus sp. CIB. The specific transcriptional regulation circuit that controls the expression of the mbd genes was investigated. The PO, PB1, and P3R promoters responsible for the expression of the mbd genes, their cognate MbdR transcriptional repressor, as well as the MbdR operator regions (ATACN10GTAT) have been characterized. The three-dimensional structure of MbdR has been solved revealing a conformation similar to that of other TetR family transcriptional regulators. The first intermediate of the catabolic pathway, i.e. 3-methylbenzoyl-CoA, was shown to act as the inducer molecule. An additional MbdR-dependent promoter, PA, which contributes to the expression of the CoA ligase that activates 3-methylbenzoate to 3-methylbenzoyl-CoA, was shown to be necessary for an efficient induction of the mbd genes. Our results suggest that the mbd cluster recruited a regulatory system based on the MbdR regulator and its target promoters to evolve a distinct central catabolic pathway that is only expressed for the anaerobic degradation of aromatic compounds that generate 3-methylbenzoyl-CoA as the central metabolite. All these results highlight the importance of the regulatory systems in the evolution and adaptation of bacteria to the anaerobic degradation of aromatic compounds.
PMCID: PMC4424350  PMID: 25795774
Bacterial Transcription; Gene Expression; Microbiology; Protein-DNA Interaction; Transcription Regulation; 3-Methylbenzoate; Azoarcus; Anaerobic Degradation
7.  Structure of the archaeal Cascade subunit Csa5 
RNA Biology  2013;10(5):762-769.
The Cascade complex for CRISPR-mediated antiviral immunity uses CRISPR RNA (crRNA) to target invading DNA species from mobile elements such as viruses, leading to their destruction. The core of the Cascade effector complex consists of the Cas5 and Cas7 subunits, which are widely conserved in prokaryotes. Cas7 binds crRNA and forms the helical backbone of Cascade. Many archaea encode a version of the Cascade complex (denoted Type I-A) that includes a Csa5 (or small) subunit, which interacts weakly with the core proteins. Here, we report the crystal structure of the Csa5 protein from Sulfolobus solfataricus. Csa5 comprises a conserved α-helical domain with a small insertion consisting of a weakly conserved β-strand domain. In the crystal, the Csa5 monomers have multimerized into infinite helical threads. At each interface is a strictly conserved intersubunit salt bridge, deletion of which disrupts multimerization. Structural analysis indicates a shared evolutionary history among the small subunits of the CRISPR effector complexes. The same α-helical domain is found in the C-terminal domain of Cse2 (from Type I-E Cascade), while the N-terminal domain of Cse2 is found in Cmr5 of the CMR (Type III-B) effector complex. As Cmr5 shares no match with Csa5, two possibilities present themselves: selective domain loss from an ancestral Cse2 to create two new subfamilies or domain fusion of two separate families to create a new Cse2 family. A definitive answer awaits structural studies of further small subunits from other CRISPR effector complexes.
PMCID: PMC3737334  PMID: 23846216
CRISPR; Csa5; structure; CRISPR interference; Cascade
The Journal of biological chemistry  2007;283(8):5118-5126.
Hel308 is a superfamily 2 helicase conserved in eukaryotes and archaea. It is thought to function in the early stages of recombination following replication fork arrest, and has a specificity for removal of the lagging strand in model replication forks. A homologous helicase constitutes the N-terminal domain of human DNA polymerase Q. The Drosophila homologue mus301 is implicated in double strand break repair and meiotic recombination. We have solved the high-resolution crystal structure of Hel308 from the crenarchaeon Sulfolobus solfataricus, revealing a five-domain structure with a central pore lined with essential DNA binding residues. The fifth domain is shown to act as a molecular brake, clamping the ssDNA extruded through the central pore of the helicase structure to limit the enzyme’s helicase activity. This provides an elegant mechanism to tune the enzyme’s processivity to its functional role. Hel308 can displace streptavidin from a biotinylated DNA molecule, suggesting that one function of the enzyme may be in the removal of bound proteins at stalled replication forks and recombination intermediates.
PMCID: PMC3434800  PMID: 18056710
9.  The structure of the cyanobactin domain of unknown function from PatG in the patellamide gene cluster 
The highly conserved domain of unknown function in the cyanobactin superfamily has a novel fold. The protein does not appear to bind the most plausible substrates, leaving questions as to its role.
Patellamides are members of the cyanobactin family of ribosomally synthesized and post-translationally modified cyclic peptide natural products, many of which, including some patellamides, are biologically active. A detailed mechanistic understanding of the biosynthetic pathway would enable the construction of a biotechnological ‘toolkit’ to make novel analogues of patellamides that are not found in nature. All but two of the protein domains involved in patellamide biosynthesis have been characterized. The two domains of unknown function (DUFs) are homologous to each other and are found at the C-termini of the multi-domain proteins PatA and PatG. The domain sequence is found in all cyanobactin-biosynthetic pathways characterized to date, implying a functional role in cyanobactin biosynthesis. Here, the crystal structure of the PatG DUF domain is reported and its binding interactions with plausible substrates are investigated.
PMCID: PMC4259220  PMID: 25484206
cyanobactins; patellamides; PatG; RiPPs
10.  Structure of the DNA repair helicase XPD 
Cell  2008;133(5):801-812.
The XPD helicase (Rad3 in Saccharomyces cerevisiae) is a component of transcription factor IIH (TFIIH), which functions in transcription initiation and Nucleotide Excision Repair in eukaryotes, catalysing DNA duplex opening localised to the transcription start site or site of DNA damage, respectively. XPD has a 5′ to 3′ polarity and the helicase activity is dependent on an iron-sulfur cluster binding domain, a feature that is conserved in related helicases such as FancJ. The xpd gene is the target of mutation in patients with xeroderma pigentosum, trichothiodystrophy and Cockayne’s syndrome, characterised by a wide spectrum of symptoms ranging from cancer susceptibility to neurological and developmental defects. The 2.25 Å crystal structure of XPD from the crenarchaeon Sulfolobus tokodaii, presented here together with detailed biochemical analyses, allows a molecular understanding of the structural basis for helicase activity and explains the phenotypes of xpd mutations in humans.
PMCID: PMC3326533  PMID: 18510925
Structural biology places a high demand on proteins both in terms of quality and quantity. Although many protein expression and purification systems have been developed, an efficient and simple system which can be easily adapted is desirable. Here, we report a new system which combines improved expression, solubility screening and purification efficiency. The system is based on two newly constructed vectors, pEHISTEV and pEHISGFPTEV derived from a pET vector. Both vectors generate a construct with an amino-terminal hexahistidine tag (His-tag). In addition, pEHISGFPTEV expresses a protein with an N-terminal His-tagged green fluorescent protein (GFP) fusion to allow rapid quantitation of soluble protein. Both vectors have a tobacco etch virus (TEV) protease cleavage site that allows for production of protein with only two additional N-terminal residues and have the same multiple cloning site which enables parallel cloning. Protein purification is a simple two-stage nickel affinity chromatography based on the His tag removal. A total of seven genes were tested using this system. Expression was optimised using pEHISGFPTEV constructs by monitoring the GFP fluorescence and the soluble target proteins were quantified using spectrophotometric analysis. All the tested proteins were purified with sufficient quantity and quality to attempt structure determination. This system has been proven to be simple and effective for structural biology. The system is easily adapted to include other vectors, tags or fusions and therefore has the potential to be broadly applicable.
PMCID: PMC3315830  PMID: 18845260
Vector; pEHISGFPTEV; pEHISTEV; Protein expression; Purification protocol; TEV protease; AKTAxpress
12.  The structure of Wza, the translocon for group 1 capsular polysaccharides in Escherichia coli, identifies a new class of outer membrane protein 
Nature  2006;444(7116):226-229.
Pathogenic bacteria frequently cloak themselves with a capsular polysaccharide layer. Escherichia coli group 1 capsules are formed from repeat-unit polysaccharides with molecular weights exceeding 100 kDa. The export of such a large polar molecule across the hydrophobic outer membrane in Gram-negative bacteria presents a formidable challenge, given that the permeability barrier of the membrane must be maintained. We describe the 2.26 Å structure of Wza, an integral outer membrane protein, that is essential for capsule export. Wza is an octamer, with a composite molecular weight of 340 kDa, and it forms an “amphora”-like structure. The protein has a large central cavity 100 Å long and 30 Å wide. The transmembrane region is a novel α-helical barrel, and is linked to three additional novel periplasmic domains, marking Wza as the representative of a new class of membrane protein. Although Wza is open to the extracellular environment, a flexible loop in the periplasmic region occludes the cavity and may regulate the opening of the channel. The structure defines the route taken by the capsular polymer as it exits the cell, using the structural data we propose a mechanism for the translocation of the large polar capsular polysaccharide.
PMCID: PMC3315050  PMID: 17086202
13.  The crystal structure of the Y140F mutant of ADP-l-glycero-d-manno-heptose 6-epimerase bound to ADP-β-d-mannose suggests a one base mechanism 
Bacteria synthesize a wide array of unusual carbohydrate molecules, which they use in a variety of ways. The carbohydrate l-glycero-d-manno-heptose is an important component of lipopolysaccharide and is synthesized in a complex series of enzymatic steps. One step involves the epimerization at the C6″ position converting ADP-d-glycero-d-manno-heptose into ADP-l-glycero-d-manno-heptose. The enzyme responsible is a member of the short chain dehydrogenase superfamily, known as ADP-l-glycero-d-manno-heptose 6-epimerase (AGME). The structure of the enzyme was known but the arrangement of the catalytic site with respect to the substrate is unclear. We now report the structure of AGME bound to a substrate mimic, ADP-β-d-mannose, which has the same stereochemical configuration as the substrate. The complex identifies the key residues and allows mechanistic insight into this novel enzyme.
PMCID: PMC2974825  PMID: 20506248
LPS biosynthesis; hydride transfer; keto sugar; carbohydrate; SDR enzymes
14.  The crystal structure of the Y140F mutant of ADP-l-glycero-d-manno-heptose 6-epimerase bound to ADP-β-d-mannose suggests a one base mechanism 
Bacteria synthesize a wide array of unusual carbohydrate molecules, which they use in a variety of ways. The carbohydrate l-glycero-d-manno-heptose is an important component of lipopolysaccharide and is synthesized in a complex series of enzymatic steps. One step involves the epimerization at the C6″ position converting ADP-d-glycero-d-manno-heptose into ADP-l-glycero-d-manno-heptose. The enzyme responsible is a member of the short chain dehydrogenase superfamily, known as ADP-l-glycero-d-manno-heptose 6-epimerase (AGME). The structure of the enzyme was known but the arrangement of the catalytic site with respect to the substrate is unclear. We now report the structure of AGME bound to a substrate mimic, ADP-β-d-mannose, which has the same stereochemical configuration as the substrate. The complex identifies the key residues and allows mechanistic insight into this novel enzyme.
PMCID: PMC2974825  PMID: 20506248
LPS biosynthesis; hydride transfer; keto sugar; carbohydrate; SDR enzymes
15.  Quantification of free cysteines in membrane and soluble proteins using a fluorescent dye and thermal unfolding 
Nature protocols  2013;8(11):10.1038/nprot.2013.128.
Cysteine is an extremely useful site for selective attachment of labels to proteins for many applications, including the study of protein structure in solution by electron paramagnetic resonance (EPR), fluorescence spectroscopy and medical imaging. The demand for quantitative data for these applications means that it is important to determine the extent of the cysteine labeling. The efficiency of labeling is sensitive to the 3D context of cysteine within the protein. Where the label or modification is not directly measurable by optical or magnetic spectroscopy, for example, in cysteine modification to dehydroalanine, assessing labeling efficiency is difficult. We describe a simple assay for determining the efficiency of modification of cysteine residues, which is based on an approach previously used to determine membrane protein stability. The assay involves a reaction between the thermally unfolded protein and a thiol-specific coumarin fluorophore that is only fluorescent upon conjugation with thiols. Monitoring fluorescence during thermal denaturation of the protein in the presence of the dye identifies the temperature at which the maximum fluorescence occurs; this temperature differs among proteins. Comparison of the fluorescence intensity at the identified temperature between modified, unmodified (positive control) and cysteine-less protein (negative control) allows for the quantification of free cysteine. We have quantified both site-directed spin labeling and dehydroalanine formation. The method relies on a commonly available fluorescence 96-well plate reader, which rapidly screens numerous samples within 1.5 h and uses <100 μg of material. The approach is robust for both soluble and detergent-solubilized membrane proteins.
PMCID: PMC3836627  PMID: 24091556
16.  Extensive DNA mimicry by the ArdA anti-restriction protein and its role in the spread of antibiotic resistance 
Nucleic Acids Research  2009;37(15):4887-4897.
The ardA gene, found in many prokaryotes including important pathogenic species, allows associated mobile genetic elements to evade the ubiquitous Type I DNA restriction systems and thereby assist the spread of resistance genes in bacterial populations. As such, ardA contributes to a major healthcare problem. We have solved the structure of the ArdA protein from the conjugative transposon Tn916 and find that it has a novel extremely elongated curved cylindrical structure with defined helical grooves. The high density of aspartate and glutamate residues on the surface follow a helical pattern and the whole protein mimics a 42-base pair stretch of B-form DNA making ArdA by far the largest DNA mimic known. Each monomer of this dimeric structure comprises three alpha–beta domains, each with a different fold. These domains have the same fold as previously determined proteins possessing entirely different functions. This DNA mimicry explains how ArdA can bind and inhibit the Type I restriction enzymes and we demonstrate that 6 different ardA from pathogenic bacteria can function in Escherichia coli hosting a range of different Type I restriction systems.
PMCID: PMC2731889  PMID: 19506028
17.  RmlC, a C3′ and C3′ carbohydrate epimerase, appears to operate via an intermediate with an unusual twist boat conformation. 
Journal of molecular biology  2006;365(1):146-159.
The striking feature of carbohydrates is their constitutional, conformational and configurational diversity. Biology has harnessed this diversity and manipulates carbohydrate residues in a variety of ways, one of which is epimerization. RmlC catalyzes the epimerization of the C3′ and C5′ positions of dTDP-6-deoxy-D-xylo-4-hexulose, forming dTDP-6-deoxy-L-lyxo-4-hexulose. RmlC is the third enzyme of the rhamnose pathway, and represents a validated anti-bacterial drug target. Although several structures of the enzyme have been reported, the mechanism and the nature of the intermediates have remained obscure. Despite its relatively small size (22 kDa), RmlC catalyses four stereospecific proton transfers and the substrate undergoes a major conformational change during the course of the transformation. Here we report the structure of RmlC from several organisms in complex with product and product mimics. We have probed site-directed mutants by assay and by deuterium exchange. The combination of structural and biochemical data has allowed us to assign key residues and identify the conformation of the carbohydrate during turnover. Clear knowledge of the chemical structure of RmlC reaction intermediates may offer new opportunities for rational drug design.
PMCID: PMC1805628  PMID: 17046787
site directed mutagenesis; X-ray crystallography; drug design; epimerization; enzyme
18.  Discovery of an Allosteric Inhibitor Binding Site in 3-Oxo-acyl-ACP Reductase from Pseudomonas aeruginosa 
ACS Chemical Biology  2013;8(11):2518-2527.
3-Oxo-acyl-acyl carrier protein (ACP) reductase (FabG) plays a key role in the bacterial fatty acid synthesis II system in pathogenic microorganisms, which has been recognized as a potential drug target. FabG catalyzes reduction of a 3-oxo-acyl-ACP intermediate during the elongation cycle of fatty acid biosynthesis. Here, we report gene deletion experiments that support the essentiality of this gene in P. aeruginosa and the identification of a number of small molecule FabG inhibitors with IC50 values in the nanomolar to low micromolar range and good physicochemical properties. Structural characterization of 16 FabG-inhibitor complexes by X-ray crystallography revealed that the compounds bind at a novel allosteric site located at the FabG subunit–subunit interface. Inhibitor binding relies primarily on hydrophobic interactions, but specific hydrogen bonds are also observed. Importantly, the binding cavity is formed upon complex formation and therefore would not be recognized by virtual screening approaches. The structure analysis further reveals that the inhibitors act by inducing conformational changes that propagate to the active site, resulting in a displacement of the catalytic triad and the inability to bind NADPH.
PMCID: PMC3833349  PMID: 24015914
19.  The Respiratory Arsenite Oxidase: Structure and the Role of Residues Surrounding the Rieske Cluster 
PLoS ONE  2013;8(8):e72535.
The arsenite oxidase (Aio) from the facultative autotrophic Alphaproteobacterium Rhizobium sp. NT-26 is a bioenergetic enzyme involved in the oxidation of arsenite to arsenate. The enzyme from the distantly related heterotroph, Alcaligenes faecalis, which is thought to oxidise arsenite for detoxification, consists of a large α subunit (AioA) with bis-molybdopterin guanine dinucleotide at its active site and a 3Fe-4S cluster, and a small β subunit (AioB) which contains a Rieske 2Fe-2S cluster. The successful heterologous expression of the NT-26 Aio in Escherichia coli has resulted in the solution of its crystal structure. The NT-26 Aio, a heterotetramer, shares high overall similarity to the heterodimeric arsenite oxidase from A. faecalis but there are striking differences in the structure surrounding the Rieske 2Fe-2S cluster which we demonstrate explains the difference in the observed redox potentials (+225 mV vs. +130/160 mV, respectively). A combination of site-directed mutagenesis and electron paramagnetic resonance was used to explore the differences observed in the structure and redox properties of the Rieske cluster. In the NT-26 AioB the substitution of a serine (S126 in NT-26) for a threonine as in the A. faecalis AioB explains a −20 mV decrease in redox potential. The disulphide bridge in the A. faecalis AioB which is conserved in other betaproteobacterial AioB subunits and the Rieske subunit of the cytochrome bc1 complex is absent in the NT-26 AioB subunit. The introduction of a disulphide bridge had no effect on Aio activity or protein stability but resulted in a decrease in the redox potential of the cluster. These results are in conflict with previous data on the betaproteobacterial AioB subunit and the Rieske of the bc1 complex where removal of the disulphide bridge had no effect on the redox potential of the former but a decrease in cluster stability was observed in the latter.
PMCID: PMC3758308  PMID: 24023621
20.  CRISPR interference: a structural perspective 
Biochemical Journal  2013;453(Pt 2):155-166.
CRISPR (cluster of regularly interspaced palindromic repeats) is a prokaryotic adaptive defence system, providing immunity against mobile genetic elements such as viruses. Genomically encoded crRNA (CRISPR RNA) is used by Cas (CRISPR-associated) proteins to target and subsequently degrade nucleic acids of invading entities in a sequence-dependent manner. The process is known as ‘interference’. In the present review we cover recent progress on the structural biology of the CRISPR/Cas system, focusing on the Cas proteins and complexes that catalyse crRNA biogenesis and interference. Structural studies have helped in the elucidation of key mechanisms, including the recognition and cleavage of crRNA by the Cas6 and Cas5 proteins, where remarkable diversity at the level of both substrate recognition and catalysis has become apparent. The RNA-binding RAMP (repeat-associated mysterious protein) domain is present in the Cas5, Cas6, Cas7 and Cmr3 protein families and RAMP-like domains are found in Cas2 and Cas10. Structural analysis has also revealed an evolutionary link between the small subunits of the type I and type III-B interference complexes. Future studies of the interference complexes and their constituent components will transform our understanding of the system.
PMCID: PMC3727216  PMID: 23805973
antiviral defence; cluster of regularly interspaced palindromic repeats (CRISPR); crystallography; evolution; protein structure; repeat-associated mysterious protein (RAMP); BhCas5c, Bacillus halodurans Cas5c; CRISPR, cluster of regularly interspaced palindromic repeats; Cas, CRISPR-associated; Cascade, CRISPR-associated complex for antiviral defence; crRNA, CRISPR RNA; dsDNA, double-stranded DNA; EcoCas3, Escherichia coli Cas3; EM, electron microscopy; HD, histidine–aspartate; MjaCas3″, Methanocaldococcus jannaschii Cas3″; PaCas6f, Pseudomonas aeruginosa Cas6f; PAM, protospacer adjacent motif; PfuCas, Pyrococcus furiosus Cas; pre-crRNA, precursor crRNA; RAMP, repeat-associated mysterious protein; RRM, RNA recognition motif; ssDNA, single-stranded DNA; SsoCas, Sulfolobus solfataricus Cas; ssRNA, single-stranded RNA; SthCas3, Streptococcus thermophilus Cas3; tracrRNA, trans-activating crRNA; TtCas, Thermus thermophilus Cas
21.  Inhibition of the PLP-dependent enzyme serine palmitoyltransferase by cycloserine: evidence for a novel decarboxylative mechanism of inactivation 
Molecular bioSystems  2010;6(9):1682-1693.
Cycloserine (CS, 4-amino-3-isoxazolidone) is a cyclic amino acid mimic that is known to inhibit many essential pyridoxal 5′-phosphate (PLP)-dependent enzymes. Two CS enantiomers are known; d-cycloserine (DCS, also known as Seromycin), is a natural product that is used to treat resistant Mycobacterium tuberculosis infections as well as neurological disorders since it is a potent NMDA receptor agonist, and l-cycloserine (LCS), is a synthetic enantiomer whose usefulness as a drug has been hampered by its inherent toxicity arising through inhibition of sphingolipid metabolism. Previous studies on various PLP-dependent enzymes revealed a common mechanism of inhibition by both enantiomers of CS; the PLP cofactor is disabled by forming a stable 3-hydroxyisoxazole/pyridoxamine 5′-phosphate (PMP) adduct at the active site where the cycloserine ring remains intact. Here we describe a novel mechanism of CS inactivation of the PLP-dependent enzyme serine palmitoyltransferase (SPT) from Sphingomonas paucimobilis. SPT catalyses the condensation of l-serine and palmitoyl-CoA, the first step in the de novo sphingolipid biosynthetic pathway. We have used a range of kinetic, spectroscopic and structural techniques to postulate that both LCS and DCS inactivate SPT by transamination to form a free pyridoxamine 5′-phosphate (PMP) and β-aminooxyacetaldehyde that remain bound at the active site. We suggest this occurs by ring opening of the cycloserine ring followed by decarboxylation. Enzyme kinetics show that inhibition is reversed by incubation with excess PLP and that LCS is a more effective SPT inhibitor than DCS. UV-visible spectroscopic data, combined with site-directed mutagenesis, suggest that a mobile Arg378 residue is involved in cycloserine inactivation of SPT.
PMCID: PMC3670083  PMID: 20445930
22.  Structure of PatF from Prochloron didemni  
The X-ray crystal structure of PatF from P. didemni was solved by the single-wavelength anomalous diffraction method to a resolution of 2.13 Å.
Patellamides are macrocyclic peptides with potent biological effects and are a subset of the cyanobactins. Cyanobactins are natural products that are produced by a series of enzymatic transformations and a common modification is the addition of a prenyl group. Puzzlingly, the pathway for patellamides in Prochloron didemni contains a gene, patF, with homology to prenylases, but patellamides are not themselves prenylated. The structure of the protein PatF was cloned, expressed, purified and determined. Prenylase activity could not be demonstrated for the protein, and examination of the structure revealed changes in side-chain identity at the active site. It is suggested that these changes have inactivated the protein. Attempts to mutate these residues led to unfolded protein.
PMCID: PMC3668578  PMID: 23722837
patellamide; cyanobactins; natural products; prenyltransferases
23.  Structure of a dimeric crenarchaeal Cas6 enzyme with an atypical active site for CRISPR RNA processing 
Biochemical Journal  2013;452(Pt 2):223-230.
The competition between viruses and hosts is played out in all branches of life. Many prokaryotes have an adaptive immune system termed ‘CRISPR’ (clustered regularly interspaced short palindromic repeats) which is based on the capture of short pieces of viral DNA. The captured DNA is integrated into the genomic DNA of the organism flanked by direct repeats, transcribed and processed to generate crRNA (CRISPR RNA) that is loaded into a variety of effector complexes. These complexes carry out sequence-specific detection and destruction of invading mobile genetic elements. In the present paper, we report the structure and activity of a Cas6 (CRISPR-associated 6) enzyme (Sso1437) from Sulfolobus solfataricus responsible for the generation of unit-length crRNA species. The crystal structure reveals an unusual dimeric organization that is important for the enzyme's activity. In addition, the active site lacks the canonical catalytic histidine residue that has been viewed as an essential feature of the Cas6 family. Although several residues contribute towards catalysis, none is absolutely essential. Coupled with the very low catalytic rate constants of the Cas6 family and the plasticity of the active site, this suggests that the crRNA recognition and chaperone-like activities of the Cas6 family should be considered as equal to or even more important than their role as traditional enzymes.
PMCID: PMC3652601  PMID: 23527601
antiviral defence; Cas6; clustered regularly interspaced short palindromic repeats (CRISPR); ribonuclease; Sulfolobus; CRISPR, clustered regularly interspaced short palindromic repeats; Cas, CRISPR-associated; crRNA, CRISPR RNA; Ni-NTA, Ni2+-nitrilotriacetate; PaCas6f, Pseudomonas aeruginosa Cas6; PfuCas6, Pyrococcus furiosus Cas6; RAMP, repeat-associated mysterious protein; RMSD, root mean square deviation; RRM, RNA-recognition motif; SAD, single-wavelength anomalous dispersion; SsoCas6, Sulfolobus solfataricus Cas6; TBE, Tris/borate/EDTA; TEV, tobacco etch virus; TtCas6e, Thermus thermophilus Cas6
24.  Wzi Is an Outer Membrane Lectin that Underpins Group 1 Capsule Assembly in Escherichia coli 
Structure(London, England:1993)  2013;21(5):844-853.
Many pathogenic bacteria encase themselves in a polysaccharide capsule that provides a barrier to the physical and immunological challenges of the host. The mechanism by which the capsule assembles around the bacterial cell is unknown. Wzi, an integral outer-membrane protein from Escherichia coli, has been implicated in the formation of group 1 capsules. The 2.6 Å resolution structure of Wzi reveals an 18-stranded β-barrel fold with a novel arrangement of long extracellular loops that blocks the extracellular entrance and a helical bundle that plugs the periplasmic end. Mutagenesis shows that specific extracellular loops are required for in vivo capsule assembly. The data show that Wzi binds the K30 carbohydrate polymer and, crucially, that mutants functionally deficient in vivo show no binding to K30 polymer in vitro. We conclude that Wzi is a novel outer-membrane lectin that assists in the formation of the bacterial capsule via direct interaction with capsular polysaccharides.
•Wzi is an 18-stranded β-barrel outer-membrane protein•Wzi has a unique N-terminal helical domain and novel extracellular loops•In vivo data show that extracellular loops are required for capsule formation•In vitro data show that these loops are required to bind K30 polymer
Wzi is a novel β-barrel outer membrane protein that is found in bacteria that make group 1 capsules. When the protein is missing or specific regions are mutated, capsule formation is impaired. Bushell et al. show that the protein binds to K30 capsular polysaccharide and suggest it templates capsule assembly.
PMCID: PMC3791409  PMID: 23623732
25.  Structure of a RING E3 ligase and ubiquitin-loaded E2 primed for catalysis 
Nature  2012;489(7414):115-120.
Ubiquitin modification is mediated by a large family of specificity determining ubiquitin E3 ligases. To facilitate ubiquitin transfer, RING E3 ligases bind both substrate and a ubiquitin E2 conjugating enzyme linked to ubiquitin via a thioester bond, but the mechanism of transfer has remained elusive. Here we report the crystal structure of the dimeric RING of RNF4 in complex with E2 (UbcH5a) linked by an isopeptide bond to ubiquitin. While the E2 contacts a single protomer of the RING, ubiquitin is folded back onto the E2 by contacts from both RING protomers. The C-terminal tail of ubiquitin is locked into an active site groove on the E2 by an intricate network of interactions, resulting in changes at the E2 active site. This arrangement is primed for catalysis as it can deprotonate the incoming substrate lysine residue and stabilise the consequent tetrahedral transition state intermediate.
PMCID: PMC3442243  PMID: 22842904

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