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1.  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.
doi:10.4161/rna.23854
PMCID: PMC3737334  PMID: 23846216
CRISPR; Csa5; structure; CRISPR interference; Cascade
2.  STRUCTURE OF THE DNA REPAIR HELICASE HEL308 REVEALS DNA BINDING AND AUTOINHIBITORY DOMAINS 
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.
doi:10.1074/jbc.M707548200
PMCID: PMC3434800  PMID: 18056710
3.  Structure of the DNA repair helicase XPD 
Cell  2008;133(5):801-812.
Summary
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.
doi:10.1016/j.cell.2008.04.029
PMCID: PMC3326533  PMID: 18510925
4.  A SIMPLE AND EFFICIENT EXPRESSION AND PURIFICATION SYSTEM USING TWO NEWLY CONSTRUCTED VECTORS 
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.
doi:10.1016/j.pep.2008.09.008
PMCID: PMC3315830  PMID: 18845260
Vector; pEHISGFPTEV; pEHISTEV; Protein expression; Purification protocol; TEV protease; AKTAxpress
5.  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.
doi:10.1038/nature05267
PMCID: PMC3315050  PMID: 17086202
6.  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.
doi:10.1042/BJ20130316
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
7.  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.
doi:10.1039/c003743e
PMCID: PMC3670083  PMID: 20445930
8.  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.
doi:10.1107/S1744309113012931
PMCID: PMC3668578  PMID: 23722837
patellamide; cyanobactins; natural products; prenyltransferases
9.  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.
doi:10.1042/BJ20130269
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
10.  Wzi Is an Outer Membrane Lectin that Underpins Group 1 Capsule Assembly in Escherichia coli 
Structure(London, England:1993)  2013;21(5):844-853.
Summary
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.
Highlights
•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.
doi:10.1016/j.str.2013.03.010
PMCID: PMC3791409  PMID: 23623732
11.  Structure of a RING E3 ligase and ubiquitin-loaded E2 primed for catalysis 
Nature  2012;489(7414):115-120.
SUMMARY
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.
doi:10.1038/nature11376
PMCID: PMC3442243  PMID: 22842904
12.  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.
doi:10.1002/pro.410
PMCID: PMC2974825  PMID: 20506248
LPS biosynthesis; hydride transfer; keto sugar; carbohydrate; SDR enzymes
13.  Structure of WbdD: a bifunctional kinase and methyltransferase that regulates the chain length of the O antigen in Escherichia coli O9a 
Molecular Microbiology  2012;86(3):730-742.
Summary
The Escherichia coli serotype O9a O-antigen polysaccharide (O-PS) is a model for glycan biosynthesis and export by the ATP-binding cassette transporter-dependent pathway. The polymannose O9a O-PS is synthesized as a polyprenol-linked glycan by mannosyltransferase enzymes located at the cytoplasmic membrane. The chain length of the O9a O-PS is tightly regulated by the WbdD enzyme. WbdD first phosphorylates the terminal non-reducing mannose of the O-PS and then methylates the phosphate, stopping polymerization. The 2.2 Å resolution structure of WbdD reveals a bacterial methyltransferase domain joined to a eukaryotic kinase domain. The kinase domain is again fused to an extended C-terminal coiled-coil domain reminiscent of eukaryotic DMPK (Myotonic Dystrophy Protein Kinase) family kinases such as Rho-associated protein kinase (ROCK). WbdD phosphorylates 2-α-d-mannosyl-d-mannose (2α-MB), a short mimic of the O9a polymer. Mutagenesis identifies those residues important in catalysis and substrate recognition and the in vivo phenotypes of these mutants are used to dissect the termination reaction. We have determined the structures of co-complexes of WbdD with two known eukaryotic protein kinase inhibitors. Although these are potent inhibitors in vitro, they do not show any in vivo activity. The structures reveal new insight into O-PS chain-length regulation in this important model system.
doi:10.1111/mmi.12014
PMCID: PMC3482155  PMID: 22970759
14.  Bacterial Mechanosensitive Channels—MscS: Evolution’s Solution to Creating Sensitivity in Function 
Annual review of biophysics  2012;41:157-177.
The discovery of mechanosensing channels has changed our understanding of bacterial physiology. The mechanosensitive channel of small conductance (MscS) is perhaps the most intensively studied of these channels. MscS has at least two states: closed, which does not allow solutes to exit the cytoplasm, and open, which allows rapid efflux of solvent and solutes. The ability to appropriately open or close the channel (gating) is critical to bacterial survival. We briefly review the science that led to the isolation and identification of MscS. We concentrate on the structure-function relationship of the channel, in particular the structural and biochemical approaches to understanding channel gating. We highlight the troubling discrepancies between the various models developed to understand MscS gating.
doi:10.1146/annurev-biophys-101211-113227
PMCID: PMC3378650  PMID: 22404681
protein structure; modeling; crystallography; EPR; mutagenesis; ion channel
15.  Structure-function relationships of the outer membrane translocon Wza investigated by cryo-electron microscopy and mutagenesis 
Journal of structural biology  2009;166(2):172-182.
Summary
The outer membrane protein, Wza from E. coli K30, forms an octameric complex that is essential for capsular polysaccharide export. Homologs of Wza are widespread in gram-negative bacterial pathogens where capsules are critical virulence determinants. Wza is unusual in that it spans the outer membrane using a barrel composed of amphipathic α-helices, rather than being a β-barrel like almost all other outer membrane channels. The transmembrane helical barrel of Wza also forms the external opening to a hydrophilic translocation pathway that spans the periplasm. Here, we have probed the structure and function of the Wza complex using both cryo-electron microscopy and mutagenesis. The helical barrel structure is stable in detergent micelles under mildly acidic conditions but is destabilised at basic pH, although the overall quaternary structure is retained. Truncation of the C-terminal region that forms the helical barrel by 4 residues has no effect on the ability of Wza to oligomerize and support capsule export, but larger truncations of 18, 24 or 35 amino acids abolish its function. The bulk of the C-terminal domain is essential for the stability and assembly of the E. coli Wza complex.
doi:10.1016/j.jsb.2009.02.005
PMCID: PMC3498625  PMID: 19236919
Outer membrane protein; capsular polysaccharide; transport; Wza; cryo-electron microscopy
16.  Structure of WbdD: a bifunctional kinase and methyltransferase that regulates the chain length of the O antigen in Escherichia coli O9a 
Molecular microbiology  2012;86(3):730-742.
Summary
The Escherichia coli serotype O9a O-antigen polysaccharide (O-PS) is a model for glycan biosynthesis and export by the ATP-binding cassette transporter-dependent pathway. The polymannose O9a O-PS is synthesized as a polyprenol-linked glycan by mannosyltransferase enzymes located at the cytoplasmic membrane. The chain length of the O9a O-PS is tightly regulated by the WbdD enzyme. WbdD first phosphorylates the terminal non-reducing mannose of the O-PS and then methylates the phosphate, stopping polymerization. The 2.2 Å resolution structure of WbdD reveals a bacterial methyltransferase domain joined to a eukaryotic kinase domain. The kinase domain is again fused to an extended C-terminal coiled-coil domain reminiscent of eukaryotic DMPK (Myotonic Dystrophy Protein Kinase) family kinases such as Rho-associated protein kinase (ROCK). WbdD phosphorylates 2-α-d-mannosyl-d-mannose (2α-MB), a short mimic of the O9a polymer. Mutagenesis identifies those residues important in catalysis and substrate recognition and the in vivo phenotypes of these mutants are used to dissect the termination reaction. We have determined the structures of co-complexes of WbdD with two known eukaryotic protein kinase inhibitors. Although these are potent inhibitors in vitro, they do not show any in vivo activity. The structures reveal new insight into O-PS chain-length regulation in this important model system.
doi:10.1111/mmi.12014
PMCID: PMC3482155  PMID: 22970759
17.  The mechanism of patellamide macrocyclization revealed by the characterization of the PatG macrocyclase domain 
Peptide macrocycles are found in many biologically active natural products. Their versatility, resistance to proteolysis and ability to traverse membranes has made them desirable molecules. Although technologies exist to synthesize such compounds, the full extent of diversity found among natural macrocycles has yet to be achieved synthetically. Cyanobactins are ribosomal peptide macrocycles encompassing an extraordinarily diverse range of ring sizes, amino acids and chemical modifications. We report the structure, biochemical characterization and initial engineering of the PatG macrocyclase domain of Prochloron sp. from the patellamide pathway that catalyzes the macrocyclization of linear peptides. The enzyme contains insertions in the subtilisin fold to allow it to recognize a three-residue signature, bind substrate in a preorganized and unusual conformation, shield an acyl-enzyme intermediate from water and catalyze peptide bond formation. The ability to macrocyclize a broad range of nonactivated substrates has wide biotechnology applications.
doi:10.1038/nsmb.2340
PMCID: PMC3462482  PMID: 22796963
18.  Crystallization, dehydration and experimental phasing of WbdD, a bifunctional kinase and methyltransferase from Escherichia coli O9a 
The optimization of WbdD crystals using a novel dehydration protocol and experimental phasing at 3.5 Å resolution by cross-crystal averaging followed by molecular replacement of electron density into a non-isomorphous 3.0 Å resolution native data set are reported.
WbdD is a bifunctional kinase/methyltransferase that is responsible for regulation of lipopolysaccharide O antigen polysaccharide chain length in Escherichia coli serotype O9a. Solving the crystal structure of this protein proved to be a challenge because the available crystals belonging to space group I23 only diffracted to low resolution (>95% of the crystals diffracted to resolution lower than 4 Å and most only to 8 Å) and were non-isomorphous, with changes in unit-cell dimensions of greater than 10%. Data from a serendipitously found single native crystal that diffracted to 3.0 Å resolution were non-isomorphous with a lower (3.5 Å) resolution selenomethionine data set. Here, a strategy for improving poor (3.5 Å resolution) initial phases by density modification and cross-crystal averaging with an additional 4.2 Å resolution data set to build a crude model of WbdD is desribed. Using this crude model as a mask to cut out the 3.5 Å resolution electron density yielded a successful molecular-replacement solution of the 3.0 Å resolution data set. The resulting map was used to build a complete model of WbdD. The hydration status of individual crystals appears to underpin the variable diffraction quality of WbdD crystals. After the initial structure had been solved, methods to control the hydration status of WbdD were developed and it was thus possible to routinely obtain high-resolution diffraction (to better than 2.5 Å resolution). This novel and facile crystal-dehydration protocol may be useful for similar challenging situations.
doi:10.1107/S0907444912029599
PMCID: PMC3447403  PMID: 22993091
WbdD; crystal dehydration
19.  Structure and mechanism of the CMR complex for CRISPR-mediated antiviral immunity 
Molecular cell  2012;45(3):303-313.
Summary
The prokaryotic Clusters of Regularly Interspaced Palindromic Repeats (CRISPR) system utilizes genomically-encoded CRISPR RNA (crRNA), derived from invading viruses and incorporated into ribonucleoprotein complexes with CRISPR-associated (CAS) proteins, to target and degrade viral DNA or RNA on subsequent infection. RNA is targeted by the CMR complex. In Sulfolobus solfataricus, this complex is composed of seven CAS protein subunits (Cmr1-7) and carries a diverse “payload” of targeting crRNA. The crystal structure of Cmr7 and low resolution structure of the complex are presented. S. solfataricus CMR cleaves RNA targets in an endonucleolytic reaction at UA dinucleotides. This activity is dependent on the 8-nucleotide repeat-derived 5′ sequence in the crRNA, but not on the presence of a proto-spacer associated motif (PAM) in the target. Both target and guide RNAs can be cleaved, although a single molecule of guide RNA can support the degradation of multiple targets.
doi:10.1016/j.molcel.2011.12.013
PMCID: PMC3381847  PMID: 22227115
20.  A Multidisciplinary Approach to Probing Enthalpy-Entropy Compensation and the Interfacial Mobility Model 
Journal of the American Chemical Society  2011;133(30):11515-11523.
In recent years, interfacial mobility has gained popularity as a model with which to rationalize both affinity in ligand binding and the often observed phenomenon of enthalpy-entropy compensation. While protein contraction and reduced mobility, as demonstrated by computational and NMR techniques respectively, have been correlated to entropies of binding for a variety of systems, to our knowledge, Raman difference spectroscopy has never been included in these analyses. Here, non-resonance Raman difference spectroscopy, isothermal titration calorimetry, and x-ray crystallography were utilized to correlate protein contraction, as demonstrated by an increase in protein interior packing and decreased residual protein movement, with trends of enthalpy-entropy compensation. These results are in accord with the interfacial mobility model, and lend additional credence to this view of protein activity.
doi:10.1021/ja1098287
PMCID: PMC3151494  PMID: 21692482
21.  Salt Bridges Regulate Both Dimer Formation and Monomeric Flexibility in HdeB and May Have a Role in Periplasmic Chaperone Function 
Journal of molecular biology  2011;415(3):538-546.
Escherichia coli and Gram-negative bacteria that live in the human gut must be able to tolerate rapid and large changes in environmental pH. Low pH irreversibly denatures and precipitates many bacterial proteins. While cytoplasmic proteins are well buffered against such swings, periplasmic proteins are not. Instead, it appears that some bacteria utilize chaperone proteins that stabilize periplasmic proteins, preventing their precipitation. Two highly expressed and related proteins, HdeA and HdeB, have been identified as acid-activated chaperones. The structure of HdeA is known and a mechanism for activation has been proposed. In this model, dimeric HdeA dissociates at low pH, and the exposed dimeric interface binds exposed hydrophobic surfaces of acid-denatured proteins, preventing their irreversible aggregation. We now report the structure and biophysical characterization of the HdeB protein. The monomer of HdeB shares a similar structure with HdeA, but its dimeric interface is different in composition and spatial location. We have used fluorescence to study the behavior of HdeB as pH is lowered, and like HdeA, it dissociates to monomers. We have identified one of the key intersubunit interactions that controls pH-induced monomerization. Our analysis identifies a structural interaction within the HdeB monomer that is disrupted as pH is lowered, leading to enhanced structural flexibility.
doi:10.1016/j.jmb.2011.11.026
PMCID: PMC3299563  PMID: 22138344
crystal structure; fluorescence measurements; hydrophobic residues; acid response; pH titration
22.  Structural and functional characterisation of a conserved archaeal RadA paralog with antirecombinase activity 
Journal of molecular biology  2009;389(4):661-673.
Summary
DNA recombinases (RecA in bacteria, Rad51 in eukarya and RadA in archaea) catalyse strand-exchange between homologous DNA molecules, the central reaction of homologous recombination, and are among the most conserved DNA repair proteins known. In bacteria, RecA is the sole protein responsible for this reaction, whereas, in eukaryotes, there are several RAD51 paralogs that cooperate to catalyse strand exchange. All archaea have at least one (and as many as four) RadA paralogs, but their function remains unclear. Here we show the three RadA paralogs encoded by the Sulfolobus solfataricus genome are expressed under normal growth conditions, and are not UV-inducible. We demonstrate that one of these proteins, Sso2452, which is representative of the large aRadC sub-family of archaeal RadA paralogs, functions as an ATPase that binds tightly to ssDNA. However, Sso2452 is not an active recombinase in vitro, and inhibits D-loop formation by RadA. We present the high-resolution crystal structure of Sso2452, which reveals key structural differences from the canonical RecA family recombinases that may explain its functional properties. The possible roles of the archaeal RadA paralogs in vivo are discussed.
doi:10.1016/j.jmb.2009.04.060
PMCID: PMC3387904  PMID: 19414020
Archaea; Recombinase; RadA; Homologous Recombination; Strand Exchange
23.  Halomethane production in plants: Structure of the biosynthetic SAM-dependent halide methyltransferase from Arabidopsis thaliana** 
A product structure of the halomethane producing enzyme in plants (Arabidopsis thaliana) is reported and a model for presentation of chloride/bromide ion to the methyl group of S-adenosyl-L-methionine (SAM) is presented to rationalise nucleophilic halide attack for halomethane production, gaseous natural products that are produced globally.
doi:10.1002/anie.201000119
PMCID: PMC3386781  PMID: 20376845
chloromethane; biohalogenation; methyltransferase; structural biology; S-adenosyl-L-methionine
24.  Structural basis for acyl acceptor specificity in the achrombactin biosynthetic enzyme AcsD 
Journal of molecular biology  2011;412(3):495-504.
Siderophores are known virulence factors and their biosynthesis is a target for new antibacterial agents. A nonribosomal peptide synthetase independent siderophore (NIS) biosynthetic pathway in Dickeya dadantii is responsible for production of the siderophore achromobactin. The D. dadantii AcsD enzyme has been shown to enantioselectively esterify citric acid with L-serine in the first committed step of achromobactin biosynthesis. The reaction occurs in two steps: stereospecific activation of citric acid by adenylation, followed by attack of the enzyme bound citryl adenylate by L-serine to give the homochiral ester. We now report a detailed characterization of the substrate profile and mechanism of the second (acyl transfer) step of AcsD enzyme. We demonstrate that the enzyme catalyzes formation of not only esters, but also amides from the citryl-adenylate intermediate. We have rationalized the substrate utilization profile for the acylation reaction by determining the first X-ray crystal structure of a product complex for this enzyme class. We have identified the residues that are important for both recognition of L-serine and catalysis of ester formation. Our hypotheses were tested by biochemical analysis of various mutants, one of which shows a reversal of specificity from the wild type with respect to non-natural substrates. This change can be rationalized on the basis of our structural data. That this change in specificity is accompanied by no loss in activity suggests that AcsD and other members of the NIS synthetase superfamily may have biotransformation potential.
doi:10.1016/j.jmb.2011.07.059
PMCID: PMC3323832  PMID: 21835184
25.  Crystal structure and silica condensing activities of silicatein α/cathepsin L chimeras 
Cathepsin L mutants with the ability to condense silica from solution have been generated and a 1.5 Å crystal structure of one of these chimeras allows us to rationalize the catalytic mechanism of silicic acid condensation.
doi:10.1039/b718264c
PMCID: PMC3326524  PMID: 18379686

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