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1.  Probing the Molecular Mechanisms in Copper Amine Oxidases by Generating Heterodimers 
Chembiochem  2015;16(4):559-564.
For some homodimeric copper amine oxidases (CuAO), there is suggestive evidence of differential activity at the two active sites implying potential cooperativity between the two monomers. To examine this phenomenon for the Arthrobacter globiformis CuAO (AGAO), we purified a heterodimeric form of the enzyme for comparison with the homodimer. The heterodimer comprises an active wild-type monomer and an inactive monomer in which an active-site tyrosine is mutated to phenylalanine (Y382F). This mutation prevents the formation of the trihydroxyphenylalanine quinone (TPQ) cofactor. A pETDuet vector and a dual fusion tag strategy was used to purify heterodimers (WT/Y382F) from homodimers. Purity was confirmed by western blot and native PAGE analyses. Spectral and kinetic studies support the view that whether there are one or two functional monomers in the dimer, the properties of each functional monomer are the same, thus indicating no communication between the active sites in this bacterial enzyme.
PMCID: PMC4497604  PMID: 25607656
Arthrobacter globiformis; cooperativity; copper amine oxidases; heterodimers
2.  Time-resolved crystallography using the Hadamard Transform 
Nature methods  2014;11(11):1131-1134.
A new method for performing time-resolved X-ray crystallographic experiments based on the Hadamard Transform is proposed and demonstrated. The time-resolution is defined by the underlying periodicity of the probe pulse sequence and the signal to noise is greatly improved compared to the fastest experiments depending on a single pulse. This approach is general and equally applicable to any spectroscopic or imaging measurement where the probe can be encoded.
PMCID: PMC4216935  PMID: 25282611
3.  The Structure of the PanD/PanZ Protein Complex Reveals Negative Feedback Regulation of Pantothenate Biosynthesis by Coenzyme A 
Chemistry & Biology  2015;22(4):492-503.
Coenzyme A (CoA) is an ubiquitous and essential cofactor, synthesized from the precursor pantothenate. Vitamin biosynthetic pathways are normally tightly regulated, including the pathway from pantothenate to CoA. However, no regulation of pantothenate biosynthesis has been identified. We have recently described an additional component in the pantothenate biosynthetic pathway, PanZ, which promotes the activation of the zymogen, PanD, to form aspartate α-decarboxylase (ADC) in a CoA-dependent manner. Here we report the structure of PanZ in complex with PanD, which reveals the structural basis for the CoA dependence of this interaction and activation. In addition, we show that PanZ acts as a CoA-dependent inhibitor of ADC catalysis. This inhibitory effect can effectively regulate the biosynthetic pathway to pantothenate, and thereby also regulate CoA biosynthesis. This represents a previously unobserved mode of metabolic regulation whereby a cofactor-utilizing protein negatively regulates the biosynthesis of the same cofactor.
Graphical Abstract
•Structure of the PanD-PanZ.AcCoA complex is reported at a resolution of 1.6 Å•Binding of AcCoA to PanZ is required to form the PanZ/PanD interface•PanZ.AcCoA activates PanD via selection of a reactive conformation of PanD•PanZ.AcCoA inhibits the activated enzyme, regulating pantothenate biosynthesis
The structure of the complex of PanD and its activating factor PanZ is determined by Monteiro et al., revealing the basis for the CoA dependence of this interaction. This CoA-dependent interaction is shown to inhibit catalysis by the activated enzyme. This is the first report of regulation of pantothenate biosynthesis.
PMCID: PMC4410942  PMID: 25910242
4.  Investigating the active centre of the Scytalidium thermophilum catalase 
S. thermophilum produces a haem-containing catalase with phenol oxidase activity which is involved in H2O2 degradation and the oxidation of some diphenolic compounds in the absence of H2O2. The dimensions or volume of the main channel are critical in determining the rate of both movement of H2O2 into the active site and dioxygen binding, as has previously been proposed for other monofunctional catalases.
Almost all monofunctional haem catalases contain a highly conserved core containing the active site, which is connected to the exterior of the enzyme by three channels. These channels have been identified as potential routes for substrate flow and product release. To further investigate the role of these molecular channels, a series of mutants of Scytalidium thermophilum catalase were generated. The three-dimensional structures of four catalase variants, N155A, V123A, V123C and V123T, have been determined at resolutions of 2.25, 1.93, 1.9 and 1.7 Å, respectively. The V123C variant contains a new covalent bond between the S atom of Cys123 and the imidazole ring of the essential His82. This variant enzyme has only residual catalase activity and contains haem b instead of the normal haem d. The H82A variant demonstrates low catalase and phenol oxidase activities (0.2 and 20% of those of recombinant wild-type catalase–phenol oxidase, respectively). The N155A and N155H variants exhibit 4.5 and 3% of the wild-type catalase activity and contain haem d, showing that Asn155 is essential for catalysis but is not required for the conversion of haem b to haem d. Structural analysis suggests that the cause of the effect of these mutations on catalysis is the disruption of the ability of dioxygen substrates to efficiently access the active site. Additional mutants have been characterized biochemically to further probe the roles of the different channels. Introducing smaller or polar side chains in place of Val123 reduces the catalase activity. The F160V, F161V and F168V mutants show a marked decrease in catalase activity but have a much lower effect on the phenol oxidase activity, despite containing substoichiometric amounts of haem.
PMCID: PMC3614159  PMID: 23545640
haem catalases; channels; covalent bonds; phenol oxidase
5.  Human Cellular Retinaldehyde-Binding Protein Has Secondary Thermal 9-cis-Retinal Isomerase Activity 
Cellular retinaldehyde-binding protein (CRALBP) chaperones 11-cis-retinal to convert opsin receptor molecules into photosensitive retinoid pigments of the eye. We report a thermal secondary isomerase activity of CRALBP when bound to 9-cis-retinal. UV/VIS and 1H-NMR spectroscopy were used to characterize the product as 9,13-dicis-retinal. The X-ray structure of the CRALBP mutant R234W:9-cis-retinal complex at 1.9 Å resolution revealed a niche in the binding-pocket for 9-cis-aldehyde different from that reported for 11-cis-retinal. Combined computational, kinetic, and structural data lead us to propose an isomerization mechanism catalyzed by a network of buried waters. Our findings highlight a specific role of water molecules in both CRALBP-assisted specificity towards 9-cis-retinal and its thermal isomerase activity yielding 9,13-dicis-retinal. Kinetic data from two point mutants of CRALBP support an essential role of Glu202 as the initial proton donor in this isomerization reaction.
PMCID: PMC3936205  PMID: 24328211
6.  In pursuit of an accurate spatial and temporal model of biomolecules at the atomistic level: a perspective on computer simulation 
The current computational techniques available for biomolecular simulation are described, and the successes and limitations of each with reference to the experimental biophysical methods that they complement are presented.
Despite huge advances in the computational techniques available for simulating biomolecules at the quantum-mechanical, atomistic and coarse-grained levels, there is still a widespread perception amongst the experimental community that these calculations are highly specialist and are not generally applicable by researchers outside the theoretical community. In this article, the successes and limitations of biomolecular simulation and the further developments that are likely in the near future are discussed. A brief overview is also provided of the experimental biophysical methods that are commonly used to probe biomolecular structure and dynamics, and the accuracy of the information that can be obtained from each is compared with that from modelling. It is concluded that progress towards an accurate spatial and temporal model of biomacromolecules requires a combination of all of these biophysical techniques, both experimental and computational.
PMCID: PMC4304696  PMID: 25615870
biomolecular simulation; computational techniques
7.  Limits of Structural Plasticity in a Picornavirus Capsid Revealed by a Massively Expanded Equine Rhinitis A Virus Particle 
Journal of Virology  2014;88(11):6093-6099.
The Picornaviridae family of small, nonenveloped viruses includes major pathogens of humans and animals. They have positive-sense, single-stranded RNA genomes, and the mechanism(s) by which these genomes are introduced into cells to initiate infection remains poorly understood. The structures of presumed uncoating intermediate particles of several picornaviruses show limited expansion and some increased porosity compared to the mature virions. Here, we present the cryo-electron microscopy structure of native equine rhinitis A virus (ERAV), together with the structure of a massively expanded ERAV particle, each at ∼17–Å resolution. The expanded structure has large pores on the particle 3-fold axes and has lost the RNA genome and the capsid protein VP4. The expanded structure thus illustrates both the limits of structural plasticity in such capsids and a plausible route by which genomic RNA might exit.
IMPORTANCE Picornaviruses are important animal and human pathogens that protect their genomic RNAs within a protective protein capsid. Upon infection, this genomic RNA must be able to leave the capsid to initiate a new round of infection. We describe here the structure of a unique, massively expanded state of equine rhinitis A virus that provides insight into how this exit might occur.
PMCID: PMC4093863  PMID: 24648455
8.  Reaction Mechanism of N-Acetylneuraminic Acid Lyase Revealed by a Combination of Crystallography, QM/MM Simulation, and Mutagenesis 
ACS Chemical Biology  2014;9(4):1025-1032.
N-Acetylneuraminic acid lyase (NAL) is a Class I aldolase that catalyzes the reversible condensation of pyruvate with N-acetyl-d-mannosamine (ManNAc) to yield the sialic acid N-acetylneuraminic acid (Neu5Ac). Aldolases are finding increasing use as biocatalysts for the stereospecific synthesis of complex molecules. Incomplete understanding of the mechanism of catalysis in aldolases, however, can hamper development of new enzyme activities and specificities, including control over newly generated stereocenters. In the case of NAL, it is clear that the enzyme catalyzes a Bi-Uni ordered condensation reaction in which pyruvate binds first to the enzyme to form a catalytically important Schiff base. The identity of the residues required for catalysis of the condensation step and the nature of the transition state for this reaction, however, have been a matter of conjecture. In order to address, this we crystallized a Y137A variant of the E. coli NAL in the presence of Neu5Ac. The three-dimensional structure shows a full length sialic acid bound in the active site of subunits A, B, and D, while in subunit C, discontinuous electron density reveals the positions of enzyme-bound pyruvate and ManNAc. These ‘snapshot’ structures, representative of intermediates in the enzyme catalytic cycle, provided an ideal starting point for QM/MM modeling of the enzymic reaction of carbon–carbon bond formation. This revealed that Tyr137 acts as the proton donor to the aldehyde oxygen of ManNAc during the reaction, the activation barrier is dominated by carbon–carbon bond formation, and proton transfer from Tyr137 is required to obtain a stable Neu5Ac-Lys165 Schiff base complex. The results also suggested that a triad of residues, Tyr137, Ser47, and Tyr110 from a neighboring subunit, are required to correctly position Tyr137 for its function, and this was confirmed by site-directed mutagenesis. This understanding of the mechanism and geometry of the transition states along the C–C bond-forming pathway will allow further development of these enzymes for stereospecific synthesis of new enzyme products.
PMCID: PMC4004234  PMID: 24521460
9.  Threonine 57 is required for the post-translational activation of Escherichia coli aspartate α-decarboxylase 
Threonine 57 is identified as the key residue required for the post-translational activation of E. coli aspartate decarboxylase. The crystal structure of the site-directed mutant T57V is reported.
Aspartate α-decarboxylase is a pyruvoyl-dependent decarboxylase required for the production of β-alanine in the bacterial pantothenate (vitamin B5) biosynthesis pathway. The pyruvoyl group is formed via the intramolecular rearrangement of a serine residue to generate a backbone ester intermediate which is cleaved to generate an N-terminal pyruvoyl group. Site-directed mutagenesis of residues adjacent to the active site, including Tyr22, Thr57 and Tyr58, reveals that only mutation of Thr57 leads to changes in the degree of post-translational activation. The crystal structure of the site-directed mutant T57V is consistent with a non-rearranged backbone, supporting the hypothesis that Thr57 is required for the formation of the ester intermediate in activation.
PMCID: PMC3975893  PMID: 24699660
aspartate decarboxylase; post-translational modification; amino-acid-derived cofactors; pyruvoyl-dependent
10.  Packaging signals in single-stranded RNA viruses: nature’s alternative to a purely electrostatic assembly mechanism 
Journal of Biological Physics  2013;39(2):277-287.
The formation of a protective protein container is an essential step in the life-cycle of most viruses. In the case of single-stranded (ss)RNA viruses, this step occurs in parallel with genome packaging in a co-assembly process. Previously, it had been thought that this process can be explained entirely by electrostatics. Inspired by recent single-molecule fluorescence experiments that recapitulate the RNA packaging specificity seen in vivo for two model viruses, we present an alternative theory, which recognizes the important cooperative roles played by RNA–coat protein interactions, at sites we have termed packaging signals. The hypothesis is that multiple copies of packaging signals, repeated according to capsid symmetry, aid formation of the required capsid protein conformers at defined positions, resulting in significantly enhanced assembly efficiency. The precise mechanistic roles of packaging signal interactions may vary between viruses, as we have demonstrated for MS2 and STNV. We quantify the impact of packaging signals on capsid assembly efficiency using a dodecahedral model system, showing that heterogeneous affinity distributions of packaging signals for capsid protein out-compete those of homogeneous affinities. These insights pave the way to a new anti-viral therapy, reducing capsid assembly efficiency by targeting of the vital roles of the packaging signals, and opens up new avenues for the efficient construction of protein nanocontainers in bionanotechnology.
PMCID: PMC3662417  PMID: 23704797
Virus assembly mechanism; RNA–protein interactions; Packaging signals; Fluorescence spectroscopy; Assembly models
11.  Sequence-Specific, RNA–Protein Interactions Overcome Electrostatic Barriers Preventing Assembly of Satellite Tobacco Necrosis Virus Coat Protein 
Journal of Molecular Biology  2013;425(6):1050-1064.
We have examined the roles of RNA–coat protein (CP) interactions in the assembly of satellite tobacco necrosis virus (STNV). The viral genomic RNA encodes only the CP, which comprises a β-barrel domain connected to a positively charged N-terminal extension. In the previous crystal structures of this system, the first 11 residues of the protein are disordered. Using variants of an RNA aptamer sequence isolated against the CP, B3, we have studied the sequence specificity of RNA-induced assembly. B3 consists of a stem–loop presenting the tetra-loop sequence ACAA. There is a clear preference for RNAs encompassing this loop sequence, as measured by the yield of T = 1 capsids, which is indifferent to sequences within the stem. The B3-containing virus-like particle has been crystallised and its structure was determined to 2.3 Å. A lower-resolution map encompassing density for the RNA has also been calculated. The presence of B3 results in increased ordering of the N-terminal helices located at the particle 3-fold axes, which extend by roughly one and a half turns to encompass residues 8–11, including R8 and K9. Under assembly conditions, STNV CP in the absence of RNA is monomeric and does not self-assemble. These facts suggest that a plausible model for assembly initiation is the specific RNA-induced stabilisation of a trimeric capsomere. The basic nature of the helical extension suggests that electrostatic repulsion between CPs prevents assembly in the absence of RNA and that this barrier is overcome by correct placement of appropriately orientated helical RNA stems. Such a mechanism would be consistent with the data shown here for assembly with longer RNA fragments, including an STNV genome. The results are discussed in light of a first stage of assembly involving compaction of the genomic RNA driven by multiple RNA packaging signal–CP interactions.
Graphical Abstract
► Sequence specificity in satellite virus assembly. ► RNA-induced conformational change in a viral CP. ► RNA binding overcomes an electrostatic barrier to CP assembly. ► RNAs containing multiple preferred stem–loops assemble more efficiently. ► Evidence supports the existence of packaging signals in single-stranded RNA genomes.
PMCID: PMC3593212  PMID: 23318955
STNV, satellite tobacco necrosis virus; CP, coat protein; ssRNA, single-stranded RNA; TCV, turnip crinkle virus; VLP, virus-like particle; svAUC, sedimentation velocity analytical ultracentrifugation; TEM, transmission electron microscopy; PDB, Protein Data Bank; PS, packaging signal; smFCS, single-molecule fluorescence correlation spectroscopy; virus assembly; RNA–protein interactions; molecular mechanism
12.  X-ray-excited optical luminescence of protein crystals: a new tool for studying radiation damage during diffraction data collection 
During X-ray irradiation protein crystals radiate energy in the form of small amounts of visible light. This is known as X-ray-excited optical luminescence (XEOL). The XEOL of several proteins and their constituent amino acids has been characterized using the microspectrophotometers at the Swiss Light Source and Diamond Light Source. XEOL arises primarily from aromatic amino acids, but the effects of local environment and quenching within a crystal mean that the XEOL spectrum of a crystal is not the simple sum of the spectra of its constituent parts. Upon repeated exposure to X-rays XEOL spectra decay non-uniformly, suggesting that XEOL is sensitive to site-specific radiation damage. However, rates of XEOL decay were found not to correlate to decays in diffracting power, making XEOL of limited use as a metric for radiation damage to protein crystals.
PMCID: PMC3370260  PMID: 22525748
13.  Isolation of an Asymmetric RNA Uncoating Intermediate for a Single-Stranded RNA Plant Virus 
Journal of Molecular Biology  2012;417(1-2):65-78.
We have determined the three-dimensional structures of both native and expanded forms of turnip crinkle virus (TCV), using cryo-electron microscopy, which allows direct visualization of the encapsidated single-stranded RNA and coat protein (CP) N-terminal regions not seen in the high-resolution X-ray structure of the virion. The expanded form, which is a putative disassembly intermediate during infection, arises from a separation of the capsid-forming domains of the CP subunits. Capsid expansion leads to the formation of pores that could allow exit of the viral RNA. A subset of the CP N-terminal regions becomes proteolytically accessible in the expanded form, although the RNA remains inaccessible to nuclease. Sedimentation velocity assays suggest that the expanded state is metastable and that expansion is not fully reversible. Proteolytically cleaved CP subunits dissociate from the capsid, presumably leading to increased electrostatic repulsion within the viral RNA. Consistent with this idea, electron microscopy images show that proteolysis introduces asymmetry into the TCV capsid and allows initial extrusion of the genome from a defined site. The apparent formation of polysomes in wheat germ extracts suggests that subsequent uncoating is linked to translation. The implication is that the viral RNA and its capsid play multiple roles during primary infections, consistent with ribosome-mediated genome uncoating to avoid host antiviral activity.
Graphical Abstract
► Native and expanded TCV cryo-electron microscopy structures reveal RNA dynamics. ► Expansion leads to the formation of pores large enough to allow exit of viral RNA. ► RNA remains resistant to nuclease, but capsid proteins become protease sensitive. ► Proteolysis causes initial extrusion of viral RNA, creating asymmetry in capsids. ► Subsequent ribosome binding prevents silencing and promotes genome extrusion.
PMCID: PMC4330335  PMID: 22306464
CP, coat protein; TCV, turnip crinkle virus; cryo-EM, cryo-electron microscopy; ssRNA, single-stranded RNA; TBSV, tomato bushy stunt virus; EDTA, ethylenediaminetetraacetic acid; 3-D, three-dimensional; VLP, virus-like particle; PDB, Protein Data Bank; CCC, cross-correlation coefficient; cryo-electron microscopy; TCV; ssRNA virus; genomic RNA structure; genome uncoating
14.  Structure of an Escherichia coli N-acetyl-d-neuraminic acid lyase mutant, E192N, in complex with pyruvate at 1.45 Å resolution 
Acta Crystallographica Section F  2009;65(Pt 11):1088-1090.
The 1.45 Å structure of E. coli N-acetyl-d-neuraminic acid lyase in complex with pyruvate in space group P212121 is reported from new low-salt crystallization conditions that will facilitate soaking experiments with substrates and inhibitors.
The structure of a mutant variant of Escherichia coli N-acetyl-d-neuraminic acid lyase (NAL), E192N, in complex with pyruvate has been determined in a new crystal form. It crystallized in space group P212121, with unit-cell parameters a = 78.3, b = 108.5, c = 148.3 Å. Pyruvate has been trapped in the active site as a Schiff base with the catalytic lysine (Lys165) without the need for reduction. Unlike the previously published crystallization conditions for the wild-type enzyme, in which a mother-liquor-derived sulfate ion is strongly bound in the catalytic pocket, the low-salt conditions described here will facilitate the determination of further E. coli NAL structures in complex with other active-site ligands.
PMCID: PMC2777032  PMID: 19923724
N-acetyl-d-neuraminic acid lyase; directed evolution; Schiff base; aldolases
15.  Structural Insight into Methyl-Coenzyme M Reductase Chemistry using Coenzyme B Analogues†,‡ 
Biochemistry  2010;49(35):7683-7693.
Methyl-coenzyme M reductase (MCR) catalyzes the final and rate-limiting step in methane biogenesis; the reduction of methyl-coenzyme M (methyl-SCoM) by coenzyme B (CoBSH) to methane and a heterodisulfide (CoBS-SCoM). Crystallographic studies show that the active site is deeply buried within the enzyme, and contains a highly reduced nickel-tetrapyrrole, coenzyme F430. Methyl-SCoM must enter the active site prior to CoBSH, as species derived from analogues of methyl-SCoM are always observed bound to the F430 nickel in the deepest part of the 30 Å long substrate channel that leads from the protein surface to the active site. The seven-carbon mercaptoalkanoyl chain of CoBSH binds within a 16 Å predominantly hydrophobic part of the channel close to F430, with the CoBSH thiolate lying closest to the nickel at a distance of 8.8 Å. It has previously been suggested that binding of CoBSH initiates catalysis by inducing a conformational change that moves methyl-SCoM closer to the nickel promoting cleavage of the C-S bond of methyl-SCoM. In order to better understand the structural role of CoBSH early in the MCR mechanism, we have determined crystal structures of MCR in complex with four different CoBSH analogues; pentanoyl-, hexanoyl-, octanoyl- and nonanoyl- derivatives of CoBSH (CoB5SH, CoB6SH, CoB8SH and CoB9SH respectively). The data presented here reveal that the shorter CoB5SH mercaptoalkanoyl chain overlays with that of CoBSH, but terminates two units short of the CoBSH thiolate position. In contrast, the mercaptoalkanoyl chain of CoB6SH adopts a different conformation, such that its thiolate is coincident with the position of the CoBSH thiolate. This is consistent with the observation that CoB6SH is a slow substrate. A labile water in the substrate channel was found to be a sensitive indicator for the presence of CoBSH and HSCoM. The longer CoB8SH and CoB9SH analogues can be accommodated in the active site through exclusion of this water. These analogues react with Ni(III)-methyl; a proposed MCR catalytic intermediate of methanogenesis. The CoB8SH thiolate is 2.6 Å closer to the nickel than that of CoBSH, but the additional carbon of CoB9SH only decreases the nickel thiolate distance a further 0.3 Å. Although the analogues did not induce any structural changes in the substrate channel, the thiolates appeared to preferentially bind at two distinct positions in the channel; one being the previously observed CoBSH thiolate position, and the other being at a hydrophobic annulus of residues that lines the channel proximal to the nickel.
PMCID: PMC3098740  PMID: 20707311
16.  Crystallization and preliminary X-ray analysis of a bifunctional catalase-phenol oxidase from Scytalidium thermophilum  
The bifunctional enzyme catalase-phenol oxidase from S. thermophilum was crystallized by the hanging-drop vapour-diffusion method in space group P21 and diffraction data were collected to 2.8 Å resolution.
Catalase-phenol oxidase from Scytalidium thermophilum is a bifunctional enzyme: its major activity is the catalase-mediated decomposition of hydrogen peroxide, but it also catalyzes phenol oxidation. To understand the structural basis of this dual functionality, the enzyme, which has been shown to be a tetramer in solution, has been purified by anion-exchange and gel-filtration chromatography and has been crystallized using the hanging-drop vapour-diffusion technique. Streak-seeding was used to obtain larger crystals suitable for X-ray analysis. Diffraction data were collected to 2.8 Å resolution at the Daresbury Synchrotron Radiation Source. The crystals belonged to space group P21 and contained one tetramer per asymmetric unit.
PMCID: PMC2675591  PMID: 19407383
Scytalidium thermophilum; Humicola insolens; catalases; phenol oxidases; catechol oxidases; CATPO
The Journal of biological chemistry  2007;282(24):17767-17776.
The accessibility of large substrates to buried enzymatic active sites is dependent upon the utilization of proteinaceous channels. The necessity of these channels in the case of small substrates is questionable as diffusion through the protein matrix is often assumed. Copper amine oxidases (CAOs) contain a buried protein-derived quinone cofactor and a mononuclear copper center that catalyze the conversion of two substrates, primary amines and molecular oxygen, to aldehydes and hydrogen peroxide respectively. The nature of molecular oxygen migration to the active site in the enzyme from Hansenula polymorpha2 (HPAO) is explored using a combination of kinetic, X-ray crystallographic and computational approaches. A crystal structure of HPAO in complex with xenon gas, which serves as an experimental probe for molecular oxygen binding sites, reveals buried regions of the enzyme suitable for transient molecular oxygen occupation. Calculated O2 free energy maps using CAO crystal structures in the absence of xenon, correspond well with later experimentally observed xenon sites in these systems, and allow the visualization of O2 migration routes of differing probabilities within the protein matrix. Site-directed mutagenesis designed to block individual routes has little effect on overall kcat/Km[O2], supporting multiple dynamic pathways for molecular oxygen to reach the active site.
PMCID: PMC3081669  PMID: 17409383
18.  Revealing low-dose radiation damage using single-crystal spectroscopy 
Journal of Synchrotron Radiation  2011;18(Pt 3):367-373.
Data on the rapid reduction of haem proteins in the X-ray beam at synchrotron sources are presented. The use of single-crystal spectroscopy to detect these changes and their implication for diffraction data collection from oxidized species is also discussed.
The structural information and functional insight obtained from X-ray crystallography can be enhanced by the use of complementary spectroscopies. Here the information that can be obtained from spectroscopic methods commonly used in conjunction with X-ray crystallography and best-practice single-crystal UV-Vis absorption data collection are briefly reviewed. Using data collected with the in situ system at the Swiss Light Source, the time and dose scales of low-dose X-ray-induced radiation damage and solvated electron generation in metalloproteins at 100 K are investigated. The effect of dose rate on these scales is also discussed.
PMCID: PMC3083913  PMID: 21525644
macromolecular crystallography; single-crystal microspectrophotometry; radiation damage; myoglobin; cytochrome c
19.  All Three Domains of the Hepatitis C Virus Nonstructural NS5A Protein Contribute to RNA Binding▿  
Journal of Virology  2010;84(18):9267-9277.
The hepatitis C virus (HCV) nonstructural protein NS5A is critical for viral genome replication and is thought to interact directly with both the RNA-dependent RNA polymerase, NS5B, and viral RNA. NS5A consists of three domains which have, as yet, undefined roles in viral replication and assembly. In order to define the regions that mediate the interaction with RNA, specifically the HCV 3′ untranslated region (UTR) positive-strand RNA, constructs of different domain combinations were cloned, bacterially expressed, and purified to homogeneity. Each of these purified proteins was probed for its ability to interact with the 3′ UTR RNA using filter binding and gel electrophoretic mobility shift assays, revealing differences in their RNA binding efficiencies and affinities. A specific interaction between domains I and II of NS5A and the 3′ UTR RNA was identified, suggesting that these are the RNA binding domains of NS5A. Domain III showed low in vitro RNA binding capacity. Filter binding and competition analyses identified differences between NS5A and NS5B in their specificities for defined regions of the 3′ UTR. The preference of NS5A, in contrast to NS5B, for the polypyrimidine tract highlights an aspect of 3′ UTR RNA recognition by NS5A which may play a role in the control or enhancement of HCV genome replication.
PMCID: PMC2937630  PMID: 20592076
20.  Structural Insights into Substrate Specificity in Variants of N-Acetylneuraminic Acid Lyase Produced by Directed Evolution 
Journal of Molecular Biology  2010;404(1):56-69.
The substrate specificity of Escherichia coli N-acetylneuraminic acid lyase was previously switched from the natural condensation of pyruvate with N-acetylmannosamine, yielding N-acetylneuraminic acid, to the aldol condensation generating N-alkylcarboxamide analogues of N-acetylneuraminic acid. This was achieved by a single mutation of Glu192 to Asn. In order to analyze the structural changes involved and to more fully understand the basis of this switch in specificity, we have isolated all 20 variants of the enzyme at position 192 and determined the activities with a range of substrates. We have also determined five high-resolution crystal structures: the structures of wild-type E. coli N-acetylneuraminic acid lyase in the presence and in the absence of pyruvate, the structures of the E192N variant in the presence and in the absence of pyruvate, and the structure of the E192N variant in the presence of pyruvate and a competitive inhibitor (2R,3R)-2,3,4-trihydroxy-N,N-dipropylbutanamide. All structures were solved in space group P21 at resolutions ranging from 1.65 Å to 2.2 Å. A comparison of these structures, in combination with the specificity profiles of the variants, reveals subtle differences that explain the details of the specificity changes. This work demonstrates the subtleties of enzyme–substrate interactions and the importance of determining the structures of enzymes produced by directed evolution, where the specificity determinants may change from one substrate to another.
PMCID: PMC3014015  PMID: 20826162
NAL, N-acetylneuraminic acid lyase; ManNAc, N-acetylmannosamine; Neu5Ac, N-acetylneuraminic acid; DPAH, (5R,6R)-7-(dipropylamino)-4,5,6-trihydroxy-2,7-dioxoheptanoic acid; DHOB, (2R,3S)-2,3-dihydroxy-4-oxo-N,N-dipropylbutanamide; THB, (2R,3R)-2,3,4-trihydroxy-N,N-dipropylbutanamide; PDB, Protein Data Bank; PEG, polyethylene glycol; directed evolution; X-ray crystallography; N-acetylneuraminic acid lyase; substrate specificity; protein engineering
21.  Exploring the Roles of the Metal Ions in Escherichia coli Copper Amine Oxidase†,‡ 
Biochemistry  2010;49(6):1268-1280.
To investigate the role of the active site copper in Escherichia coli copper amine oxidase (ECAO), we initiated a metal-substitution study. Copper reconstitution of ECAO (Cu-ECAO) restored only ∼12% wild-type activity as measured by kcat(amine). Treatment with EDTA, to remove exogenous divalent metals, increased Cu-ECAO activity but reduced the activity of wild-type ECAO. Subsequent addition of calcium restored wild-type ECAO and further enhanced Cu-ECAO activities. Cobalt-reconstituted ECAO (Co-ECAO) showed lower but significant activity. These initial results are consistent with a direct electron transfer from TPQ to oxygen stabilized by the metal. If a Cu(I)-TPQ semiquinone mechanism operates, then an alternative outer-sphere electron transfer must also exist to account for the catalytic activity of Co-ECAO. The positive effect of calcium on ECAO activity led us to investigate the peripheral calcium binding sites of ECAO. Crystallographic analysis of wild-type ECAO structures, determined in the presence and absence of EDTA, confirmed that calcium is the normal ligand of these peripheral sites. The more solvent exposed calcium can be easily displaced by mono- and divalent cations with no effect on activity, whereas removal of the more buried calcium ion with EDTA resulted in a 60−90% reduction in ECAO activity and the presence of a lag phase, which could be overcome under oxygen saturation or by reoccupying the buried site with various divalent cations. Our studies indicate that binding of metal ions in the peripheral sites, while not essential, is important for maximal enzymatic activity in the mature enzyme.
PMCID: PMC2817917  PMID: 20052994
22.  A comparative analysis of the fluorescence properties of the wild-type and active site mutants of the hepatitis C virus autoprotease NS2-3 
Biochimica et Biophysica Acta  2010;1804(1):212-222.
Hepatitis C virus encodes an autoprotease, NS2-3, which is required for processing of the viral polyprotein between the non-structural NS2 and NS3 proteins. This protease activity is vital for the replication and assembly of the virus and therefore represents a target for the development of anti-viral drugs. The mechanism of this auto-processing reaction is not yet clear but the protease activity has been shown to map to the C-terminal region of NS2 and the N-terminal serine protease region of NS3. The NS2-3 precursor can be expressed in Escherichia coli as inclusion bodies, purified as denatured protein and refolded, in the presence of detergents and the divalent metal ion zinc, into an active form capable of auto-cleavage. Here, intrinsic tryptophan fluorescence has been used to assess refolding in the wild-type protein and specific active site mutants. We also investigate the effects on protein folding of alterations to the reaction conditions that have been shown to prevent auto-cleavage. Our data demonstrate that these active site mutations do not solely affect the cleavage activity of the HCV NS2-3 protease but significantly affect the integrity of the global protein fold.
PMCID: PMC2824112  PMID: 19853065
HCV, Hepatitis C virus; NS, non-structural; JFH-1, Japanese fulminant hepatitis C virus genotype 2a isolate; WT, wild-type; GdnHCl, guanidine hydrochloride; DTT, dithiothreitol; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; CD, circular dichroism; Hepatitis C virus; NS2-3 autoprotease; Mutagenesis; Refolding; Tryptophan fluorescence; Acrylamide quenching
23.  A new on-axis multimode spectrometer for the macromolecular crystallography beamlines of the Swiss Light Source 
Journal of Synchrotron Radiation  2009;16(Pt 2):173-182.
Complementary techniques greatly aid the interpretation of macromolecule structures to yield functional information, and can also help to track radiation-induced changes. A new on-axis spectrometer being integrated into the macromolecular crystallography beamlines of the Swiss Light Source is presented.
X-ray crystallography at third-generation synchrotron sources permits tremendous insight into the three-dimensional structure of macromolecules. Additional information is, however, often required to aid the transition from structure to function. In situ spectroscopic methods such as UV–Vis absorption and (resonance) Raman can provide this, and can also provide a means of detecting X-ray-induced changes. Here, preliminary results are introduced from an on-axis UV–Vis absorption and Raman multimode spectrometer currently being integrated into the beamline environment at X10SA of the Swiss Light Source. The continuing development of the spectrometer is also outlined.
PMCID: PMC2651763  PMID: 19240329
single-crystal microspectrophotometry; kinetic crystallography; structural enzymology; radiation damage; macromolecular crystallography; complementary techniques
24.  Structure of a xenon derivative of Escherichia coli copper amine oxidase: confirmation of the proposed oxygen-entry pathway 
The structure of a xenon derivative of E. coli copper amine oxidase confirms the pathway of oxygen entry to the buried active site proposed for this class of enzymes.
The mechanism of molecular oxygen entry into the buried active site of the copper amine oxidase family has been investigated in several family members using biochemical, structural and in silico methods. These studies have revealed a structurally conserved β-sandwich which acts as a hydrophobic reservoir from which molecular oxygen can take several species-specific preferred pathways to the active site. Escherichia coli copper amine oxidase (ECAO) possesses an extra N-terminal domain that lies close to one entrance to the β-sandwich. In order to investigate whether the presence of this domain alters molecular oxygen entry in this enzyme, xenon was used as a molecular oxygen binding-site probe. The resulting 2.5 Å resolution X-ray crystal structure reveals xenon bound in similar positions to those observed in xenon-derivative crystal structures of other family members, suggesting that the N-terminal domain does not affect oxygen entry and that the E. coli enzyme takes up oxygen in a similar manner to the rest of the copper amine oxidase family.
PMCID: PMC2593700  PMID: 19052360
copper amine oxidase; xenon derivatives; oxygen entry
25.  The crystal structure of cytochrome P460 of Nitrosomonas europaea reveals a novel cytochrome fold and heme-protein cross-link† 
Biochemistry  2007;46(28):8340-8349.
We have determined the 1.8 Å X-ray crystal structure of a mono-heme c-type cytochrome, cytochrome P460, from Nitrosomonas europea. The chromophore possesses unusual spectral properties analogous to those of the catalytic heme P460 of hydroxylamine oxidoreductase (HAO), the only known heme in biology to withdraw electrons from substrate coordinated to the iron. The analysis reveals a homodimeric structure and elucidates a new c-type cytochrome fold that is predominantly β-sheet. In addition to the two cysteine thioether links to the porphyrin typical of c-type hemes, there is a third proteinaceous link involving a conserved lysine. The covalent bond is between the lysine side-chain nitrogen and the 13′-meso carbon of the heme, which following cross-link formation is sp3 hybridized demonstrating loss of conjugation at this position within the porphyrin. The structure has implications for the analogous tyrosine-heme meso carbon cross-link observed in HAO.
PMCID: PMC2527454  PMID: 17583915

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