Search PubMed⌕ Search

Biomedical subjects

Elspeth Garman

Publications and source records attributed to Elspeth Garman.

At least 19 recordsLinked to original sources
PubMed2007

Crystal structure and function of the zinc uptake regulator FurB from Mycobacterium tuberculosis.

Members of the ferric/zinc uptake regulator (Fur/Zur) family are the central metal-dependent regulator proteins in many Gram-negative and -positive bacteria. They are responsible for the control of a wide variety of basic physiological processes and the expression of important virulence factors in human pathogens. Therefore, Fur has gathered significant interest as a potential target for novel antibiotics. Here we report the crystal structure of FurB from Mycobacterium tuberculosis at a resolution of 2.7A, and we present biochemical and spectroscopic data that allow us to propose the functional role of this protein. Although the overall fold of FurB with an N-terminal DNA binding domain and a C-terminal dimerization domain is conserved among the Zur/Fur family, large differences in the spatial arrangement of the two domains with respect to each other can be observed. The biochemical and spectroscopic analysis presented here reveals that M. tuberculosis FurB is Zn(II)-dependent and is likely to control genes involved in the bacterial zinc uptake. The combination of the structural, spectroscopic, and biochemical results enables us to determine the structural basis for functional differences in this important family of bacterial regulators.

Bacterial Proteins↗
PubMed2007

Cryocrystallography of macromolecules: practice and optimization.

Techniques for flash-cooling protein crystals to around 100K (-173 degrees C) for data collection have developed enormously in the last decade, to the extent that cryocrystallography is now standard practice. The main advantage of these methods is the vastly reduced rate of radiation damage to protein crystals in the X-ray beam at cryogenic temperatures over room temperature, extending their lifetimes so that complete datasets can be collected from a single crystal. The practical application of the techniques has become somewhat anecdotal and rather fixed within individual laboratories. This chapter gives step-by-step guidelines for flash-cooling crystals and some of the rationale for the recommended procedures. Optimization of the entire cryoprotocol can give substantial improvements to both the resolution and quality of the data, often resulting in more straight-forward structure solution and subsequent model refinement. Attention to seemingly insignificant details can have a real impact on the usefulness of the final dataset, and are thus worth addressing.

Cryoprotective Agents↗
PubMed2007

X-ray data collection from macromolecular crystals.

Instruments, methods, and software for modern macromolecular crystallography is becoming so effective that molecular biologists often can solve structures from their crystals by working "without a license." In this chapter, the authors attempt to demystify some of the apparatus and techniques by providing a roadmap. Current methods for collecting X-ray diffraction data from macromolecular crystals are described. The principles of operation of the required X-ray sources, optics, goniometers, and detectors are outlined, and a typical data collection protocol is presented. Optimization of data quality is a pivotal stage in the whole crystallographic process, so much attention is given to the detailed setting up of the experiment. This is followed by a summary of the basic ideas behind the diffraction image-processing packages and their application to data reduction. Despite the increasingly "black box" nature of these computer programs, understanding how they extract the intensities, errors, and indices from the data can make subsequent structure solution and refinement much easier.

Crystallization↗
PubMed2006

HDAC6-p97/VCP controlled polyubiquitin chain turnover.

HDAC6 is a unique cytoplasmic deacetylase capable of interacting with ubiquitin. Using a combination of biophysical, biochemical and biological approaches, we have characterized the ubiquitin-binding domain of HDAC6, named ZnF-UBP, and investigated its biological functions. These studies show that the three Zn ion-containing HDAC6 ZnF-UBP domain presents the highest known affinity for ubiquitin monomers and mediates the ability of HDAC6 to negatively control the cellular polyubiquitin chain turnover. We further show that HDAC6-interacting chaperone, p97/VCP, dissociates the HDAC6-ubiquitin complexes and counteracts the ability of HDAC6 to promote the accumulation of polyubiquitinated proteins. We propose that a finely tuned balance of HDAC6 and p97/VCP concentrations determines the fate of ubiquitinated misfolded proteins: p97/VCP would promote protein degradation and ubiquitin turnover, whereas HDAC6 would favour the accumulation of ubiquitinated protein aggregates and inclusion body formation.

3T3 Cells↗
PubMed2006

The structures of frataxin oligomers reveal the mechanism for the delivery and detoxification of iron.

Defects in the mitochondrial protein frataxin are responsible for Friedreich ataxia, a neurodegenerative and cardiac disease that affects 1:40,000 children. Here, we present the crystal structures of the iron-free and iron-loaded frataxin trimers, and a single-particle electron microscopy reconstruction of a 24 subunit oligomer. The structures reveal fundamental aspects of the frataxin mechanism. The trimer has a central channel in which one atom of iron binds. Two conformations of the channel with different metal-binding affinities suggest that a gating mechanism controls whether the bound iron is delivered to other proteins or transferred to detoxification sites. The trimer constitutes the basic structural unit of the 24 subunit oligomer. The architecture of this oligomer and several features of the trimer structure demonstrate striking similarities to the iron-storage protein ferritin. The data reveal how stepwise assembly provides frataxin with the structural flexibility to perform two functions: metal delivery and detoxification.

Amino Acid Sequence↗
PubMed2005

"Zn-link": a metal-sharing interface that organizes the quaternary structure and catalytic site of the endoribonuclease, RNase E.

Ribonuclease E is an essential hydrolytic endonuclease in Escherichia coli, and it plays a central role in maintaining the balance and composition of the messenger RNA population. The enzyme is also required for rRNA and tRNA processing. We have shown earlier that the highly conserved catalytic domain of E. coli RNase E is a homotetramer [Callaghan, A. J. et al. (2003) Biochemistry 42, 13848-13855]. Here, we report that this quaternary organization requires zinc. Two protomers share a single zinc ion, and quantitative analysis indicates that each protein contributes two cysteine thiols toward the coordination of the metal. The candidate cysteines are part of a motif that is conserved in the RNase E protein family, and mutation of these residues causes the partial loss of zinc, the complete disruption of the tetramer into dimers, and effective catalytic inactivation. However, these mutations do not affect RNA binding. The tetramer can be artificially maintained by disulfide bond formation, which fully displaces the zinc but largely preserves the catalytic activity. Thus, catalytic activity does not require zinc directly but does require the quaternary structure, for which the metal is essential. We propose that the RNase E tetramer has two nonequivalent subunit interfaces, one of which is mediated by a single, tetrathiol-zinc complex, which we refer to as a "Zn-link" motif. One or both interfaces organize the active site, which is distinct from the primary site of RNA binding.

Amino Acid Motifs↗
PubMed2005

A new method for predetermining the diffraction quality of protein crystals: using SOAP as a selection tool.

A microscope for quantitative analysis of the birefringence properties of samples is introduced. The microscope is used to measure variations in the slow optical axis position (SOAP) across hen egg-white lysozyme, glucose isomerase and fibronectin crystals. By comparing these variations with indicators of diffraction quality, it is shown that the optical properties of a protein crystal provide a non-invasive method of determining crystal diffraction quality before any X-ray data collection is attempted.

Protein Conformation↗
PubMed2004

Crystallization, microPIXE and preliminary crystallographic analysis of the complex between the third KH domain of hnRNP K and single-stranded DNA.

hnRNP K is one of the major proteins found in hnRNP particles which are ribonucleoprotein complexes containing proteins and pre-mRNA. hnRNP K contains hnRNP K homology (KH) domains which bind both CT-rich single-stranded DNA (ssDNA) and CU-rich ssRNA. Co-crystallization of the third KH domain of human hnRNP K with a 15-mer ssDNA gave rod-shaped crystals belonging to the trigonal space group P3(1)21 (unit-cell parameters a = 54.0, c = 149.7 A) and diffracting to 2.4 A resolution. MicroPIXE (proton-induced X-ray emission) experiments showed that the crystals contained the complex and that the phosphorus to sulfur atomic ratio was consistent with the asymmetric unit containing three KH3 domains per 15-mer ssDNA. This was confirmed by structure solution by molecular replacement.

Cloning, Molecular↗
PubMed2004

Controlling influenza by inhibiting the virus's neuraminidase.

Despite the fact that influenza is a disease which affects millions of people, sometimes with fatal consequences. there has not, until recently, been any drug effective against all strains. Vaccines may be relatively or totally ineffective, so drugs are needed. Random screening of many thousands of compounds by pharmaceutical companies has resulted in only two compounds, amantadine and rimantidine, which target the M2 ion channel on the virus. These drugs have major disadvantages. Knowledge of the crystal structure of influenza virus neuraminidase, on the other hand, has allowed the rational design of four "plug-drugs" which bind to the active site of flu neuraminidase and stop replication of the virus. Two of these compounds. Relenza and Tamiflu, are now being used worldwide and, although effective when used properly, suffer from problems of delivery. They need to be given very soon after infection to be effective, they only inhibit the influenza virus and none of the other respiratory agents which cause flu-like symptoms, and they are very expensive.

Animals↗
PubMed2003

Glucocorticoid receptor-like Zn(Cys)4 motifs in BslI restriction endonuclease.

BslI restriction endonuclease cleaves the symmetric sequence CCN(7)GG (where N=A, C, G or T). The enzyme is composed of two subunits, alpha and beta, that form a heterotetramer (alpha(2)beta(2)) in solution. The alpha subunit is believed to be responsible for DNA recognition, while the beta subunit is thought to mediate cleavage. Here, for the first time, we provide experimental evidence that BslI binds Zn(II). Specifically, using X-ray absorption spectroscopic analysis we show that the alpha subunit of BslI contains two Zn(Cys)(4)-type zinc motifs similar to those in the DNA-binding domain of the glucocorticoid receptor. This conclusion is supported by genetic analysis of the zinc-binding motifs, whereby amino acid substitutions in the zinc finger motifs are demonstrated to abolish or impair cleavage activity. An additional putative zinc-binding motif was identified in the beta subunit, consistent with the X-ray absorption data. These data were corroborated by proton induced X-ray emission measurements showing that full BslI contains at least three fully occupied Zn sites per alpha/beta heterodimer. On the basis of these data, we propose a role for the BslI Zn motifs in protein-DNA as well as protein-protein interactions.

Binding Sites↗
PubMed2003

Heavy-atom derivatization.

Most of the standard methods of solving macromolecular structures involve producing a protein crystal that is derivatized by an anomalous scatterer or heavy atom (MIR, SIRAS, MAD, SAD etc.). The theoretical methodology which underpins the extraction of phase information from such derivatives is widely available in the literature. In addition, there are comprehensive sources of information on the chemistry of heavy-atom compounds and the ligands with which they are known to interact, as well as the Heavy Atom Databank accessible on the World Wide Web. This contribution therefore aims to provide some information on the less well documented practical problems of firstly deciding on an overall strategy for derivatization and secondly performing the physical manipulations involved in producing heavy-atom derivatives from native protein crystals and then cryocooling them. Ways to optimize the chances of isomorphous unit cells are suggested. Methods of determining whether or not the heavy atom is bound are outlined, including the powerful technique of PIXE (particle-induced X-ray emission).

Crystallization↗
PubMed2003

The three-dimensional structure of the N-acetylglucosamine-6-phosphate deacetylase, NagA, from Bacillus subtilis: a member of the urease superfamily.

The enzyme N-acetylglucosamine-6-phosphate deacetylase, NagA, catalyzes the hydrolysis of the N-acetyl group of GlcNAc-6-P to yield glucosamine 6-phosphate and acetate, the first committed step in the biosynthetic pathway to amino-sugar-nucleotides. It is classified into carbohydrate esterase family CE-9 (see afmb.cnrs-mrs.fr/CAZY/). Here we report the cloning, expression, and three-dimensional structure (Protein Data Bank code 1un7) determination by x-ray crystallography of the Bacillus subtilis NagA at a resolution of 2.0 A. The structure presents two domains, a (beta/alpha)(8) barrel enclosing the active center and a small beta barrel domain. The structure is dimeric, and the substrate phosphate coordination at the active center is provided by an Arg/His pair contributed from the second molecule of the dimer. Both the overall structure and the active center bear a striking similarity to the urease superfamily with two metals involved in substrate binding and catalysis. PIXE (Proton-Induced x-ray Emission) data show that iron is the predominant metal in the purified protein. We propose a catalytic mechanism involving proton donation to the leaving group by aspartate, nucleophilic attack by an Fe-bridged hydroxide, and stabilization of the carbonyl oxygen by one of the two Fe atoms of the pair. We believe that this is the first sugar deacetylase to utilize this fold and catalytic mechanism.

Amidohydrolases↗
PubMed2003

'Cool' crystals: macromolecular cryocrystallography and radiation damage.

Macromolecular crystals commonly suffer rapid radiation damage during room temperature X-ray data collection. Therefore, data are now routinely collected with the sample held at around 100K, significantly reducing secondary radiation damage, and usually resulting in higher resolution and better quality data. At synchrotron sources, the frequent observation of radiation damage even at cryotemperatures has prompted the development of exciting new experiments aimed at characterising and reducing this damage, and using it for structure determination and enzymatic studies. Current research into cryotechniques seeks to understand the basic physical and chemical processes involved in flash-cooling and radiation damage, which should eventually enable the rational optimisation of cryoprotocols.

Algorithms↗
PubMed2003

Architecture of a protein central to iron homeostasis: crystal structure and spectroscopic analysis of the ferric uptake regulator.

Iron is an essential element for almost all organisms, although an overload of this element results in toxicity because of the formation of hydroxyl radicals. Consequently, most living entities have developed sophisticated mechanisms to control their intracellular iron concentration. In many bacteria, including the opportunistic pathogen Pseudomonas aeruginosa, this task is performed by the ferric uptake regulator (Fur). Fur controls a wide variety of basic physiological processes including iron uptake systems and the expression of exotoxin A. Here, we present the first crystal structure of Fur from P. aeruginosa in complex with Zn2+ determined at a resolution of 1.8 A. Furthermore, X-ray absorption spectroscopic measurements and microPIXE analysis were performed in order to characterize the distinct zinc and iron binding sites in solution. The combination of these complementary techniques enables us to present a model for the activation and DNA binding of the Fur protein.

Amino Acid Sequence↗
PubMed2002

Investigation of possible free-radical scavengers and metrics for radiation damage in protein cryocrystallography.

The introduction of highly intense wiggler and undulator beamlines has reintroduced the problem of X-ray radiation damage in protein crystals even at cryogenic temperatures. Several metrics for monitoring radiation damage are considered and unit-cell volume expansion is systematically investigated using crystals of three different types, but it is found to be too variable to be a useful metric. Radical scavengers of secondary radiation damage are investigated as possible mitigating agents. Styrene is found to be ineffective. A method of spectroscopically measuring the radiation damage with a microspectrophotometer was used and, in conjunction with crystallographic data, provided tentative but suggestive evidence for the efficacy of ascorbate as a free-radical scavenging agent in cryocooled hen egg-white lysozyme crystals.

Animals↗
PubMed2002

Blu-Ice and the Distributed Control System: software for data acquisition and instrument control at macromolecular crystallography beamlines.

The Blu-Ice and Distributed Control System (DCS) software packages were developed to provide unified control over the disparate hardware resources available at a macromolecular crystallography beamline. Blu-Ice is a user interface that provides scientific experimenters and beamline support staff with intuitive graphical tools for collecting diffraction data and configuring beamlines for experiments. Blu-Ice communicates with the hardware at a beamline via DCS, an instrument-control and data-acquisition package designed to integrate hardware resources in a highly heterogeneous networked computing environment. Together, Blu-Ice and DCS provide a flexible platform for increasing the ease of use, the level of automation and the remote accessibility of beamlines. Blu-Ice and DCS are currently installed on four Stanford Synchrotron Radiation Laboratory crystallographic beamlines and are being implemented at sister light sources.

Crystallography, X-Ray↗