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The bacterium, L. pneumophila, causes a severe form of pneumonia known as Legionnaires' disease. The pathogenicity of this bacterium depends on the secretion of over 300 effector proteins into the host. One such effector, WipA, drew attention for its reliance on a chaperone complex (needed to ensure protein functionality) for its secretion and its unknown role in pathogenicity. Two of the Macromolecular Crystallography (MX) beamlines (I02 and I04) at Diamond Light Source were used to initiate the study and solve the crystal structure of a large fragment of WipA. The structure showed that the protein possessed a serine/threonine phosphatase fold that surprisingly targeted tyrosine-phoshorylated peptides. Additionally, it was hypothesised that WipA could transition from a homo-dimeric state to a hetero-dimeric state to interact with a tyrosine-phosphorylated host target. The discoveries will help to clarify the molecular mechanisms underpinning Legionella pathogenicity and could aid the development of inhibitors. Legionnaires' disease can be lethal among vulnerable members of a population, and is particularly dangerous during hospital outbreaks. One of the main sources of Legionella infection is from water vapour in air-conditioning systems, boilers, hot baths and showers. Once a host breathes in the infected vapour, the bacteria enter the lungs and seek out white blood cells, known as macrophages. The bacteria become engulfed by the macrophages, but instead of being killed by them, the bacteria cleverly employ a type IVb secretion system to secrete a huge array of effector proteins into the host cell in order to highjack the host's cellular machinery and ensure bacterial survival. The major fragment of WipA was successfully crystallised by removing both termini of the sequence that predicted unstructured regions. The crystals were initially analysed at Diamond's I02 and I04 beamlines, while the work was completed at the PetraIII P13 beamline at the European Molecular Biology Laboratory in Hamburg. At Diamond, the Microfocus MX beamline (I04) was used for crystal screening and I02 was used for collecting diffraction data. Dr Nikos Pinotsis is a post-doctoral researcher in structural and molecular biology in the laboratory of Professor Gabriel Waksman at the Institute of Structural and Molecular Biology at Birkbeck/UCL and co-investigator of the study. He explained their approach, "Even though the size of the WipA crystals was sufficient for single crystal diffraction experiments, most of them displayed multiple and/or disordered lattices, therefore extensive screening was critical in successfully acquiring high resolution and quality data. We optimised our data-collection strategies to a beam size that fitted sufficiently the shorter of the crystal dimensions and optimised the beam by maximising beam and exposure time while minimising radiation damage." First of its class The structure of the WipA fragment exhibited a phosphatase fold mounted on a helical hairpin, which was the first of its class. The core of this structure resembled a serine/threonine protein phosphatase, but biochemical experiments unexpectedly showed that it had a preference for tyrosine-phosphorylated substrates. This surprising observation had only been noted once before among several hundred similar phosphatases. Dr Pinotsis explained the relevance of the findings: "Once the host target for dephosphorylation by WipA is known, inhibitors could be designed to inhibit that interaction. While it is possible that these findings might lead to the design of antibacterials, they mostly contribute to a general understanding of a bacterium's survival and pathogenicity which is a very important first step to establish long-lasting treatments against infections." The team plans to identify the WipA target within host cells and will continue to explore further protein-protein interactions to provide a more complete view of the infection mechanism of Legionella. Explore further: 3-D image of bacterial machine that injects toxins into cells and spreads antibiotic resistance More information: Nikos Pinotsis et al. Structure of the WipA protein reveals a novel tyrosine protein phosphatase effector from Legionella pneumophila, Journal of Biological Chemistry (2017). DOI: 10.1074/jbc.M117.781948


Clark A.R.,Institute of Structural and Molecular Biology | Lubsen N.H.,Radboud University Nijmegen | Slingsby C.,Institute of Structural and Molecular Biology
International Journal of Biochemistry and Cell Biology | Year: 2012

α-Crystallin, a major component of the eye lens cytoplasm, is a large multimer formed from two members of the small heat shock protein (sHsp) family. Inherited crystallin mutations are a common cause of childhood cataract, whereas miscellaneous changes to the long-lived crystallins cause age-related cataract, the most common cause of blindness worldwide. Newly formed eye lens cells use proteostasis to deal with the consequences of mutations, whereas mature lens cells, devoid of the ATP-driven folding and degradation machines, are hypothesized to have the α-crystallin "holdase" chaperone function to prevent protein aggregation. We discuss the impact of truncating and missense mutations on α-crystallin, based on recent progress towards determining sHsp 3D structure. Dominant missense mutations to the "α-crystallin domain" of αA- (HSPB4) or αB-crystallin (HSPB5) occur on residues predicted to facilitate domain dynamics. αB-Crystallin is also expressed in striated muscle and mutations cause myopathy. The impact on these cellular cytoplasms is compared where sHsp multimer partners and metabolic constraints are different. Selected inherited mutations of the lens β- and γ-crystallins are considered in the context of their possible dependence on the "holdase" chaperone function of α-crystallin. Looking at discrete changes to specific crystallin polypeptide chains that can function as chaperone or substrate provide insights into the workings of a cytoplasmic proteostatic system. These observations provide a framework for validating the function of α-crystallin as a chaperone, or as a lens space filler adapted from a chaperone function. Understanding the mechanistic role of α-crystallins will aid progress in research into age-related cataract and adult-onset myopathy. This article is part of a Directed Issue entitled: Small HSPs in physiology and pathology.


Slingsby C.,Institute of Structural and Molecular Biology | Wistow G.J.,U.S. National Institutes of Health
Progress in Biophysics and Molecular Biology | Year: 2014

The vertebrate lens evolved to collect light and focus it onto the retina. In development, the lens grows through massive elongation of epithelial cells possibly recapitulating the evolutionary origins of the lens. The refractive index of the lens is largely dependent on high concentrations of soluble proteins called crystallins. All vertebrate lenses share a common set of crystallins from two superfamilies (although other lineage specific crystallins exist). The α-crystallins are small heat shock proteins while the β- and γ-crystallins belong to a superfamily that contains structural proteins of uncertain function. The crystallins are expressed at very high levels in lens but are also found at lower levels in other cells, particularly in retina and brain. All these proteins have plausible connections to maintenance of cytoplasmic order and chaperoning of the complex molecular machines involved in the architecture and function of cells, particularly elongated and post-mitotic cells. They may represent a suite of proteins that help maintain homeostasis in such cells that are at risk from stress or from the accumulated insults of aging. © 2014 Elsevier Ltd.


Maurer S.P.,Cancer Research UK Research Institute | Maurer S.P.,Cell Biology and Biophysics Unit | Fourniol F.J.,Cancer Research UK Research Institute | Bohner G.,Cancer Research UK Research Institute | And 3 more authors.
Cell | Year: 2012

Growing microtubule ends serve as transient binding platforms for essential proteins that regulate microtubule dynamics and their interactions with cellular substructures. End-binding proteins (EBs) autonomously recognize an extended region at growing microtubule ends with unknown structural characteristics and then recruit other factors to the dynamic end structure. Using cryo-electron microscopy, subnanometer single-particle reconstruction, and fluorescence imaging, we present a pseudoatomic model of how the calponin homology (CH) domain of the fission yeast EB Mal3 binds to the end regions of growing microtubules. The Mal3 CH domain bridges protofilaments except at the microtubule seam. By binding close to the exchangeable GTP-binding site, the CH domain is ideally positioned to sense the microtubule's nucleotide state. The same microtubule-end region is also a stabilizing structural cap protecting the microtubule from depolymerization. This insight supports a common structural link between two important biological phenomena, microtubule dynamic instability and end tracking. © 2012 Elsevier Inc.


Waksman G.,Institute of Structural and Molecular Biology | Orlova E.V.,Institute of Structural and Molecular Biology
Current Opinion in Microbiology | Year: 2014

Type IV secretion (T4S) systems are large dynamic nanomachines that transport DNAs and/or proteins through the membranes of bacteria. Because of their complexity and multi-protein organisation, T4S systems have been extremely challenging to study structurally. However in the past five years significant milestones have been achieved by X-ray crystallography and cryo-electron microscopy. This review describes some of the more recent advances: the structures of some of the protein components of the T4S systems and the complete core complex structure that was determined using electron microscopy. © 2013 The Authors.


Dumoux M.,Birkbeck, University of London | Clare D.K.,Institute of Structural and Molecular Biology | Saibil H.R.,Institute of Structural and Molecular Biology | Hayward R.D.,Birkbeck, University of London
Traffic | Year: 2012

Chlamydiae are obligate intracellular bacterial pathogens that replicatewithin a specialized membrane-bound compartment, termed an 'inclusion'. The inclusion membrane is a critical host-pathogen interface, yet the extent of its interaction with cellular organelles and the origin of this membrane remain poorly defined. Here we show that the host endoplasmic reticulum (ER) is specifically recruited to the inclusion, and that key rough ER (rER) proteins are enriched on and translocated into the inclusion. rER recruitment is a Chlamydia-orchestrated process that occurs independently of host trafficking. Generation of infectious progeny requires an intact ER, since ER vacuolation early during infection stalls inclusion development, whereas disruption post ER recruitment bursts the inclusion. Electron tomography and immunolabelling of Chlamydia-infected cells reveal 'pathogen synapses' at which ordered arrays of chlamydial type III secretion complexes connect to the inclusion membrane only at rER contact sites. Our data show a supramolecular assembly involved in pathogen hijack of a key host organelle. © 2012 John Wiley & Sons A/S.


Moores C.A.,Institute of Structural and Molecular Biology
Cell Cycle | Year: 2010

The microtubule-based mitotic spindle orchestrates chromosome segregation, facilitated by many microtubule-associated proteins. Kinesin-5 proteins are important components of the cell division machinery, and are involved in generation of mitotic spindle bipolarity by cross-linking microtubules. Kinesin-5s are members of the ATP- and MT-dependent kinesin superfamily of molecular motors. Human kinesin-5 is also a target for small molecule inhibitors that specifically bind to the motor domain and are currently in cancer clinical trials. The regulatory mechanisms that control kinesin-5 activity during mitosis and the effects of regulation on the kinesin-5-microtubule interaction remain unknown. Recent work has focused on a kinesin-5 specific region within the motor domain, loop5, as a potential regulatory switch. Kinesin-5-specific small molecule inhibitors bind beneath loop5, loop5 is rearranged when kinesin-5 binds to microtubules and residues adjacent to loop5 are subject to cell cycle-dependent tyrosine phosphorylation which could affect its conformation. It will be essential to consider these studies, which shed light on potential kinesin-5 regulatory mechanisms, as part of efforts to develop clinically effective kinesin-5 inhibitors. © 2010 Landes Bioscience.


Slingsby C.,Institute of Structural and Molecular Biology | Wistow G.J.,U.S. National Institutes of Health | Clark A.R.,Institute of Structural and Molecular Biology
Protein Science | Year: 2013

The camera eye lens of vertebrates is a classic example of the re-engineering of existing protein components to fashion a new device. The bulk of the lens is formed from proteins belonging to two superfamilies, the α-crystallins and the βγ-crystallins. Tracing their ancestry may throw light on the origin of the optics of the lens. The α-crystallins belong to the ubiquitous small heat shock proteins family that plays a protective role in cellular homeostasis. They form enormous polydisperse oligomers that challenge modern biophysical methods to uncover the molecular basis of their assembly structure and chaperone-like protein binding function. It is argued that a molecular phenotype of a dynamic assembly suits a chaperone function as well as a structural role in the eye lens where the constraint of preventing protein condensation is paramount. The main cellular partners of α-crystallins, the β- and γ-crystallins, have largely been lost from the animal kingdom but the superfamily is hugely expanded in the vertebrate eye lens. Their structures show how a simple Greek key motif can evolve rapidly to form a complex array of monomers and oligomers. Apart from remaining transparent, a major role of the partnership of α-crystallins with β- and γ-crystallins in the lens is to form a refractive index gradient. Here, we show some of the structural and genetic features of these two protein superfamilies that enable the rapid creation of different assembly states, to match the rapidly changing optical needs among the various vertebrates. © 2013 The Protein Society.


Slingsby C.,Institute of Structural and Molecular Biology | Clark A.R.,Institute of Structural and Molecular Biology
Structure | Year: 2013

A crystal structure of a yeast small heat shock protein reported by Hanazono and colleagues in this issue of Structure reveals the versatility of the α-crystallin domain dimer for building assemblies of different size and symmetry. The domains assemble into a vessel filled with hydrophobic sequence extensions enriched with phenylalanines. © 2013 Elsevier Ltd. All Rights Reserved.


Hospenthal M.K.,Medical Research Council | Hospenthal M.K.,Institute of Structural and Molecular Biology | Mevissen T.E.T.,Medical Research Council | Komander D.,Medical Research Council
Nature Protocols | Year: 2015

Protein ubiquitination is a versatile protein modification that regulates virtually all cellular processes. This versatility originates from polyubiquitin chains, which can be linked in eight distinct ways. The combinatorial complexity of eight linkage types in homotypic (one chain type per polymer) and heterotypic (multiple linkage types per polymer) chains poses significant problems for biochemical analysis. Here we describe UbiCRest, in which substrates (ubiquitinated proteins or polyubiquitin chains) are treated with a panel of linkage-specific deubiquitinating enzymes (DUBs) in parallel reactions, followed by gel-based analysis. UbiCRest can be used to show that a protein is ubiquitinated, to identify which linkage type(s) are present on polyubiquitinated proteins and to assess the architecture of heterotypic polyubiquitin chains. DUBs used in UbiCRest can be obtained commercially; however, we include details for generating a toolkit of purified DUBs and for profiling their linkage preferences in vitro. UbiCRest is a qualitative method that yields insights into ubiquitin chain linkage types and architecture within hours, and it can be performed on western blotting quantities of endogenously ubiquitinated proteins. © 2015 Nature America, Inc. All rights reserved.

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