scholarly journals Monitoring conformational changes during the catalytic cycle of OpuAA, the ATPase subunit of the ABC transporter OpuA from Bacillus subtilis

2008 ◽  
Vol 412 (2) ◽  
pp. 233-244 ◽  
Author(s):  
Carsten Horn ◽  
Stefan Jenewein ◽  
Britta Tschapek ◽  
Werner Bouschen ◽  
Sabine Metzger ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Abc Transporter ◽  
Conformational Changes ◽  
Compatible Solutes ◽  
Three Dimensional ◽  
Fine Tuning ◽  
Transport Systems ◽  
Dimensional Structure ◽  
Atpase Subunit ◽  
Accessory Domain

The ABC transporter (ATP-binding-cassette transporter) OpuA is one of five membrane transport systems in Bacillus subtilis that mediate osmoprotection by importing compatible solutes. Just like all bacterial and archaeal ABC transporters that catalyse the import of substrates, OpuA (where Opu is osmoprotectant uptake) is composed of an ATPase subunit (OpuAA), a transmembrane subunit (OpuAB) and an extracellular substrate-binding protein (OpuAC). In contrast with many well-known ABC-ATPases, OpuAA is composed not only of a catalytic and a helical domain but also of an accessory domain located at its C-terminus. The paradigm of such an architecture is MalK, the ABC-ATPase of the maltose importer of Escherichia coli, for which detailed structural and functional information is available. In the present study, we have applied solution FRET (Förster resonance energy transfer) techniques using two single cysteine mutants to obtain initial structural information on the architecture of the OpuAA dimer in solution. Analysing our results in detail and comparing them with the existing MalK structures revealed that the catalytic and helical domains adopted an arrangement similar to those of MalK, whereas profound differences in the three-dimensional orientation of the accessory domain, which contains two CBS (cystathionine β-synthetase) domains, were observed. These results shed new light on the role of this accessory domain present in a certain subset of ABC-ATPase in the fine-tuning of three-dimensional structure and biological function.

scholarly journals The three-dimensional structure of a class-Pi glutathione S-transferase complexed with glutathione: the active-site hydration provides insights into the reaction mechanism

1998 ◽  
Vol 333 (3) ◽  
pp. 811-816 ◽  
Author(s):  
Antonio PÁRRAGA ◽  
Isabel GARCÍA-SÁEZ ◽  
Sinead B. WALSH ◽  
Timothy J. MANTLE ◽  
Miquel COLL
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Water Molecules ◽  
X Ray Diffraction ◽  
Glutathione S Transferase ◽  
X Ray ◽  
The Right

The structure of mouse liver glutathione S-transferase P1-1 complexed with its substrate glutathione (GSH) has been determined by X-ray diffraction analysis. No conformational changes in the glutathione moiety or in the protein, other than small adjustments of some side chains, are observed when compared with glutathione adduct complexes. Our structure confirms that the role of Tyr-7 is to stabilize the thiolate by hydrogen bonding and to position it in the right orientation. A comparison of the enzyme–GSH structure reported here with previously described structures reveals rearrangements in a well-defined network of water molecules in the active site. One of these water molecules (W0), identified in the unliganded enzyme (carboxymethylated at Cys-47), is displaced by the binding of GSH, and a further water molecule (W4) is displaced following the binding of the electrophilic substrate and the formation of the glutathione conjugate. The possibility that one of these water molecules participates in the proton abstraction from the glutathione thiol is discussed.

scholarly journals Viral Genome Conformations and Contacts across Different Lifecycle Stages

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 109
Author(s):  
Peter G. Stockley ◽  
Nikesh Patel ◽  
Emma L. Wroblewski ◽  
Andrew J. P. Scott ◽  
Carlos P. Mata ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Drug Targets ◽  
Three Dimensional ◽  
Host Cells ◽  
Dimensional Structure ◽  
Viral Genomes ◽  
Starting Point ◽  
B Virus ◽  
Infection Mechanisms ◽  
Conformational Memory

Single-stranded RNA viral genomes (gRNA) are dynamic molecules that permit packaging into virions and their subsequent extrusion during infection. For viruses with such genomes, we discovered a previously unsuspected mechanism that regulates their assembly. This regulation is the result of multiple cognate coat protein (CP)–gRNA contacts distributed across the RNA. Collectively, these interactions make the assembly highly efficient and specific. The regions of the gRNA packaging signals (PSs) driving this assembly are potential drug targets, whilst the manipulation of PS–CP contacts with nonviral RNA cargos is a route towards bespoke virus-like particles. Infectivity depends on the virions being able to transfer their gRNAs into host cells. The starting point for this transfer appears to be an encapsidated RNA with a defined three-dimensional structure, especially around the PSs. A combination of asymmetric cryo-electron microscopy structure determination and X-ray synchrotron footprinting were used to define these contacts and structures in a number of viral examples, including hepatitis B virus and enteroviruses. These tools allow us to look beyond the outer CP layer of the virion shell and to see the functional, asymmetric components that regulate viral infectivity. This revealed yet more unexpected aspects of critical infection mechanisms, such as the RNA conformational changes required for encapsidation, the details of PS–CP contacts regulating the assembly, and the conformational “memory” imposed by encapsidation.

Structures of the substrate-binding protein provide insights into the multiple compatible solute binding specificities of the Bacillus subtilis ABC transporter OpuC

2011 ◽  
Vol 436 (2) ◽  
pp. 283-289 ◽  
Author(s):  
Yang Du ◽  
Wei-Wei Shi ◽  
Yong-Xing He ◽  
Yi-Hu Yang ◽  
Cong-Zhao Zhou ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Abc Transporter ◽  
Binding Protein ◽  
Substrate Binding ◽  
Compatible Solutes ◽  
Binding Pocket ◽  
Compatible Solute ◽  
Structural Basis ◽  
Binding Property ◽  
Substrate Binding Protein

The compatible solute ABC (ATP-binding cassette) transporters are indispensable for acquiring a variety of compatible solutes under osmotic stress in Bacillus subtilis. The substrate-binding protein OpuCC (Opu is osmoprotectant uptake) of the ABC transporter OpuC can recognize a broad spectrum of compatible solutes, compared with its 70% sequence-identical paralogue OpuBC that can solely bind choline. To explore the structural basis of this difference of substrate specificity, we determined crystal structures of OpuCC in the apo-form and in complex with carnitine, glycine betaine, choline and ectoine respectively. OpuCC is composed of two α/β/α globular sandwich domains linked by two hinge regions, with a substrate-binding pocket located at the interdomain cleft. Upon substrate binding, the two domains shift towards each other to trap the substrate. Comparative structural analysis revealed a plastic pocket that fits various compatible solutes, which attributes the multiple-substrate binding property to OpuCC. This plasticity is a gain-of-function via a single-residue mutation of Thr94 in OpuCC compared with Asp96 in OpuBC.

scholarly journals Structure of Isolated Nucleocapsids from Venezuelan Equine Encephalitis Virus and Implications for Assembly and Disassembly of Enveloped Virus

2003 ◽  
Vol 77 (1) ◽  
pp. 659-664 ◽  
Author(s):  
Angel Paredes ◽  
Kathy Alwell-Warda ◽  
Scott C. Weaver ◽  
Wah Chiu ◽  
Stanley J. Watowich
Keyword(s):  
Conformational Changes ◽  
Rna Virus ◽  
Three Dimensional ◽  
Venezuelan Equine Encephalitis Virus ◽  
Encephalitis Virus ◽  
Dimensional Structure ◽  
Icosahedral Symmetry ◽  
Venezuelan Equine Encephalitis ◽  
Equine Encephalitis Virus ◽  
Enveloped Virus

ABSTRACT Venezuelan equine encephalitis virus (VEEV) is an important human and equine pathogen in the Americas, with widespread reoccurring epidemics extending from South America to the southern United States. Most troubling, VEEV has been made into a weapon by several countries and is currently restricted by the Centers for Disease Control and Prevention as a potential biological warfare and terrorism agent. To facilitate the development of antiviral compounds, the structure of the nucleocapsid isolated from VEEV has been determined by electron cryomicroscopy and image reconstruction and represents the first three-dimensional structure of a nucleocapsid isolated from a single-stranded enveloped RNA virus. The isolated VEEV nucleocapsid undergoes significant reorganization relative to its structure within VEEV. However, the isolated nucleocapsid clearly exhibits T=4 icosahedral symmetry, and its characteristic nucleocapsid hexons and pentons are preserved. The diameter of the isolated nucleocapsid is ∼11.5% larger than that of the nucleocapsid within VEEV, with radial expansion being greatest near the hexons. Significantly, this is the first direct structural evidence showing that a simple enveloped virus undergoes large conformational changes during maturation, suggesting that the lipid bilayer and the transmembrane proteins of simple enveloped viruses provide the energy necessary to reorganize the nucleocapsid during maturation.

scholarly journals Streptococcal phosphotransferase system imports unsaturated hyaluronan disaccharide derived from host extracellular matrices

2018 ◽  
Author(s):  
Sayoko Oiki ◽  
Yusuke Nakamichi ◽  
Yukie Maruyama ◽  
Bunzo Mikami ◽  
Kousaku Murata ◽  
...  
Keyword(s):  
Abc Transporter ◽  
Molecular System ◽  
Bacterial Species ◽  
Three Dimensional ◽  
Amino Sugar ◽  
Extracellular Matrices ◽  
Dimensional Structure ◽  
Phosphotransferase System ◽  
Streptobacillus Moniliformis ◽  
Genes Encoding

ABSTRACTCertain bacterial species target the polysaccharide glycosaminoglycans (GAGs) of animal extracellular matrices for colonization and/or infection. GAGs such as hyaluronan and chondroitin sulfate consist of repeating disaccharide units of uronate and amino sugar residues, and are depolymerized to unsaturated disaccharides by bacterial extracellular or cell-surface polysaccharide lyase. The disaccharides are degraded and metabolized by cytoplasmic enzymes such as unsaturated glucuronyl hydrolase, isomerase, and reductase. The genes encoding these enzymes are assembled to form a GAG genetic cluster. Here, we demonstrate theStreptococcus agalactiaephosphotransferase system (PTS) for import of unsaturated hyaluronan disaccharide.S. agalactiaeNEM316 was found to depolymerize and assimilate hyaluronan, whereas its mutant with a disruption in PTS genes included in the GAG cluster was unable to grow on hyaluronan, while retaining the ability to depolymerize hyaluronan. Using toluene-treated wild-type cells, the PTS import activity of unsaturated hyaluronan disaccharide was significantly higher than that observed in the absence of the substrate. In contrast, the PTS mutant was unable to import unsaturated hyaluronan disaccharide, indicating that the corresponding PTS is the only importer of fragmented hyaluronan, which is suitable for PTS to phosphorylate the substrate at the C-6 position. The three-dimensional structure of streptococcal EIIA, one of the PTS components, was found to contain a Rossman-fold motif by X-ray crystallization. Docking of EIIA with another component EIIB by modeling provided structural insights into the phosphate transfer mechanism. This study is the first to identify the substrate (unsaturated hyaluronan disaccharide) recognized and imported by the streptococcal PTS.IMPORTANCE (118/120 words)The PTS identified in this work imports sulfate group-free unsaturated hyaluronan disaccharide as a result of the phosphorylation of the substrate at the C-6 position.S. agalactiaecan be indigenous to animal hyaluronan-rich tissues owing to the bacterial molecular system for fragmentation, import, degradation, and metabolism of hyaluronan. Distinct from hyaluronan, most GAGs, which are sulfated at the C-6 position, are unsuitable for PTS due to its inability to phosphorylate the substrate. More recently, we have identified a solute-binding protein-dependent ABC transporter in a pathogenicStreptobacillus moniliformisas an importer of sulfated and non-sulfated fragmented GAGs without any substrate modification. Our findings regarding PTS and ABC transporter shed light on bacterial clever colonization/infection system targeting various animal GAGs.

scholarly journals Crystal Structure of the Human Natural Killer Cell Activating Receptor KIR2DS2 (CD158j)

2003 ◽  
Vol 197 (7) ◽  
pp. 933-938 ◽  
Author(s):  
Xavier Saulquin ◽  
Louis N. Gastinel ◽  
Eric Vivier
Keyword(s):  
T Cell ◽  
Natural Killer ◽  
Conformational Changes ◽  
Killer Cell ◽  
Three Dimensional ◽  
Cell Receptor ◽  
Amino Acid Identity ◽  
T Cell Subsets ◽  
Dimensional Structure ◽  
Human Natural Killer Cell

Killer cell Ig-like receptors (KIRs) regulate the function of human natural killer and T cell subsets. A feature of the KIR locus is the clustering of homologous genes encoding for inhibitory and activating KIR. Inhibitory and activating KIR differ for ligand specificities and/or affinities. In particular, we show here with KIR tetramers that activating KIR2DS2 does not bind HLA-Cw3 molecules recognized by inhibitory KIR2DL2, despite 99% extracellular amino acid identity. We also report the 2.3-Å structure of KIR2DS2, which reveals subtle displacements of two residues (Tyr45 and Gln71) involved in the interaction of KIR2DL2 with HLA-Cw3. These results show that KIR molecules cannot tolerate any variability in their three-dimensional structure without altering their MHC class I recognition capacities. Therefore, the mode of recognition used by KIR largely differs from the conformational changes that characterize T cell receptor or NKG2D interaction with their respective ligands.

scholarly journals Conformational Changes of Spo0F along the Phosphotransfer Pathway

2005 ◽  
Vol 187 (24) ◽  
pp. 8221-8227 ◽  
Author(s):  
Kottayil I. Varughese
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Conformational Changes ◽  
Solution Structure ◽  
Three Dimensional ◽  
Bound State ◽  
Dimensional Structure ◽  
Single Domain Protein ◽  
Secondary Messenger ◽  
Domain Protein ◽  
Apo State

ABSTRACT Spo0F is a secondary messenger in the sporulation phosphorelay, and its structure has been characterized crystallographically in the apo-state, in the metal-bound state, and in an interacting state with a phosphotransferase. Additionally, the solution structure of the molecule has been characterized by nuclear magnetic resonance techniques in the unliganded state and in complex with beryllofluoride. Spo0F is a single-domain protein with a well-defined three-dimensional structure, but it is capable of adapting to specific conformations for catching and releasing the phosphoryl moiety. This commentary deals with the conformational fluctuations of the molecule as it moves from an apo-state to a metal-coordinated state, to a phosphorylated state, and then to a phosphoryl-transferring state.

scholarly journals Sulfate-Binding Protein (Sbp) from Xanthomonas citri: Structure and Functional Insights

2017 ◽  
Vol 30 (7) ◽  
pp. 578-588 ◽  
Author(s):  
Cristiane Tambascia Pereira ◽  
Cássia Roesler ◽  
Jéssica Nascimento Faria ◽  
Melissa Regina Fessel ◽  
Andrea Balan
Keyword(s):  
Abc Transporter ◽  
Structural Changes ◽  
Binding Protein ◽  
Functional Characterization ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Xanthomonas Citri ◽  
Canker Disease

The uptake and transport of sulfate in bacteria is mediated by an ATP-binding cassette transporter (ABC transporter) encoded by sbpcysUWA genes, whose importance has been widely demonstrated due to their relevance in cysteine synthesis and bacterial growth. In Xanthomonas citri, the causative agent of canker disease, the expression of components from this ABC transporter and others related to uptake of organic sulfur sources has been shown during in vitro growth cultures. In this work, based on gene reporter and proteomics analyses, we showed the activation of the promoter that controls the sbpcysUWA operon in vitro and in vivo and the expression of sulfate-binding protein (Sbp), a periplasmic-binding protein, indicating that this protein plays an important function during growth and that the transport system is active during Citrus sinensis infection. To characterize Sbp, we solved its three-dimensional structure bound to sulfate at 1.14 Å resolution and performed biochemical and functional characterization. The results revealed that Sbp interacts with sulfate without structural changes, but the interaction induces a significant increasing of protein thermal stability. Altogether, the results presented in this study show the evidence of the functionality of the ABC transporter for sulfate in X. citri and its relevance during infection.

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