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1.
Nat Genet ; 52(8): 778-789, 2020 08.
Article in English | MEDLINE | ID: mdl-32661416

ABSTRACT

Although DNA methylation is a key regulator of gene expression, the comprehensive methylation landscape of metastatic cancer has never been defined. Through whole-genome bisulfite sequencing paired with deep whole-genome and transcriptome sequencing of 100 castration-resistant prostate metastases, we discovered alterations affecting driver genes that were detectable only with integrated whole-genome approaches. Notably, we observed that 22% of tumors exhibited a novel epigenomic subtype associated with hypermethylation and somatic mutations in TET2, DNMT3B, IDH1 and BRAF. We also identified intergenic regions where methylation is associated with RNA expression of the oncogenic driver genes AR, MYC and ERG. Finally, we showed that differential methylation during progression preferentially occurs at somatic mutational hotspots and putative regulatory regions. This study is a large integrated study of whole-genome, whole-methylome and whole-transcriptome sequencing in metastatic cancer that provides a comprehensive overview of the important regulatory role of methylation in metastatic castration-resistant prostate cancer.


Subject(s)
DNA Methylation/genetics , Prostatic Neoplasms/genetics , Aged , Aged, 80 and over , Carcinogenesis/genetics , Epigenomics/methods , Gene Expression Regulation, Neoplastic/genetics , Genome/genetics , Humans , Male , Middle Aged , Mutation/genetics , Prospective Studies , Sequence Analysis, DNA/methods , Exome Sequencing/methods , Whole Genome Sequencing/methods
2.
Clin Cancer Res ; 25(8): 2450-2457, 2019 04 15.
Article in English | MEDLINE | ID: mdl-30573691

ABSTRACT

PURPOSE: Carcinomas originate from epithelial tissues, which have apical (luminal) and basal orientations. The degree of luminal versus basal differentiation in cancer has been shown to be biologically important in some carcinomas and impacts treatment response. EXPERIMENTAL DESIGN: Although prior studies have focused on individual cancer types, we used a modified clinical-grade classifier (PAM50) to subtype 8,764 tumors across 22 different carcinomas into luminal A, luminal B, and basal-like tumors. RESULTS: We found that all epithelial tumors demonstrated similar gene expression-based luminal/basal subtypes. As expected, basal-like tumors were associated with increased expression of the basal markers KRT5/6 and KRT14, and luminal-like tumors were associated with increased expression of the luminal markers KRT20. Luminal A tumors consistently had improved outcomes compared with basal across many tumor types, with luminal B tumors falling between the two. Basal tumors had the highest rates of TP53 and RB1 mutations and copy number loss. Luminal breast, cervical, ovarian, and endometrial tumors had increased ESR1 expression, and luminal prostate, breast, cervical, and bladder tumors had increased androgen receptor (AR) expression. Furthermore, luminal B tumors had the highest rates of AR and ESR1 mutations and had increased sensitivity in vitro to bicalutamide and tamoxifen. Luminal B tumors were more sensitive to gemcitabine, and basal tumors were more sensitive to docetaxel. CONCLUSIONS: This first pan-carcinoma luminal/basal subtyping across epithelial tumors reveals global similarities across carcinomas in the transcriptome, genome, clinical outcomes, and drug sensitivity, emphasizing the biological and translational importance of these luminal versus basal subtypes.


Subject(s)
Carcinoma, Basal Cell/diagnosis , Carcinoma, Basal Cell/genetics , Carcinoma/diagnosis , Carcinoma/genetics , Genetic Association Studies , Genetic Predisposition to Disease , Genomics , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Biomarkers, Tumor , Carcinoma/drug therapy , Carcinoma, Basal Cell/drug therapy , Cell Line, Tumor , Computational Biology/methods , Drug Resistance, Neoplasm/genetics , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/drug effects , Genetic Association Studies/methods , Genomics/methods , Humans , Kaplan-Meier Estimate , Mutation , Prognosis , Transcriptome
4.
Cell ; 174(3): 758-769.e9, 2018 07 26.
Article in English | MEDLINE | ID: mdl-30033370

ABSTRACT

While mutations affecting protein-coding regions have been examined across many cancers, structural variants at the genome-wide level are still poorly defined. Through integrative deep whole-genome and -transcriptome analysis of 101 castration-resistant prostate cancer metastases (109X tumor/38X normal coverage), we identified structural variants altering critical regulators of tumorigenesis and progression not detectable by exome approaches. Notably, we observed amplification of an intergenic enhancer region 624 kb upstream of the androgen receptor (AR) in 81% of patients, correlating with increased AR expression. Tandem duplication hotspots also occur near MYC, in lncRNAs associated with post-translational MYC regulation. Classes of structural variations were linked to distinct DNA repair deficiencies, suggesting their etiology, including associations of CDK12 mutation with tandem duplications, TP53 inactivation with inverted rearrangements and chromothripsis, and BRCA2 inactivation with deletions. Together, these observations provide a comprehensive view of how structural variations affect critical regulators in metastatic prostate cancer.


Subject(s)
Genomic Structural Variation/genetics , Prostatic Neoplasms/genetics , Aged , Aged, 80 and over , BRCA2 Protein/metabolism , Cyclin-Dependent Kinases/metabolism , DNA Copy Number Variations , Exome , Gene Expression Profiling/methods , Genomics/methods , Humans , Male , Middle Aged , Mutation , Neoplasm Metastasis/genetics , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Receptors, Androgen/genetics , Receptors, Androgen/metabolism , Tandem Repeat Sequences/genetics , Tumor Suppressor Protein p53/metabolism , Whole Genome Sequencing/methods
5.
Nature ; 538(7623): 60-65, 2016 Oct 06.
Article in English | MEDLINE | ID: mdl-27654919

ABSTRACT

In Gram-negative bacteria, outer membrane transporters import nutrients by coupling to an inner membrane protein complex called the Ton complex. The Ton complex consists of TonB, ExbB, and ExbD, and uses the proton motive force at the inner membrane to transduce energy to the outer membrane via TonB. Here, we structurally characterize the Ton complex from Escherichia coli using X-ray crystallography, electron microscopy, double electron-electron resonance (DEER) spectroscopy, and crosslinking. Our results reveal a stoichiometry consisting of a pentamer of ExbB, a dimer of ExbD, and at least one TonB. Electrophysiology studies show that the Ton subcomplex forms pH-sensitive cation-selective channels and provide insight into the mechanism by which it may harness the proton motive force to produce energy.


Subject(s)
Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Escherichia coli/chemistry , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Proton-Motive Force , Crystallography, X-Ray , Escherichia coli/ultrastructure , Escherichia coli Proteins/ultrastructure , Hydrogen-Ion Concentration , Membrane Proteins/ultrastructure , Multiprotein Complexes/ultrastructure
6.
Structure ; 24(6): 965-976, 2016 06 07.
Article in English | MEDLINE | ID: mdl-27161977

ABSTRACT

Incorporation of lipopolysaccharide (LPS) into the outer membrane of Gram-negative bacteria is essential for viability, and is accomplished by a two-protein complex called LptDE. We solved crystal structures of the core LptDE complexes from Yersinia pestis, Klebsiella pneumoniae, Pseudomonas aeruginosa, and a full-length structure of the K. pneumoniae LptDE complex. Our structures adopt the same plug and 26-strand ß-barrel architecture found recently for the Shigella flexneri and Salmonella typhimurium LptDE structures, illustrating a conserved fold across the family. A comparison of the only two full-length structures, SfLptDE and our KpLptDE, reveals a 21° rotation of the LptD N-terminal domain that may impart flexibility on the trans-envelope LptCAD scaffold. Utilizing mutagenesis coupled to an in vivo functional assay and molecular dynamics simulations, we demonstrate the critical role of Pro231 and Pro246 in the function of the LptD lateral gate that allows partitioning of LPS into the outer membrane.


Subject(s)
Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/metabolism , Gram-Negative Bacteria/metabolism , Lipopolysaccharides/metabolism , Binding Sites , Crystallography, X-Ray , Gram-Negative Bacteria/chemistry , Models, Molecular , Protein Binding , Protein Domains , Protein Structure, Secondary
7.
PLoS One ; 9(4): e95808, 2014.
Article in English | MEDLINE | ID: mdl-24752441

ABSTRACT

Many fluorescent proteins have been created to act as genetically encoded biosensors. With these sensors, changes in fluorescence report on chemical states in living cells. Transition metal ions such as copper, nickel, and zinc are crucial in many physiological and pathophysiological pathways. Here, we engineered a spectral series of optimized transition metal ion-binding fluorescent proteins that respond to metals with large changes in fluorescence intensity. These proteins can act as metal biosensors or imaging probes whose fluorescence can be tuned by metals. Each protein is uniquely modulated by four different metals (Cu2+, Ni2+, Co2+, and Zn2+). Crystallography revealed the geometry and location of metal binding to the engineered sites. When attached to the extracellular terminal of a membrane protein VAMP2, dimeric pairs of the sensors could be used in cells as ratiometric probes for transition metal ions. Thus, these engineered fluorescent proteins act as sensitive transition metal ion-responsive genetically encoded probes that span the visible spectrum.


Subject(s)
Luminescent Proteins/chemistry , Animals , Cobalt/chemistry , Cobalt/metabolism , Copper/chemistry , Copper/metabolism , Crystallography, X-Ray , Fluorescence Resonance Energy Transfer , Luminescent Proteins/metabolism , Nickel/chemistry , Nickel/metabolism , PC12 Cells , Protein Structure, Secondary , Rats , Vesicle-Associated Membrane Protein 2/chemistry , Vesicle-Associated Membrane Protein 2/metabolism , Zinc/chemistry , Zinc/metabolism
9.
Biochem Soc Trans ; 40(6): 1503-6, 2012 Dec 01.
Article in English | MEDLINE | ID: mdl-23176506

ABSTRACT

Purified phage lysins present an alternative to traditional antibiotics and work by hydrolysing peptidoglycan. Phage lysins have been developed against Gram-positive pathogens such as Bacillus anthracis and Streptococcus pneumoniae, where the peptidoglycan layer is exposed on the cell surface. Addition of the lysin to a bacterial culture results in rapid death of the organism. Gram-negative bacteria are resistant to phage lysins because they contain an outer membrane that protects the peptidoglycan from degradation. We solved crystal structures of a Yersinia pestis outer-membrane protein and the bacteriocin that targets it, which informed engineering of a bacterial-phage hybrid lysin that can be transported across the outer membrane to kill specific Gram-negative bacteria. This work provides a template for engineering phage lysins against a wide variety of bacterial pathogens.


Subject(s)
Anti-Bacterial Agents/chemistry , Bacteriocins/chemistry , Viral Proteins/chemistry , Yersinia pestis/drug effects , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/metabolism , Bacteriocins/genetics , Bacteriophages/enzymology , Drug Design , Models, Molecular , Protein Conformation , Protein Engineering , Protein Structure, Secondary , Protein Structure, Tertiary , Receptors, Cell Surface/metabolism , Viral Proteins/genetics , Viral Proteins/pharmacology , Yersinia pestis/metabolism
10.
Proc Natl Acad Sci U S A ; 109(25): 9857-62, 2012 Jun 19.
Article in English | MEDLINE | ID: mdl-22679291

ABSTRACT

Bacterial pathogens are becoming increasingly resistant to antibiotics. As an alternative therapeutic strategy, phage therapy reagents containing purified viral lysins have been developed against gram-positive organisms but not against gram-negative organisms due to the inability of these types of drugs to cross the bacterial outer membrane. We solved the crystal structures of a Yersinia pestis outer membrane transporter called FyuA and a bacterial toxin called pesticin that targets this transporter. FyuA is a ß-barrel membrane protein belonging to the family of TonB dependent transporters, whereas pesticin is a soluble protein with two domains, one that binds to FyuA and another that is structurally similar to phage T4 lysozyme. The structure of pesticin allowed us to design a phage therapy reagent comprised of the FyuA binding domain of pesticin fused to the N-terminus of T4 lysozyme. This hybrid toxin kills specific Yersinia and pathogenic E. coli strains and, importantly, can evade the pesticin immunity protein (Pim) giving it a distinct advantage over pesticin. Furthermore, because FyuA is required for virulence and is more common in pathogenic bacteria, the hybrid toxin also has the advantage of targeting primarily disease-causing bacteria rather than indiscriminately eliminating natural gut flora.


Subject(s)
Bacteriophages/metabolism , Gram-Negative Bacteria/virology , Mucoproteins/metabolism , Bacterial Proteins/chemistry , Bacteriocins/chemistry , Bacteriophages/physiology , Cell Membrane/metabolism , Cryoelectron Microscopy , Models, Molecular , Mucoproteins/chemistry , Protein Conformation , Protein Engineering , Protein Transport , Receptors, Cell Surface/chemistry
11.
Structure ; 20(7): 1233-43, 2012 Jul 03.
Article in English | MEDLINE | ID: mdl-22658748

ABSTRACT

Intimins and invasins are virulence factors produced by pathogenic Gram-negative bacteria. They contain C-terminal extracellular passenger domains that are involved in adhesion to host cells and N-terminal ß domains that are embedded in the outer membrane. Here, we identify the domain boundaries of an E. coli intimin ß domain and use this information to solve its structure and the ß domain structure of a Y. pseudotuberculosis invasin. Both ß domain structures crystallized as monomers and reveal that the previous range of residues assigned to the ß domain also includes a protease-resistant domain that is part of the passenger. Additionally, we identify 146 nonredundant representative members of the intimin/invasin family based on the boundaries of the highly conserved intimin and invasin ß domains. We then use this set of sequences along with our structural data to find and map the evolutionarily constrained residues within the ß domain.


Subject(s)
Adhesins, Bacterial/chemistry , Enterohemorrhagic Escherichia coli/chemistry , Escherichia coli Proteins/chemistry , Recombinant Fusion Proteins/chemistry , Yersinia pseudotuberculosis/chemistry , Adhesins, Bacterial/genetics , Adhesins, Bacterial/metabolism , Amino Acid Sequence , Bacterial Adhesion , Conserved Sequence , Crystallography, X-Ray , Enterohemorrhagic Escherichia coli/metabolism , Enterohemorrhagic Escherichia coli/pathogenicity , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Evolution, Molecular , Models, Molecular , Molecular Sequence Data , Plasmids , Protein Structure, Secondary , Protein Structure, Tertiary , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Virulence Factors/chemistry , Virulence Factors/genetics , Virulence Factors/metabolism , Yersinia pseudotuberculosis/metabolism , Yersinia pseudotuberculosis/pathogenicity
12.
J Mol Biol ; 415(1): 128-42, 2012 Jan 06.
Article in English | MEDLINE | ID: mdl-22094314

ABSTRACT

Autotransporters are secreted proteins produced by pathogenic Gram-negative bacteria. They consist of a membrane-embedded ß-domain and an extracellular passenger domain that is sometimes cleaved and released from the cell surface. We solved the structures of three noncleavable mutants of the autotransporter EspP to examine how it promotes asparagine cyclization to cleave its passenger. We found that cyclization is facilitated by multiple factors. The active-site asparagine is sterically constrained to conformations favorable for cyclization, while electrostatic interactions correctly orient the carboxamide group for nucleophilic attack. During molecular dynamics simulations, water molecules were observed to enter the active site and to form hydrogen bonds favorable for increasing the nucleophilicity of the active-site asparagine. When the activated asparagine attacks its main-chain carbonyl carbon, the resulting oxyanion is stabilized by a protonated glutamate. Upon cleavage, this proton could be transferred to the leaving amine group, helping overcome a significant energy barrier. Together, these findings provide insight into factors important for asparagine cyclization, a mechanism broadly used for protein cleavage.


Subject(s)
Asparagine/chemistry , Asparagine/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Serine Endopeptidases/chemistry , Serine Endopeptidases/metabolism , Catalysis , Catalytic Domain , Cyclization , Escherichia coli/metabolism , Glutamic Acid/chemistry , Glutamic Acid/metabolism , Hydrogen Bonding , Models, Molecular , Molecular Dynamics Simulation , Protein Structure, Tertiary , Protons , Static Electricity , Water/chemistry , Water/metabolism
13.
Structure ; 19(11): 1672-82, 2011 Nov 09.
Article in English | MEDLINE | ID: mdl-22078566

ABSTRACT

Ail is an outer membrane protein from Yersinia pestis that is highly expressed in a rodent model of bubonic plague, making it a good candidate for vaccine development. Ail is important for attaching to host cells and evading host immune responses, facilitating rapid progression of a plague infection. Binding to host cells is important for injection of cytotoxic Yersinia outer proteins. To learn more about how Ail mediates adhesion, we solved two high-resolution crystal structures of Ail, with no ligand bound and in complex with a heparin analog called sucrose octasulfate. We identified multiple adhesion targets, including laminin and heparin, and showed that a 40 kDa domain of laminin called LG4-5 specifically binds to Ail. We also evaluated the contribution of laminin to delivery of Yops to HEp-2 cells. This work constitutes a structural description of how a bacterial outer membrane protein uses a multivalent approach to bind host cells.


Subject(s)
Bacterial Adhesion , Bacterial Outer Membrane Proteins/chemistry , Sucrose/analogs & derivatives , Virulence Factors/chemistry , Yersinia pestis , Amino Acid Sequence , Bacterial Outer Membrane Proteins/metabolism , Bacterial Outer Membrane Proteins/physiology , Bacterial Proteins/metabolism , Cell Line , Crystallography, X-Ray , Extracellular Matrix/metabolism , Extracellular Matrix Proteins/metabolism , Host-Pathogen Interactions , Humans , Laminin/metabolism , Membrane Proteins/metabolism , Molecular Chaperones/metabolism , Molecular Sequence Data , Protein Binding , Protein Structure, Tertiary , Sequence Alignment , Sucrose/chemistry , Surface Properties , Virulence Factors/metabolism , Virulence Factors/physiology
14.
Annu Rev Microbiol ; 64: 43-60, 2010.
Article in English | MEDLINE | ID: mdl-20420522

ABSTRACT

TonB-dependent transporters (TBDTs) are bacterial outer membrane proteins that bind and transport ferric chelates, called siderophores, as well as vitamin B(12), nickel complexes, and carbohydrates. The transport process requires energy in the form of proton motive force and a complex of three inner membrane proteins, TonB-ExbB-ExbD, to transduce this energy to the outer membrane. The siderophore substrates range in complexity from simple small molecules such as citrate to large proteins such as serum transferrin and hemoglobin. Because iron uptake is vital for almost all bacteria, expression of TBDTs is regulated in a number of ways that include metal-dependent regulators, σ/anti-σ factor systems, small RNAs, and even a riboswitch. In recent years, many new structures of TBDTs have been solved in various states, resulting in a more complete understanding of siderophore selectivity and binding, signal transduction across the outer membrane, and interaction with the TonB-ExbB-ExbD complex. However, the transport mechanism is still unclear. In this review, we summarize recent progress in understanding regulation, structure, and function in TBDTs and questions remaining to be answered.


Subject(s)
Bacteria/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Membrane Proteins/genetics , Membrane Proteins/metabolism , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Bacteria/genetics , Biological Transport , Carbohydrate Metabolism , Energy Metabolism , Iron/metabolism , Models, Biological , Nickel/metabolism , Proton-Motive Force , Siderophores/metabolism , Vitamin B 12/metabolism
15.
Nat Struct Mol Biol ; 14(12): 1214-20, 2007 Dec.
Article in English | MEDLINE | ID: mdl-17994105

ABSTRACT

Autotransporters are virulence factors produced by Gram-negative bacteria. They consist of two domains, an N-terminal 'passenger' domain and a C-terminal beta-domain. beta-domains form beta-barrel structures in the outer membrane while passenger domains are translocated into the extracellular space. In some autotransporters, the two domains are separated by proteolytic cleavage. Using X-ray crystallography, we solved the 2.7-A structure of the post-cleavage state of the beta-domain of EspP, an autotransporter produced by Escherichia coli strain O157:H7. The structure consists of a 12-stranded beta-barrel with the passenger domain-beta-domain cleavage junction located inside the barrel pore, approximately midway between the extracellular and periplasmic surfaces of the outer membrane. The structure reveals an unprecedented intra-barrel cleavage mechanism and suggests that two conformational changes occur in the beta-domain after cleavage, one conferring increased stability on the beta-domain and another restricting access to the barrel pore.


Subject(s)
Escherichia coli Proteins/chemistry , Serine Endopeptidases/chemistry , Crystallography, X-Ray , Escherichia coli O157/chemistry , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Hydrolysis , Point Mutation , Protein Conformation , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Static Electricity
16.
EMBO J ; 26(10): 2594-604, 2007 May 16.
Article in English | MEDLINE | ID: mdl-17464289

ABSTRACT

Colicin Ia is a 69 kDa protein that kills susceptible Escherichia coli cells by binding to a specific receptor in the outer membrane, colicin I receptor (70 kDa), and subsequently translocating its channel forming domain across the periplasmic space, where it inserts into the inner membrane and forms a voltage-dependent ion channel. We determined crystal structures of colicin I receptor alone and in complex with the receptor binding domain of colicin Ia. The receptor undergoes large and unusual conformational changes upon colicin binding, opening at the cell surface and positioning the receptor binding domain of colicin Ia directly above it. We modelled the interaction with full-length colicin Ia to show that the channel forming domain is initially positioned 150 A above the cell surface. Functional data using full-length colicin Ia show that colicin I receptor is necessary for cell surface binding, and suggest that the receptor participates in translocation of colicin Ia across the outer membrane.


Subject(s)
Colicins/chemistry , Colicins/metabolism , Colicins/genetics , Crystallography, X-Ray , Molecular Weight , Protein Structure, Secondary , Protein Structure, Tertiary , Protein Transport , Spectrum Analysis, Raman
17.
EMBO J ; 26(7): 1942-52, 2007 Apr 04.
Article in English | MEDLINE | ID: mdl-17347646

ABSTRACT

Bacterial autotransporters are comprised of an N-terminal 'passenger domain' and a C-terminal beta barrel ('beta domain') that facilitates transport of the passenger domain across the outer membrane. Following translocation, the passenger domains of some autotransporters are cleaved by an unknown mechanism. Here we show that the passenger domain of the Escherichia coli O157:H7 autotransporter EspP is released in a novel autoproteolytic reaction. After purification, the uncleaved EspP precursor underwent proteolytic processing in vitro. An analysis of protein topology together with mutational studies strongly suggested that the reaction occurs inside the beta barrel and revealed that two conserved residues, an aspartate within the beta domain (Asp(1120)) and an asparagine (Asn(1023)) at the P1 position of the cleavage junction, are essential for passenger domain cleavage. Interestingly, these residues were also essential for the proteolytic processing of two distantly related autotransporters. The data strongly suggest that Asp(1120) and Asn(1023) form an unusual catalytic dyad that mediates self-cleavage through the cyclization of the asparagine. Remarkably, a very similar mechanism has been proposed for the maturation of eukaryotic viral capsids.


Subject(s)
Escherichia coli O157/metabolism , Escherichia coli/metabolism , Evolution, Molecular , Protein Processing, Post-Translational , Amino Acid Sequence , Asparagine/metabolism , Aspartic Acid/metabolism , Catalysis , Conserved Sequence , DNA Mutational Analysis , Detergents , Escherichia coli O157/chemistry , Escherichia coli O157/isolation & purification , Models, Biological , Molecular Sequence Data , Protein Precursors/metabolism , Protein Structure, Tertiary , Solutions , Succinimides/metabolism
18.
Curr Protoc Protein Sci ; Chapter 17: Unit 17.9, 2007 Feb.
Article in English | MEDLINE | ID: mdl-18429311

ABSTRACT

Over the last 20 years, the use of X-ray crystallography has become a viable technique for the structure determination of integral membrane proteins. However, standard crystallizaton protocols must be modified to account for difficulties involved in handling membrane proteins, which arise primarily from having detergent present. This unit provides protocols that can be used to crystallize a purified membrane protein, including detergent exchange, sample concentration, initial screening using a crystallization robot, and finally, optimization of crystallization conditions to obtain diffraction-quality crystals. These protocols were established for outer membrane proteins, but can be used for inner membrane proteins as well. Advice on alternative protocols, detergent selection, and optimization of crystallization conditions is provided.


Subject(s)
Membrane Proteins/chemistry , Crystallization , Crystallography, X-Ray , Protein Conformation
19.
Mol Microbiol ; 58(4): 945-58, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16262782

ABSTRACT

Bacterial autotransporters are proteins that contain a small C-terminal 'beta domain' that facilitates translocation of a large N-terminal 'passenger domain' across the outer membrane (OM) by an unknown mechanism. Here we used EspP, an autotransporter produced by Escherichia coli 0157:H7, as a model protein to gain insight into the transport reaction. Initially we found that the passenger domain of a truncated version of EspP (EspPDelta1-851) was translocated efficiently across the OM. Blue Native polyacrylamide gel electrophoresis, analytical ultracentrifugation and other biochemical methods showed that EspPDelta1-851 behaves as a compact monomer and strongly suggest that the channel formed by the beta domain is too narrow to accommodate folded polypeptides. Surprisingly, we found that a folded protein domain fused to the N-terminus of EspPDelta1-851 was efficiently translocated across the OM. Further analysis revealed that the passenger domain of wild-type EspP also folds at least partially in the periplasm. To reconcile these data, we propose that the EspP beta domain functions primarily to target and anchor the protein and that an external factor transports the passenger domain across the OM.


Subject(s)
Escherichia coli O157/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Protein Folding , Protein Structure, Tertiary , Protein Transport , Serine Endopeptidases/chemistry , Serine Endopeptidases/metabolism , Blotting, Western , Cell Membrane/metabolism , Electrophoresis, Polyacrylamide Gel , Escherichia coli , Escherichia coli Proteins/genetics , Protein Subunits , Recombinant Fusion Proteins/metabolism , Sequence Deletion , Serine Endopeptidases/genetics
20.
Infect Immun ; 70(12): 7156-60, 2002 Dec.
Article in English | MEDLINE | ID: mdl-12438401

ABSTRACT

IroN was recently identified in the extracellular pathogenic Escherichia coli strain CP9. In this study experimental evidence demonstrating that IroN mediates utilization of the siderophore enterobactin was obtained, thereby establishing IroN as a catecholate siderophore receptor. In a mouse model of ascending urinary tract infection the presence of iroN contributed significantly to CP9's ability to colonize the mouse bladder, kidneys, and urine, evidence that IroN is a urovirulence factor. However, growth in human urine ex vivo and adherence to uroepithelial cells in vitro were equivalent for an isogenic mutant deficient in IroN (CP82) and its wild-type parent (CP9). Taken together, these findings establish that IroN is a siderophore receptor and a urovirulence factor. However, uncertainty exists as to the mechanism(s) via which IroN contributes to urovirulence.


Subject(s)
Bacterial Outer Membrane Proteins/metabolism , Enterobactin/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/pathogenicity , Receptors, Cell Surface/metabolism , Urinary Tract Infections/microbiology , Animals , Bacterial Adhesion , Bacterial Outer Membrane Proteins/genetics , Cells, Cultured , Disease Models, Animal , Escherichia coli/growth & development , Escherichia coli Infections/microbiology , Escherichia coli Proteins/genetics , Female , Humans , Mice , Molecular Sequence Data , Rats , Receptors, Cell Surface/genetics , Urinary Bladder/cytology , Urinary Bladder/microbiology , Urine/microbiology , Virulence
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