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Baltimore, MD, United States

Berry A.A.,University of Maryland Baltimore County | Yang Y.,Imperial College London | Pakharukova N.,University of Turku | Garnett J.A.,Imperial College London | And 13 more authors.
PLoS Pathogens | Year: 2014

Enteroaggregative Escherichia coli (EAEC) is a leading cause of acute and persistent diarrhea worldwide. A recently emerged Shiga-toxin-producing strain of EAEC resulted in significant mortality and morbidity due to progressive development of hemolytic-uremic syndrome. The attachment of EAEC to the human intestinal mucosa is mediated by aggregative adherence fimbria (AAF). Using X-ray crystallography and NMR structures, we present new atomic resolution insight into the structure of AAF variant I from the strain that caused the deadly outbreak in Germany in 2011, and AAF variant II from archetype strain 042, and propose a mechanism for AAF-mediated adhesion and biofilm formation. Our work shows that major subunits of AAF assemble into linear polymers by donor strand complementation where a single minor subunit is inserted at the tip of the polymer by accepting the donor strand from the terminal major subunit. Whereas the minor subunits of AAF have a distinct conserved structure, AAF major subunits display large structural differences, affecting the overall pilus architecture. These structures suggest a mechanism for AAF-mediated adhesion and biofilm formation. Binding experiments using wild type and mutant subunits (NMR and SPR) and bacteria (ELISA) revealed that despite the structural differences AAF recognize a common receptor, fibronectin, by employing clusters of basic residues at the junction between subunits in the pilus. We show that AAF-fibronectin attachment is based primarily on electrostatic interactions, a mechanism not reported previously for bacterial adhesion to biotic surfaces. © 2014 Berry et al. Source


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Paragon Bioservices | Date: 2008-10-21

Pharmaceutical products, namely, medical diagnostic reagents for use in immuno purification, imaging and assays, bacteriological culture media for medical use. Pharmaceutical research and product development services for others, namely, preclinical contract research, current good manufacturing and product development for pharmaceutical and biotechnology companies.


Paragon Bioservices | Entity website

Working as an outsourcing partner, and with a long-standing tradition of quality and service, Paragon expands the capabilities of pharmaceutical companies, biotechnology companies and academic laboratories involved in identifying, developing and producing monoclonal antibodies, therapeutic proteins, disease biomarkers, vaccines and reagents for diagnostics. Our Success Responsibility, duty, passion for science, overall optimism about the future, and our collective need to contribute is what makes Paragon successful among its peers ...


Paragon Bioservices | Entity website

Bob Roche Principal, Robert Roche Associates LLC Mr. Roche is the founding member of Robert Roche Associates LLC, a consulting firm providing guidance to the pharmaceutical and healthcare industries ...


Miller M.S.,Monash Institute of Pharmaceutical Sciences | Miller M.S.,Johns Hopkins University | Schmidt-Kittler O.,Johns Hopkins University | Schmidt-Kittler O.,Sanofi S.A. | And 14 more authors.
Oncotarget | Year: 2014

We report two crystal structures of the wild-type phosphatidylinositol 3-kinase a (PI3Kα) heterodimer refined to 2.9 Å and 3.4 Å resolution: the first as the free enzyme, the second in complex with the lipid substrate, diC4-PIP2, respectively. The first structure shows key interactions of the N-terminal SH2 domain (nSH2) and iSH2 with the activation loop that suggest a mechanism by which the enzyme is inhibited in its basal state. In the second structure, the lipid substrate binds in a positively charged pocket adjacent to the ATP-binding site, bordered by the P-loop, the activation loop and the iSH2 domain. An additional lipid-binding site was identified at the interface of the ABD, iSH2 and kinase domains. The ability of PI3Kα to bind an additional PIP2 molecule was confirmed in vitro by fluorescence quenching experiments. The crystal structures reveal key differences in the way the nSH2 domain interacts with wild-type p110a and with the oncogenic mutant p110aH1047R. Increased buried surface area and two unique salt-bridges observed only in the wild-type structure suggest tighter inhibition in the wild-type PI3Kα than in the oncogenic mutant. These differences may be partially responsible for the increased basal lipid kinase activity and increased membrane binding of the oncogenic mutant. Source

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