Characterization of a high-affinity complex between the bacterial outer membrane protein FhuA and the phage T5 protein pb5.
Binding of bacteriophage T5 to Escherichia coli cells is mediated by specific interactions between the receptor-binding protein pb5 (67.8 kDa) and the outer membrane iron-transporter FhuA. A histidine-tagged form of pb5 was overproduced and purified. Isolated pb5 is monomeric and organized mostly as beta-sheets (51%). pb5 functionality was attested in vivo by its ability to impair infection of E. coli cells by phage T5 and Phi80, and to prevent growth of bacteria on iron-ferrichrome as unique iron source. pb5 was functional in vitro, since addition of an equimolar concentration of pb5 to purified FhuA prevented DNA release from phage T5. However, pb5 alone was not sufficient for the conversion of FhuA into an open channel. Direct interaction of pb5 with FhuA was demonstrated by isolating a pb5/FhuA complex using size-exclusion chromatography. The stoichiometry, 1 mol of pb5/1 mol of FhuA, was deduced from its molecular mass, established by analytical ultracentrifugation after determination of the amount of bound detergent. SDS-PAGE and differential scanning calorimetry experiments highlighted the great stability of the complex: (i) it was not dissociated by 2% SDS even when the temperature was raised to 70 degrees C; (ii) thermal denaturation of the complex occurred at 85 degrees C, while pb5 and FhuA were denatured at 45 degrees C and 74 degrees C, respectively. The stability of the complex renders it suitable for high-resolution structural studies, allowing future analysis of conformational changes into both FhuA and pb5 upon adsorption of the virus to its host.
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Institut de Biochimie et Biophysique Moléculaire et Cellulaire, UMR CNRS 8619, Université Paris Sud, Bât 430, 91 405 Orsay Cedex, France.
Janmot
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le Maire
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Desmadril
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Bonhivers
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Letellier
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Boulanger
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D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004355:Drug Stability; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000821:virology; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D007473:Ion Channels / Q000737:chemistry; D046911:Macromolecular Substances; D011489:Protein Denaturation; D011991:Receptors, Virus / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry; D013604:T-Phages / Q000737:chemistry / Q000235:genetics; D013696:Temperature; D013816:Thermodynamics; D014461:Ultracentrifugation; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics
D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; C050900:FhuA protein, E coli; D007473:Ion Channels; D046911:Macromolecular Substances; D011991:Receptors, Virus; D011994:Recombinant Proteins; D014764:Viral Proteins; C067129:oad protein, Bacteriophage T5