title: Thiamine diphosphate binds to intermediates in the assembly of adenovirus fiber knob trimers in Escherichia coli.
abstract: Assembly of the adenovirus (Ad) homotrimeric fiber protein is nucleated by its C-terminal knob domain, which itself can trimerize when expressed as a recombinant protein fragment. The non-interlocked, globular structure of subunits in the knob trimer implies that trimers assemble from prefolded monomers through a dimer intermediate, but these intermediates have not been observed and the mechanism of assembly therefore remains uncharacterized. Here we report that expression of the Ad serotype 2 (Ad2) knob was toxic for thi- strains of Escherichia coli, which are defective in de novo synthesis of thiamine (vitamin B1). Ad2 knob trimers isolated from a thi+ strain copurified through multiple chromatography steps with a small molecule of mass equivalent to that of thiamine diphosphate (ThDP). Mutant analysis did not implicate any specific site for ThDP binding. Our results suggest that ThDP may associate with assembly intermediates and become trapped in assembled trimers, possibly within one of several large cavities that are partially solvent-accessible or buried completely within the trimer interior.
authors.affiliation: Biology Department, Brookhaven National Laboratory, Upton, New York 11973, USA .
authors.forename: Ryan
authors.initials: R
authors.lastname: Schulz
authors.forename: Yian-Biao
authors.initials: YB
authors.lastname: Zhang
authors.forename: Chang-Jun
authors.initials: CJ
authors.lastname: Liu
authors.forename: Paul
authors.initials: P
authors.lastname: Freimuth
mesh_terms: D000256:Adenoviridae / Q000378:metabolism*; D001665:Binding Sites; D036022:Capsid Proteins / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D013058:Mass Spectrometry; D008958:Models, Molecular; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D013835:Thiamine Pyrophosphate / Q000378:metabolism*
chemical_list: D036022:Capsid Proteins; D011994:Recombinant Proteins; C018315:hexon capsid protein, Adenovirus; D013835:Thiamine Pyrophosphate