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Thiamine diphosphate binds to intermediates in the assembly of adenovirus fiber knob trimers in Escherichia coli
Author(s) -
Schulz Ryan,
Zhang YianBiao,
Liu ChangJun,
Freimuth Paul
Publication year - 2007
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.072805007
Subject(s) - trimer , dimer , chemistry , escherichia coli , thiamine , mutant , monomer , recombinant dna , stereochemistry , biophysics , biochemistry , biology , gene , organic chemistry , polymer
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.