Interactions among the A and T units of an ECF-type biotin transporter analyzed by site-specific crosslinking

• Published in: PloS one 2011-12, Vol.6 (12), p.e29087-e29087
• Main Authors: Neubauer, Olivia, Reiffler, Christin, Behrendt, Laura, Eitinger, Thomas
• Format: Article
• Language: English
• Subjects: ABC transporters; Acids; Adenosine triphosphatase; Amino acids; Analysis; ATPases; Biology; Biotin; Catalytic oxidation; Coupling; Crosslinking; Cysteine; Cysteine - chemistry; Cystine; E coli; Electrophoretic Mobility Shift Assay; Escherichia coli; Lactose; Polymer crosslinking; Prokaryotes; Proteins; Proto-Oncogene Proteins c-myc - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Residues; Signatures; Symporters - chemistry; Symporters - metabolism
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0029087

Energy-coupling factor (ECF) transporters are a huge group of micronutrient importers in prokaryotes. They are composed of a substrate-specific transmembrane protein (S component) and a module consisting of a moderately conserved transmembrane protein (T component) and two ABC ATPase domains (A components). Modules of A and T units may be dedicated to a specific S component or shared by many different S units in an organism. The mode of subunit interactions in ECF transporters is largely unknown. BioMNY, the focus of the present study, is a biotin transporter with a dedicated AT module. It consists of the S unit BioY, the A unit BioM and the T unit BioN. Like all T units, BioN contains two three-amino-acid signatures with a central Arg residue in a cytoplasmic helical region. Our previous work had demonstrated a central role of the two motifs in T units for stability and function of BioMNY and other ECF transporters. Here we show by site-specific crosslinking of pairs of mono-cysteine variants that the Ala-Arg-Ser and Ala-Arg-Gly signatures in BioN are coupling sites to the BioM ATPases. Analysis of 64 BioN-BioM pairs uncovered interactions of both signatures predominantly with a segment of ~13 amino acid residues C-terminal of the Q loop of BioM. Our results further demonstrate that portions of all BioN variants with single Cys residues in the two signatures are crosslinked to homodimers. This finding may point to a dimeric architecture of the T unit in BioMNY complexes.