Interactions among the A and T units of an ECF-type biotin transporter analyzed by site-specific crosslinking.
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.
Institut für Biologie/Mikrobiologie, Humboldt-Universität zu Berlin, Berlin, Germany.
Olivia
O
Neubauer
Christin
C
Reiffler
Laura
L
Behrendt
Thomas
T
Eitinger
D003545:Cysteine / Q000737:chemistry; D024202:Electrophoretic Mobility Shift Assay; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D027981:Symporters / Q000737:chemistry / Q000378:metabolism*
D016271:Proto-Oncogene Proteins c-myc; D011994:Recombinant Proteins; D027981:Symporters; C119981:biotin transporter; D003545:Cysteine