Disulfide cross-linking reveals a site of stable interaction between C-terminal regulatory domains of the two MalK subunits in the maltose transport complex.
Understanding the structure and function of the ATP-binding cassette (ABC) transporters is very important because defects in ABC transporters lie at the root of several serious diseases including cystic fibrosis. MalK, the ATP-binding cassette of the maltose transporter of Escherichia coli, is distinct from most other ATP-binding cassettes in that it contains an additional C-terminal regulatory domain. The published structure of a MalK dimer is elongated with C-terminal domains at opposite poles (Diederichs, K., Diez, J., Greller, G., Muller, C., Breed, J., Schnell, C., Vonrhein, C., Boos, W., and Welte, W. (2000) EMBO J. 19, 5951-5961). Some uncertainty exists as to whether the orientation of MalK in the dimer structure is correct. Superpositioning of the N-terminal domains of MalK onto the ATP-binding domains of an alternate ABC dimer, in which ATP is bound along the dimer interface between Walker A and LSGGQ motifs, places both N- and C-terminal domains of MalK along the dimer interface. Consistent with this model, a cysteine substitution at position 313 in the C-terminal domain of an otherwise cysteine-free MalK triggered disulfide bond formation between two MalK subunits in an intact maltose transporter. Disulfide bond formation did not inhibit the function of the transporter, suggesting that the C-terminal domains of MalK remain in close proximity throughout the transport cycle. Enzyme IIAglc still inhibited the ATPase activity of the disulfide-linked transporter indicating that the mechanism of inducer exclusion was unaffected. These data support a model for ATP hydrolysis in which the C-terminal domains of MalK remain in contact whereas the N-terminal domains of MalK open and close to allow nucleotide binding and dissociation.
Samanta
Susmita
S
Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas 77030, USA.
Ayvaz
Tulin
T
Reyes
Moriama
M
Shuman
Howard A
HA
Chen
Jue
J
Davidson
Amy L
AL
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers; D004220:Disulfides / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008320:Maltose / Q000378:metabolism*; D008958:Models, Molecular; D009004:Monosaccharide Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D017931:DNA Primers; D004220:Disulfides; D029968:Escherichia coli Proteins; C461639:MalF protein, E coli; C079123:MalG protein, E coli; C068021:MalK protein, Bacteria; C489398:MalK protein, E coli; D009004:Monosaccharide Transport Proteins; D021122:Protein Subunits; C461599:maltose transport system, E coli; D008320:Maltose; D000251:Adenosine Triphosphatases