The hydroxyl group of S685 in Walker A motif and the carboxyl group of D792 in Walker B motif of NBD1 play a crucial role for multidrug resistance protein folding and function.
Structural analysis of MRP1-NBD1 revealed that the Walker A S685 forms hydrogen-bond with the Walker B D792 and interacts with magnesium and the beta-phosphate of the bound ATP. We have found that substitution of the D792 with leucine resulted in misfolding of the protein. In this report we tested whether substitution of the S685 with residues that prevent formation of this hydrogen-bond would also cause misfolding. Indeed, substitution of the S685 with residues potentially preventing formation of this hydrogen-bond resulted in misfolding of the protein. In addition, some substitutions that might form hydrogen-bond with D792 also yielded immature protein. All these mutants are temperature-sensitive variants. However, these complex-glycosylated mature mutants prepared from the cells grown at 27 degrees C still significantly affect ATP binding and ATP-dependent solute transport. In contrast, substitution of the S685 with threonine yielded complex-glycosylated mature protein that is more active than the wild-type MRP1, indicating that the interaction between the hydroxyl group of 685 residue and the carboxyl group of D792 plays a crucial role for the protein folding and the interactions of the hydroxyl group at 685 with magnesium and the beta-phosphate of the bound ATP play an important role for ATP-binding and ATP-dependent solute transport.
Yang
Runying
R
Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, 13400 East Shea Boulevard, Scottsdale, AZ 85259, USA.
Scavetta
Robert
R
Chang
Xiu-Bao
XB
D000255:Adenosine Triphosphate / Q000378:metabolism; D020816:Amino Acid Motifs; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D006224:Cricetinae; D017931:DNA Primers; D006860:Hydrogen Bonding; D008958:Models, Molecular; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding
D000255:Adenosine Triphosphate; D017931:DNA Primers; D027425:ATP-Binding Cassette, Sub-Family C Proteins; C421109:multidrug resistance-associated protein 1