title: Glutamine residues in Q-loops of multidrug resistance protein MRP1 contribute to ATP binding via interaction with metal cofactor.
abstract: Structural analyses of bacterial ATP-binding-cassette transporters revealed that the glutamine residue in Q-loop plays roles in interacting with: 1) a metal cofactor to participate in ATP binding; 2) a putative catalytic water molecule to participate in ATP hydrolysis; 3) other residues to transmit the conformational changes between nucleotide-binding-domains and transmembrane-domains, in ATP-dependent solute transport. We have mutated the glutamines at 713 and 1375 to asparagine, methionine or leucine to determine the functional roles of these residues in Q-loops of MRP1. All these single mutants significantly decreased Mg·ATP binding and increased the K(m) (Mg·ATP) and V(max) values in Mg·ATP-dependent leukotriene-C4 transport. However, the V(max) values of the double mutants Q713N/Q1375N, Q713M/Q1375M and Q713L/Q1375L were lower than that of wtMRP1, implying that the double mutants cannot efficiently bind Mg·ATP. Interestingly, MRP1 has higher affinity for Mn·ATP than for Mg·ATP and the Mn·ATP-dependent leukotriene-C4 transport activities of Q713N/Q1375N and Q713M/Q1375M are significantly higher than that of wtMRP1. All these results suggest that: 1) the glutamine residues in Q-loops contribute to ATP-binding via interaction with a metal cofactor; 2) it is most unlikely that these glutamine residues would play crucial roles in ATP hydrolysis and in transmitting the conformational changes between nucleotide-binding-domains and transmembrane-domains.
authors.affiliation: Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Mayo Clinic, Scottsdale, AZ, USA.
authors.forename: Runying
authors.initials: R
authors.lastname: Yang
authors.forename: Yue-xian
authors.initials: YX
authors.lastname: Hou
authors.forename: Chase A
authors.initials: CA
authors.lastname: Campbell
authors.forename: Kanagaraj
authors.initials: K
authors.lastname: Palaniyandi
authors.forename: Qing
authors.initials: Q
authors.lastname: Zhao
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Bordner
authors.forename: Xiu-bao
authors.initials: XB
authors.lastname: Chang
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D017931:DNA Primers; D018432:Drug Resistance, Multiple; D005973:Glutamine / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D019920:Photoaffinity Labels; D018411:Spodoptera
chemical_list: D000255:Adenosine Triphosphate; D017931:DNA Primers; D005973:Glutamine; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D019920:Photoaffinity Labels; C421109:multidrug resistance-associated protein 1