Modulation of ATPase Activity by Physical Disengagement of the ATP-binding Domains of an ABC Transporter, the Histidine Permease

ABSTRACT The yeast cadmium factor (Ycf1p) is a vacuolar ATP binding cassette (ABC) transporter required for heavy metal and drug detoxification. Cluster analysis shows that Ycf1p is strongly related to the human multidrug-associated protein (MRP1) and cystic fibrosis transmembrane conductance regulator and therefore may serve as an excellent model for the study of eukaryotic ABC transporter structure and function. Identifying intramolecular interactions in these transporters may help to elucidate energy transfer mechanisms during transport. To identify regions in Ycf1p that may interact to couple ATPase activity to substrate binding and/or movement across the membrane, we sought intragenic suppressors of ycf1 mutations that affect highly conserved residues presumably involved in ATP binding and/or hydrolysis. Thirteen intragenic second-site suppressors were identified for the D777N mutation which affects the invariant Asp residue in the Walker B motif of the first nucleotide binding domain (NBD1). Two of the suppressor mutations (V543I and F565L) are located in the first transmembrane domain (TMD1), nine (A1003V, A1021T, A1021V, N1027D, Q1107R, G1207D, G1207S, S1212L, and W1225C) are found within TMD2, one (S674L) is in NBD1, and another one (R1415G) is in NBD2, indicating either physical proximity or functional interactions between NBD1 and the other three domains. The original D777N mutant protein exhibits a strong defect in the apparent affinity for ATP and V max of transport. The phenotypic characterization of the suppressor mutants shows that suppression does not result from restoring these alterations but rather from a change in substrate specificity. We discuss the possible involvement of Asp777 in coupling ATPase activity to substrate binding and/or transport across the membrane.

Download Full-text

The Walker B motif of the second nucleotide-binding domain (NBD2) of CFTR plays a key role in ATPase activity by the NBD1–NBD2 heterodimer

Biochemical Journal ◽

10.1042/bj20060968 ◽

2006 ◽

Vol 401 (2) ◽

pp. 581-586 ◽

Cited By ~ 29

Author(s):

Fiona L. L. Stratford ◽

Mohabir Ramjeesingh ◽

Joanne C. Cheung ◽

Ling-JUN Huan ◽

Christine E. Bear

Keyword(s):

Atpase Activity ◽

Atp Hydrolysis ◽

Nucleotide Binding ◽

Vital Role ◽

Atp Binding ◽

Primary Role ◽

Binding Domains ◽

Membrane Spanning ◽

Atp Binding And Hydrolysis

CFTR (cystic fibrosis transmembrane conductance regulator), a member of the ABC (ATP-binding cassette) superfamily of membrane proteins, possesses two NBDs (nucleotide-binding domains) in addition to two MSDs (membrane spanning domains) and the regulatory ‘R’ domain. The two NBDs of CFTR have been modelled as a heterodimer, stabilized by ATP binding at two sites in the NBD interface. It has been suggested that ATP hydrolysis occurs at only one of these sites as the putative catalytic base is only conserved in NBD2 of CFTR (Glu1371), but not in NBD1 where the corresponding residue is a serine, Ser573. Previously, we showed that fragments of CFTR corresponding to NBD1 and NBD2 can be purified and co-reconstituted to form a heterodimer capable of ATPase activity. In the present study, we show that the two NBD fragments form a complex in vivo, supporting the utility of this model system to evaluate the role of Glu1371 in ATP binding and hydrolysis. The present studies revealed that a mutant NBD2 (E1371Q) retains wild-type nucleotide binding affinity of NBD2. On the other hand, this substitution abolished the ATPase activity formed by the co-purified complex. Interestingly, introduction of a glutamate residue in place of the non-conserved Ser573 in NBD1 did not confer additional ATPase activity by the heterodimer, implicating a vital role for multiple residues in formation of the catalytic site. These findings provide the first biochemical evidence suggesting that the Walker B residue: Glu1371, plays a primary role in the ATPase activity conferred by the NBD1–NBD2 heterodimer.

Download Full-text

Interaction of the Nucleotide Binding Domains and Regulation of the ATPase Activity of the Human Retina Specific ABC Transporter, ABCR†

Biochemistry ◽

10.1021/bi052059u ◽

2006 ◽

Vol 45 (11) ◽

pp. 3813-3823 ◽

Cited By ~ 9

Author(s):

Esther E. Biswas-Fiss

Keyword(s):

Atpase Activity ◽

Abc Transporter ◽

Nucleotide Binding ◽

Human Retina ◽

Binding Domains

Download Full-text

Insect ATP-Binding Cassette (ABC) Transporters: Roles in Xenobiotic Detoxification and Bt Insecticidal Activity

International Journal of Molecular Sciences ◽

10.3390/ijms20112829 ◽

2019 ◽

Vol 20 (11) ◽

pp. 2829 ◽

Cited By ~ 9

Author(s):

Chao Wu ◽

Swapan Chakrabarty ◽

Minghui Jin ◽

Kaiyu Liu ◽

Yutao Xiao

Keyword(s):

Abc Transporter ◽

Conformational Changes ◽

Abc Transporters ◽

Insecticidal Activity ◽

Atp Hydrolysis ◽

Atp Binding ◽

Bt Toxin ◽

Xenobiotic Detoxification ◽

Binding Domains ◽

Atp Binding Cassette

ATP-binding cassette (ABC) transporters, a large class of transmembrane proteins, are widely found in organisms and play an important role in the transport of xenobiotics. Insect ABC transporters are involved in insecticide detoxification and Bacillus thuringiensis (Bt) toxin perforation. The complete ABC transporter is composed of two hydrophobic transmembrane domains (TMDs) and two nucleotide binding domains (NBDs). Conformational changes that are needed for their action are mediated by ATP hydrolysis. According to the similarity among their sequences and organization of conserved ATP-binding cassette domains, insect ABC transporters have been divided into eight subfamilies (ABCA–ABCH). This review describes the functions and mechanisms of ABC transporters in insecticide detoxification, plant toxic secondary metabolites transport and insecticidal activity of Bt toxin. With improved understanding of the role and mechanisms of ABC transporter in resistance to insecticides and Bt toxins, we can identify valuable target sites for developing new strategies to control pests and manage resistance and achieve green pest control.

Download Full-text

Functional asymmetry of the ATP-binding-cassettes of the ABC transporter TAP is determined by intrinsic properties of the nucleotide binding domains

European Journal of Biochemistry ◽

10.1046/j.1432-1327.2001.02406.x ◽

2001 ◽

Vol 268 (17) ◽

pp. 4776-4786 ◽

Cited By ~ 25

Author(s):

Oliver Daumke ◽

Michael R. Knittler

Keyword(s):

Abc Transporter ◽

Nucleotide Binding ◽

Functional Asymmetry ◽

Atp Binding ◽

Intrinsic Properties ◽

Binding Domains ◽

Atp Binding Cassettes

Download Full-text

Exploring conformational equilibria of a heterodimeric ABC transporter

eLife ◽

10.7554/elife.20236 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 42

Author(s):

M Hadi Timachi ◽

Cedric AJ Hutter ◽

Michael Hohl ◽

Tufa Assafa ◽

Simon Böhm ◽

...

Keyword(s):

Binding Site ◽

High Temperatures ◽

Abc Transporter ◽

Thermotoga Maritima ◽

Nucleotide Binding ◽

Atp Binding ◽

Molecular Feature ◽

Atp Binding Site ◽

Binding Domains ◽

Conformational Equilibria

ABC exporters pump substrates across the membrane by coupling ATP-driven movements of nucleotide binding domains (NBDs) to the transmembrane domains (TMDs), which switch between inward- and outward-facing (IF, OF) orientations. DEER measurements on the heterodimeric ABC exporter TM287/288 from Thermotoga maritima, which contains a non-canonical ATP binding site, revealed that in the presence of nucleotides the transporter exists in an IF/OF equilibrium. While ATP binding was sufficient to partially populate the OF state, nucleotide trapping in the pre- or post-hydrolytic state was required for a pronounced conformational shift. At physiologically high temperatures and in the absence of nucleotides, the NBDs disengage asymmetrically while the conformation of the TMDs remains unchanged. Nucleotide binding at the degenerate ATP site prevents complete NBD separation, a molecular feature differentiating heterodimeric from homodimeric ABC exporters. Our data suggest hydrolysis-independent closure of the NBD dimer, which is further stabilized as the consensus site nucleotide is committed to hydrolysis.

Download Full-text

The Human ATP-Binding Cassette (ABC) Transporter Superfamily

Genome Research ◽

10.1101/gr.184901 ◽

2001 ◽

Vol 11 (7) ◽

pp. 1156-1166

Author(s):

Michael Dean ◽

Andrey Rzhetsky ◽

Rando Allikmets

Keyword(s):

Abc Transporter ◽

Complete Characterization ◽

Model Organisms ◽

Atp Binding ◽

Evolutionary Analysis ◽

Inherited Disease ◽

New Members ◽

Binding Domains ◽

Human Disorders ◽

Atp Binding Cassette

The ATP-binding cassette (ABC) transporter superfamily contains membrane proteins that translocate a variety of substrates across extra- and intra-cellular membranes. Genetic variation in these genes is the cause of or contributor to a wide variety of human disorders with Mendelian and complex inheritance, including cystic fibrosis, neurological disease, retinal degeneration, cholesterol and bile transport defects, anemia, and drug response. Conservation of the ATP-binding domains of these genes has allowed the identification of new members of the superfamily based on nucleotide and protein sequence homology. Phylogenetic analysis is used to divide all 48 known ABC transporters into seven distinct subfamilies of proteins. For each gene, the precise map location on human chromosomes, expression data, and localization within the superfamily has been determined. These data allow predictions to be made as to potential functions or disease phenotypes associated with each protein. In this paper, we review the current state of knowledge on all human ABC genes in inherited disease and drug resistance. In addition, the availability of the completeDrosophila genome sequence allows the comparison of the known human ABC genes with those in the fly genome. The combined data enable an evolutionary analysis of the superfamily. Complete characterization of all ABC from the human genome and from model organisms will lead to important insights into the physiology and the molecular basis of many human disorders.

Download Full-text