Cooperative, ATP-dependent Association of the Nucleotide Binding Cassettes during the Catalytic Cycle of ATP-binding Cassette Transporters

Abstract

ATP-binding cassette (ABC) transporters harvest the energy present in cellular ATP to drive the translocation of a structurally diverse set of solutes across the membrane barriers of eubacteria, archaebacteria, and eukaryotes. The positively cooperative ATPase activity (Hill coefficient, 1.7) of a model soluble cassette of known structure, MJ0796, from Methanococcus jannaschii indicates that at least two binding sites participate in the catalytic reaction. Mutation of the catalytic base in MJ0796, E171Q, produced a cassette that can bind but not efficiently hydrolyze ATP. The equivalent mutation (E179Q) in a homologous cassette, MJ1267, had an identical effect. Both mutant cassettes formed dimers in the presence of ATP but not ADP, indicating that the energy of ATP binding is first coupled to the transport cycle through a domain association reaction. The non-hydrolyzable nucleotides adenosine 5-(␤,␥-imino)triphosphate and adenosine 5-3-O-(thio)triphosphate were poor analogues of ATP in terms of their ability to promote dimerization. Moreover, inclusion of MgCl 2 , substitution of KCl for NaCl, or alterations in the polarity of the side chain at the catalytic base all weakened the ATP-dependent dimer, suggesting that electrostatic interactions are critical for the association reaction. Thus, upon hydrolysis of bound ATP and the release of product, both electrostatic and conformational changes drive the cassettes apart, providing a second opportunity to couple free energy changes to the transport reaction. ATP-binding cassette (ABC)1 transporters are ubiquitous membrane proteins that couple ATP hydrolysis to the energydependent transport of a wide variety of molecules across lipid bilayers. They comprise the single largest gene family in several sequenced prokaryotic genomes (1). Mutations in human ABC transporters underlie diseases such as cystic fibrosis, hypercholesterolemia, adrenoleukodystrophy, and Stargardt's disease, while multidrug resistance in cancer cells and infectious microorganisms often arises from the overexpression of ABC transporters that serve as drug efflux pumps (2, 3).The ABC transporters share an invariant domain organization of two conserved cytoplasmic nucleotide binding cassettes associated with two transmembrane (TM) domains (2). The cassettes contain three highly conserved motifs required for nucleotide binding and hydrolysis: the Walker A site (GX 4 GK(S/T), where X ϭ any residue) and the Walker B site (RX 6 -8 ⌽ 4 D, where ⌽ ϭ hydrophobic residue) (4), which reside in the ␣␤ core of the cassette (5-11), and the LSGGQ signature sequence (1, 2), which lies more toward the periphery of the cassette in an ␣-helical subdomain (5-11). The TM domains that mediate the movement of the structurally diverse solutes exhibit less sequence conservation (1, 2). The organization of prokaryotic transporter operons and of single polypeptide chain transporters suggests that the minimal functional unit consists of at least two cassettes and two TM domains (5).The crystal structures of a homodimeric half...

The motor domains of ABC-transporters

“…However, recently, some interesting findings have contributed enormously to this field. First, Moody et al (2002) proposed that the Glu residue at the end of the Walker B sequence is the general or catalytic base of the NBD. This idea is based on the lack of measured ATPase activity when the Glu in question is mutated to Gln in MJ0796 and MJ1267 .…”

Intracellular peptide transporters in human – compartmentalization of the “peptidome”

“…The second lobe (arm II) forms an α-helical structure containing the C-loop and the Q-loop motif [28]. It was shown that in the presence of MgATP, the NBDs dimerize [9,36,57,63,82]. In this state, two ATP molecules are bound at the interface of two monomers and sandwiched between the Walker A and B motifs of one NBD and the C-loop of the opposite NBD.…”

Characterization of the LSGGQ and H Motifs from the Escherichia coli Lipid A Transporter MsbA

“…Bacterial efflux ABC transporters transport a variety of solutes † This work was supported by grant GM070642 from the NIH. ATP (25;26). Also in the E. coli MsbA crystal structure, the NBD conserved Walker A domain is unresolved and the LSGGQ motif appears to be out of position for ATP hydrolysis since it must act in conjunction with the Walker A domain on the opposite monomer in order to bind ATP (Fig.…”