Nucleotide-dependent Allostery within the ABC Transporter ATP-binding Cassette

Abstract

ATP-binding cassette (ABC)3 transporters couple hydrolysis of ATP to vectorial translocation of substrates across cellular membranes, typically against a concentration gradient. These integral membrane proteins are involved in the transport of a wide range of substrates, including ions, sugars, amino acids, peptides, proteins, lipids, and xenotoxins, and form one of the largest protein families across all species (1). Many ABC transporters are central to important biomedical problems, including resistance of cancers, microbes, and parasites to cytotoxic drugs, as well as human genetic diseases such cystic fibrosis and Stargardt disease (2). Understanding the molecular mechanism of these proteins is necessary for the rational design of agents to control their transport function. The architecture of ABC transporters comprises a conserved core structure of two transmembrane domains (TMDs) and two cytosolic ABCs, also known as nucleotide-binding domains (NBDs). Most prokaryotic ABC transporters are importers with the core TMDs and NBDs expressed as separate subunits, whereas most eukaryotic ABC transporters are exporters that commonly have a single polypeptide for the core structure, with each NBD C-terminal to each TMD. The primary sequences of the NBDs are generally highly conserved and contain the Walker A and B consensus motifs for ATP binding (3) and the "LSGG(Q)" motif or "signature sequence," which also interacts directly with ATP (4) (see Fig. 1A). The TMDs, which contain cytosolic as well as membrane-spanning regions, are less well conserved, reflecting their role in substrate binding and translocation.Notwithstanding recent advances in structural studies, crucial questions remain concerning how ATP binding and hydrolysis in the NBDs are coupled to protein conformational changes in the TMDs, which drive vectorial transmembrane substrate movement. Many protein monomers are composed of relatively independent subdomains, which are able to change conformation cooperatively and move as rigid bodies, thus providing the structural flexibility that enables the functioning of the enzyme (5). In other P-loop NBDs such as Ras, myosin, RecA, and F 1 -ATPase, NBD intradomain motions have been found, or are generally thought, to be central to the mechanism of the oligomeric complex, mediating the transduction of the free energy of ATP hydrolysis to functional changes in the protein conformation (6, 7). Crystallographic studies of the ABC ATPase have revealed a bilobal design (see Fig. 1A), in which a region comprising three ␣-helices, known as the helical domain (HD), is flexibly attached to the rest of the NBD and rotates as a rigid body (8 -10). The HD contains the LSGG signature sequence, which interacts with the nucleotide bound to the Walker A and B residues of the opposite monomer, thus forming two active sites in the NBD dimer (Fig. 1A). Enzymological studies have suggested that the HD mediates interdomain allosteric communication between the ATPase active site and the * The costs of publication of this article we...

Dynamics of α-helical subdomain rotation in the intact maltose ATP-binding cassette transporter

“…In addition, structural comparisons of isolated NBDs reveal a putative motion within each NBD, characterized by a rotation of the α-helical subdomain relative to the RecA-like subdomain upon ATP binding and hydrolysis (5)(6)(7)(8). This motion is also supported by molecular dynamic studies (10,11) and the rotation is suggested to transmit conformational changes to the transmembrane domains (16). Interestingly, the helical subdomain (14,39) is the most variable region of the NBDs (40,41) and might have evolved to accommodate the structural constraints imposed by the TMDs in ABC transporters.…”

“…Each NBD consists of a RecA-like subdomain, found in numerous ATPases (4), and an α-helical subdomain that is specific to the ABC family (5). Crystallographic studies (5)(6)(7)(8) and molecular dynamic simulations (9,10), performed on isolated NBDs, suggest that the helical subdomain rotates toward the RecA-like subdomain in response to ATP binding. This rotation positions the ABC family signature motif to interact with nucleotide across the dimer interface so that the NBDs can close to hydrolyze ATP.…”

Structure and mechanism of ATP-dependent phospholipid transporters

“…However, it has been shown that some of these transporters can be partially uncoupled in reconstituted systems [81,82]. A quest to understand the uncoupling process has led to extensive evidence that the two NBDs bind and hydrolyze ATP in a cooperative manner [83][84][85][86].…”

Kinetics of the Association/Dissociation Cycle of an ATP-binding Cassette Nucleotide-binding Domain

“…2) Constant contact model. The NBDs remain in contact during the hydrolysis cycle, and the power stroke results from smaller conformational changes at the NBD-dimer interface (8,(13)(14)(15)17).…”