Understanding the Role of ABC Transporters

sgene

Last updated 2025-04-27

1. Introduction

The ABCs are the largest family of proteins found in all living organisms, from Archaea to humans, constituting about 4% of the genome in certain bacteria like Escherichia coli. Eukaryotic genomes contain numerous ABC proteins, characterized by a consensus nucleotide-binding domain (NBD) of around 100 amino acids. This domain features several structural motifs, including the Walker A and B motifs, the ABC1 signature, and various loops marked by specific amino acids.

ABC proteins are involved in a range of physiological functions, particularly in transport processes. The text aims to discuss the major physiological, biochemical functions, and structural properties of well-known ABC transporters, specifically highlighting Sav1866, Pdr5p, and P-gp.

2. Structure of ABC Transporters

Every ABC transporter molecule is composed of, or linked to, one or two cytoplasmic adenosine triphosphate (ATP)-binding domains known as nucleotide-binding domains (NBDs), along with one or two transmembrane domains (TMDs). Typically, each TMD consists of six transmembrane spans (TMSs) interspersed with three extracytoplasmic loops (ECL1, ECL2, ECL3) and two intracytoplasmic loops (ICL2, ICL3). The combination of one TMD with one NBD creates a half-size ABC transporter, which operates as either homo- or heterodimers. Therefore, the essential functional structure of an ABC transporter is regarded as TMD–NBD/TMD–NBD or NBD–TMD/NBD–TMD.

In bacteria, it is common for two NBDs to be paired with two TMDs, either as four individual subunits encoded within the same operon or in various combinations of combined subunits.

Other proteins may also associate with these transporters. The most notable associated bacterial protein is the periplasmic solute-binding receptor, which is located in the periplasm of Gram-negative bacteria, while in Gram-positive bacteria, it typically exists as a lipoprotein attached to the outer membrane surface through electrostatic interactions.

The three domains of bacterial ABC uptake transporters—the periplasmic-binding receptor, the cytoplasmic NBD, and the membrane TMD—are thought to have evolved from a common ancestral ABC transporter that already contained these three proteins. However, through the course of evolution, the sequence of the periplasmic solute-binding receptors has diverged more quickly than that of the TMDs, whereas the NBDs have shown the least divergence. Consequently, all NBDs are homologous, but this does not apply to the TMDs or the receptors.

In eukaryotic organisms, two TMDs and two NBDs are frequently, though not always, found together in a single entity referred to as a full-sized ABC transporter. The topological relationship between NBDs and TMDs can vary; certain systems may include additional TM spans as well as extracytoplasmic domains thought to have regulatory roles.

3. Functions of ABC Transporters

The ABC transporters found in Archaea, as well as in the plasma membranes of Gram-negative and Gram-positive organisms, show significant differences in their structure and makeup. The range of substrates they transport—including sugars, amino acids, lipids, ions, polysaccharides, peptides, proteins, toxins, drugs, antibiotics, xenobiotics, and other metabolites—reflects the variations in the periplasmic sensor and TMD that dictate the specificity and transport direction (either efflux or influx).

Although all eukaryotic ABC transporters function primarily as effluxers, each comprising a TMD fused to an NBD, not all are directly responsible for substrate movement. For example, in the cystic fibrosis transmembrane conductance regulator (CFTR) and the sulfonylurea receptor (SUR), ATP hydrolysis seems to be associated with regulating the opening and closing of ion channels managed by the ABC protein itself or by other proteins.

Nevertheless, the presence of both NBD and TMD in all ABC transporters indicates a fundamental coupling mechanism for both efflux and influx, regardless of the substrate being transported. Interestingly, even distantly related proteins can employ an NBD to facilitate various non-transport functions such as DNA repair, protein elongation, or the regulation of RNase activities.

4. ABC Transporters in Human Health

A significant motivation for researching mammalian ABC transporters lies in their role in human diseases. Numerous Mendelian disorders and complex genetic conditions are linked to ABC transporters, including:

Cystic fibrosis
Adrenoleukodystrophy
Stargardt disease
Tangier disease
Immune deficiencies
Progressive familial intrahepatic cholestasis
Dublin–Johnson syndrome
Pseudoxanthoma elasticum
Persistent hyperinsulinemic hypoglycemia of infancy (due to focal adenomatous hyperplasia)
X-linked sideroblastosis and anemia
Age-related macular degeneration
Familial hypoapoproteinemia
Fundus flavimaculatis
Retinitis pigmentosa
Cone-rod dystrophy

Common occurrences of resistance to plant, fungal, or mammalian pathogens, as well as to chemotherapy treatments for cancer, are often due to the induction or activating mutations of pleiotropic drug resistance (Pdrp), multiple drug resistance (MDR), or multidrug resistance-related protein (MRP) transporters.

5. Structure and Biochemical Mechanism

In 2010, more than a dozen high-resolution X-ray structures of functional ABC transporters were accessible, significantly aiding in the development of a unified understanding of the transport catalytic mechanism. Recent evaluations at the electron microscopy level of purified bacterial (BmrA from B. subtilis) and fungal (Pdr5p from S. cerevisiae) drug efflux ABC transporters have led to a remarkably consistent set of findings.

In both instances, the fundamental structural unit appears to consist of four interconnected NBDs, corresponding to either two full-size Pdr5p or four half-size BmrA. The NBDs are connected to the TMDs via four separate stalks. Each NBD maintains a fixed orientation at a 90-degree angle in relation to its adjacent NBDs. This suggests the potential for coordinated rotational movements of the NBDs, indicating some degree of flexibility in the stalks. No intramolecular or intramembrane pores were detected, even though there is ample space (or chamber) between the four stalks that converge at their NBD tips.

The characteristics of the stalks, TMD, and NBD were further clarified through the examination of well-defined structures of complete dimeric ABC transporters from both bacteria and eukaryotes, such as:

The phospholipid flippase MsbA from E. coli and Staphylococcus typhimurium,
The vitamin B12 transporter BtuCD from E. coli,
The metal-chelate importer HI1470/1 from Haemophilus influenzae,
The drug exporter Sav1866 from S. aureus,
The *mammalian multidrug exporter P-gp.

The arrangements of the membrane and stalk domains were found to vary across the different structures analyzed. This variability is expected, given the diverse species studied, co-crystallized ligands, numbers of transmembrane segments (TMS), and functions of the proteins.

Notably, the interaction between the NBD and TMD was found to differ:

In MsbA, via a long and intricate intracytoplasmic loop (ICL),
In BtuCD, through a short L-shaped ICL connecting TMS 6 and 7,
In Sav1866, via ICL3 and ICL6.

A significant observation from the X-ray structures is that two ATP molecules bind at the interface of the two NBDs. Each nucleotide-binding site consists of a Walker A motif from one monomer and an ABC1 C motif from the other monomer, arranged in a head-to-tail fashion of the two interacting NBD monomers.

5.1 Sav1866 Structure

Given the high variability in ABC transporter structures, we focus on key structural features of the bacterial Sav1866 and mammalian P-gp multidrug exporters, as reported by Kaspar Locher (2006) and Goffrey Chang (2009), respectively.

The homodimeric Sav1866 transporter consists of two identical subunits, each containing a nucleotide-binding domain (NBD) fused with a transmembrane domain (TMD) formed by six transmembrane α-helices. It facilitates the export of a diverse array of unrelated pharmaceuticals, including anticancer agents like doxorubicin and vinblastine.

Two coupling α-helices, situated in the intracellular loops ICL3 and ICL6, interact with grooves from the two NBDs. This structural feature is preserved across all ABC transporters, though the amino acid composition varies.

The individual TMSs from the two TMDs interlock, creating two symmetrical membrane bundles, each with six mixed TMS made up of two membrane α-helices from one TMD and four from the other. A central cavity between the TMDs, oriented outward, is interpreted as an open pocket for drug extrusion.

5.2 P-Glycoprotein (P-gp) Structure

P-glycoprotein (P-gp) is a complete NBD–TMD–NBD–TMD transporter that contributes to multidrug resistance in both mouse and human cells, linked to the unsuccessful treatment of cancer patients undergoing chemotherapy.

The mouse P-gp was expressed in yeast (Pichia pastoris), purified, and crystallized. The structure, resolved at 3.8 Å, represents a nucleotide-free, inward-facing conformation organized into two symmetrical halves, each containing an NBD and a transmembrane bundle.

Each transmembrane bundle includes six mixed TMs where segments from the two TMDs intertwine. A substantial drug-binding pocket, approximately 6000 cubic angstroms in size, is located at the junction of the two TM bundles, accessible to both the inner leaflet of the membrane and the cytoplasm.

A total of 73 aromatic and hydrophobic amino acid residues, facing this cavity, are solvent-accessible and contribute to the overlapping binding of three distinct ABC inhibitors, with only two amino acids being common across all three binding sites. Two openings are fully accessible to the inner phospholipid bilayer, allowing the entry of hydrophobic drugs that diffuse passively across the membrane.

6. Phylogeny

The various groups of ABC proteins facilitate the transport of a diverse range of substrates against their concentration gradients by utilizing the energy released from ATP hydrolysis performed by the nucleotide-binding domain (NBD). In bacteria, the substrates are either brought into or expelled from the cell. In eukaryotic organisms, only extracytoplasmic exporters, which move substrates either out of the cell or into organelles, have been recognized.

A total of 173 phylogenetic families have been identified and categorized within the transporter classification (TC) system, which was established by Milton Saier in San Diego. These families typically correspond to substrate specificity and include both efflux and influx transporters.

Since 1995, the computational analysis of numerous complete genome sequences has made it possible to discover thousands of new ABC proteins. For example, among the 49 ABC proteins found in the human genome, five primary transporter families have been identified and designated as:

ABCA (or ABC1),
ABCB (or MDR, which includes the well-known P-gp mentioned below),
ABCC (or MRP),
ABCD (or ALD),
ABCG (or WHITE).

This classification is consistent with the TC system.

The genome of S. cerevisiae contains 32 ABC proteins, of which 22 are linked to TMDs. The largest ABC family in yeast, Pdrp, was identified in 1997 by Anabelle Decottignies and André Goffeau, and it has since been shown to exist in all fungi and plants.

This family is absent in the animal kingdom. In contrast, the sizable human and mouse ABCA family, identified in 1994 by Giovanna Chimini, is not found in fungal genomes.

7. Substrate Specificity of the Multidrug Exporters and Reversal Agents

One of the most fascinating challenges in modern biochemistry is understanding how the transmembrane domains (TMDs) interact with their respective substrates. A particularly striking aspect of this issue is the seemingly non-specific nature of the yeast Pdr5p and human P-glycoprotein (Pgp), which are capable of transporting hundreds of distinct chemicals, seemingly contradicting the well-known lock-and-key model outlined in textbooks.

It has now been established that at least three partially overlapping drug-binding sites function in multidrug resistance (MDR), multidrug resistance-associated protein (MRP), and Pdrp transporters, with drug binding being influenced by:

The hydrogen-bonding characteristics of acceptors,
The presence of aromatic structures,
Hydrophobicity, and
The surface area of the substrates.

Since 1980, significant efforts have been undertaken to discover inhibitors that can reverse MDR in cancer cells or Pdrp in fungi. More than 30 compounds with enigmatic names and structures have been identified as inhibitors of the cancer-associated efflux pumps:

ABCB1 (MDR),
ABCC1 (MRP),
ABCG2 (BCRP), as well as
The fungal Pdrp transporters.

These inhibitors may function in several ways:

As pseudosubstrates,
As competitive blockers of ATP binding,
As non-competitive inhibitors acting on distant sites, or
Indirectly by influencing metabolic processes that impact efflux.

The range of identified drug pump inhibitors suggests the potential to develop effective and specific agents against anticancer and antifungal pumps. Nonetheless, there are currently no ABC pump inhibitors utilized in clinical settings.

It is evident that many pump inhibitors—including antimalarial quinine derivatives and the immunosuppressant FK506—affect various physiological functions. Furthermore, ABC drug pumps appear to play crucial roles in important biological processes unrelated to drug efflux, such as the:

Development,
Differentiation, and
Maturation of immune cells.

This likely accounts for the numerous side effects associated with these inhibitors. Until the molecular structure and mechanisms of drug efflux are thoroughly understood, the development of anti-MDR or Pdrp agents will likely remain ineffective and reliant on random screening. The responsibility now lies with scientists investigating fundamental mechanisms.

7.1 Promising Clinical Alternatives

Nevertheless, some unconventional clinical treatments have been proposed. For example:

Low doses of ibuprofen, a powerful anti-inflammatory medication, have been shown to inhibit azole efflux from Candida albicans.
This has led to suggestions for its use in combination therapy for fungal infections alongside fluconazole.

Additionally, alternative approaches such as:

Modulating the expression of drug transporters, or
Enhancing the uptake of anticancer drugs

...are also being actively explored in current biomedical research.

8. Conclusion

The upcoming challenges in the exploration of ATP-binding cassette (ABC) transporters are both vast and significant. Despite extensive research, numerous elements of the evolutionary background of this extensive and widespread protein family remain largely undiscovered.

To advance the field, several key areas must be addressed:

Improved heterologous overexpression systems need to be developed to support deeper biochemical and mechanistic investigations.
Additional atomic-level structures are essential to fully reveal the conformational changes that occur during the ATP-driven transport cycle.
The diverse mechanisms of ABC transporters across bacteria, fungi, plants, and animals require comprehensive clarification.
Targeted inhibitors for both ABC drug importers and exporters should be identified through high-throughput screening methods.
The physiological processes associated with ABC-linked diseases must be investigated further, particularly through the use of mouse knockout models.
New interfering RNA systems should be explored to enable specific inhibition of ABC transporter expression.
Finally, there is a pressing need for the development of genetic therapies targeting ABC-related diseases.

The complexity and importance of ABC transporters in health, disease, and drug resistance underscore the necessity of continued, multidisciplinary research. Bridging the gap between molecular mechanisms and clinical applications remains a central goal for the future of ABC transporter studies.