A Tale of Two Transporters

Courtesy of Kaspar Locher B12 TRANSPORTER EXPOSED: Ribbon diagram of the B12 transporter BtuCD from E. coli, with cyclotetravanadate bound at the ATP-binding sites. The transporter is assembled from four subunits, two each of the membrane spanning BtuC (yellow and red) and the cytoplasmic BtuD (green and blue). The ATP binding cassette transporters epitomize nature's ability to re-use a successful protein motif. With diverse membrane-spanning regions, but highly conserved soluble ATP binding domains, the ABC transporters serve a wide variety of functions and are defunct in disorders such as Tangier disease and cystic fibrosis (see All in the Superfamily). Small wonder then, that the first high-resolution structures of this largest family of transporters were greeted with great acclaim. But there was puzzlement as well; the structures did not agree. "The initial MsbA structure came as a surprise ... although it was undoubtedly the result of an impressive tour de force," writes Kaspar Locher, of the Swiss Federal Institute of Technology in an E-mail interview. In the first of this issue's Hot Papers, Geoffrey Chang and Christopher Roth of the Scripps Research Institute, La Jolla, Calif., crystallized a lipid exporter known as MsbA from Escherichia coli.1 MsbA facilitates the export of lipid A, a critical outer membrane component, across the inner plasma membrane. Since it is essential for cell growth, MsbA is a potential target for new antibiotics. This issue's second Hot Paper, published several months later, described the structure of the vitamin B12 transporter BtuCD, also from E. coli.2 Locher crystallized the protein while in the laboratory of Chang's former mentor, Douglas Rees, at the California Institute of Technology. BtuCD imports vitamin B12. But the significance of these structures goes well beyond their roles in bacterial physiology. ABC transporters have great clinical importance, as they mediate multidrug resistance in bacteria and mammals. Bacterial ABC transporters severely limit antibiotic effectiveness by exporting them as fast as they enter the cell. In human cancers, ABC transporters export chemotherapy drugs, resulting in higher mortality rates. Because of the high homology within this family, the MsbA and BtuCD structures provide critical new tools with which to attack these major health problems. While the BtuCD structure closely matched researchers' predictions, the MsbA structure did not. Its transmembrane region contained the requisite 12 *-helix bundle, but its ATP binding regions were unlike any previously proposed model. Since then, researchers have been working to understand these differences and how they relate to 20 years of accumulated biochemical and genetic evidence. GREAT EXPECTATIONS Since 1982, more than a thousand ABC transporters have been cloned and sequenced in bacteria, yeast, human, and more. Diversity is their hallmark. Their substrates vary and include single ions, simple solutes such as sugars and amino acids, and large lipids and peptides. With their diverse mechanisms, some can import necessary nutrients into the cell, while others export harmful toxins. All use ATP hydrolysis, however, to power substrate transport across the membrane. Data derived from the Science Watch/Hot Papers database and the Web of Science (ISI, Philadelphia) show that Hot Papers are cited 50 to 100 times more often than the average paper of the same type and age. G. Chang, C.B. Roth, "Structure of MsbA from E. coli: A homolog of the multidrug resistance ATP binding cassette (ABC) transporters," Science, 293:1793-1800, 2001. (Cited in 148 papers) K. Locher et al., "The E. coli BtuCD structure: A framework for ABC transporter architecture and mechanism," Science, 296:1091-8, 2002. (Cited in 87 papers) All ABC transporters contain two hydrophobic transmembrane domains (TMDs), and two soluble nucleotide-binding domains (NBDs). Each NBD contains a single ATP binding site. This basic architecture stems from a remarkably large number of blueprints. Some ABC transporters are built from four separate polypeptides that associate into a functional tetramer. Others form homodimers, composed of two identical subunits each containing a TMD fused to an NBD. Still others, including human multi-drug resistance protein 1, contain all four components within a single polypeptide. Previous work on these transporters had led to certain expectations about how their structures would look. Several individual NBD polypeptides had already been crystallized. The highly similar monomer structures reflect their high sequence similarity. Each dimeric structure, however, seemed to show a different location for the interface between the two monomers. Since the Hot Papers' publication, studies have solidified widespread agreement that the NBDs are arranged with their ATP binding sites facing one another.3,4 This arrangement fits biochemical studies, which indicate a high degree of cooperativity between the ATP binding sites. Moreover, crosslinking and genetic studies show which residues are likely to be in contact at the dimer interface. Yet, in the MsbA structure, the NBDs face in opposite directions and are nowhere near one another. MsbA is composed of two identical monomers, each containing one TMD and one NBD. In the crystal structure, the MsbA dimer looks like an inverted V. The two TMDs interact at the top within the membrane, while the two NBDs lie at the ends of the V in the cytoplasm, separated by a distance of about 50 angstroms. PROTEIN SNAPSHOTS "It took me by surprise," says Chang. And while he says he's "always open to the possibility that it could be an artifact of crystallization," he offers another potential explanation. Membrane transporters must under- go a "tremendous amount of conformational change" before they can carry their substrates across the membrane. The MsbA structure might represent a "snapshot" of the transporter in the midst of such changes. He points out that the biochemical studies have focused almost exclusively on ATP binding, which represents only a "very small slice of what [these transporters] can do." Supporting this notion, Chang's recently published structure of an MsbA homolog from Vibrio cholerae shows a closed conformation, with the NBDs much closer together, providing "another snapshot" of the transporter's mechanism.5 Reprinted with permission from AAAS TRANSPORT IN PROGRESS: In this potential model for lipid A transport by msbA, (1) the triggering of ATP hydrolysis accompanies lipid A binding to the chamber in the transmembrane (TM ) domain. (2) The chamber closes and lipid A translocates. Interaction between the nucleotide binding domains (NBDs) is possible. (3) The chamber opens and lipid A is released to the outer bilayer leaflet. In contrast to MsbA, the BtuCD structure matched researchers' predictions more closely. Here, the NBDs are situated in very close contact, with the ATP binding sites facing each other. Other features suggest that native interactions are more intact. For example, says Rees, the BtuCD dimer's transmembrane domains show "very definite and intimate interactions" with one another, while those of the MsbA structure appear somewhat tenuous. Rather than dismiss the MsbA dimer as an artifact, however, he suggests that the differences may arise because MsbA, an exporter, is likely to have a very different mechanism from BtuCD, an importer. Other researchers express stronger doubts that the MsbA structure represents an authentic dimer. Christopher Higgins, of the Medical Research Council's Clinical Sciences Centre, Imperial College, UK, agrees that the dimer interface between the TMDs is too small. In addition, he says, thermodynamic calculations suggest that the monomers' tilt angles would be highly unstable in a membrane environment. He and his coworkers recently published a study in which they mapped experimentally determined chemical crosslinks from a well-studied ABC transporter, P-glycoprotein, onto the MsbA structure.6 They found that the structural data matched the biochemical data only if the NBD domains were rotated with the ATP binding sites facing each other, as in BtuCD. Another long time investigator in this field, Amy Davidson, of Baylor College of Medicine, Houston, has raised similar doubts about the MsbA dimer. The BtuCD structure does contain one feature that was not entirely expected. Instead of twelve membrane spans, its transmembrane region contains a complicated arrangement of 20 helices. Yet, both Rees and Chang point out that the TMDs' sequences are much less conserved than those of the NBDs. Since the former are responsible for substrate handling, this lack of conservation probably reflects very different substrate specificities, in turn reflected in different structures they say. Most agree on one point. More structures are needed, both of new ABC transporters, and of the same transporters in the presence of substrates, ATP, and ATP analogs, which can trap the transporters at specific stages of the transport mechanism. According to Higgins, the BtuCD structure is important but "does not tell us much new." Instead, it presents a "great framework for moving forward" with further studies to enhance understanding of these transporters. Megan M. Stephan (dr.meganstephan@att.net) is a freelance writer in Cheshire, Conn. References 1. G. Chang, C.B. Roth, "Structure of MsbA from E. coli: A homolog of the multidrug resistance ATP binding cassette (ABC) transporters," Science, 293:1793-1800, 2001. (Cited in 148 papers) 2. K. Locher et al., "The E. coli BtuCD structure: A framework for ABC transporter architecture and mechanism," Science, 296:1091-8, 2002. (Cited in 87 papers) 3. P.C. Smith et al., "ATP binding to the motor domain from an ABC transporter drive formation of a nucleotide sandwich dimer," Mol Cell, 10:139-49, 2002. 4. E.E. Fetsch, A.L. Davidson, "Vanadate-catalyzed photocleavage of the signature motif of an ATP-binding cassette (ABC) transporter," Proc Natl Acad Sci, 99:9685-90, 2002. 5. G. Chang, "Structure of MsbA from Vibrio cholera: A multidrug resistance ABC transporter homolog in a closed conformation," J Mol Biol, 330:419-30, 2003. 6. D.R. Stenham et al., "An atomic detail model for the human ATP binding cassette transporter P-glycoprotein derived from disulphide cross-linking and homology modeling," FASEB J, published online Oct. 16, 2003. function sendData() { document.frm.pathName.value = location.pathname; result = false if (document.frm.score[0].checked) result = true; if (document.frm.score[1].checked) result = true; if (document.frm.score[2].checked) result = true; if (document.frm.score[3].checked) result = true; if (document.frm.score[4].checked) result = true; if (!result) alert("Please select") return result; } .myradio{background-color : #94bee5} Please indicate on a 1 - 5 scale how strongly you would recommend this article to your colleagues? Not recommended 1 2 3 4 5 Highly recommended Please register your vote

A Tale of Two Transporters

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Courtesy of Kaspar Locher
B12 TRANSPORTER EXPOSED: Ribbon diagram of the B12 transporter BtuCD from E. coli, with cyclotetravanadate bound at the ATP-binding sites. The transporter is assembled from four subunits, two each of the membrane spanning BtuC (yellow and red) and the cytoplasmic BtuD (green and blue).