The E. coli BtuCD Structure: A Framework for ABC Transporter Architecture and Mechanism

Abstract

Materials and methodsThe sequences for the different transporters were subcloned into various pET vectors (Novagen), which attached poly---histidine tags at the amino---or carboxy---terminus. The constructs were expressed in E. coli BL21 (DE3) (Stratagene) in a 50---liter fermenter at 37°C and were induced using 1 mM IPTG. BtuC and BtuD were coexpressed from a single plasmid, with the BtuC subunit containing an NH 2 ---terminal decahistidine tag. BtuCD protein was solubilized in 1% dodecyl---N,N---dimethylamineoxide (LDAO) and purified using Ni---NTA metal affinity chromatography (Qiagen), followed by gel filtration. Crystals were grown by mixing BtuCD protein (20 mg/ml) with an equal volume of reservoir solution (100 mM Tris pH 8.0, 300 mM magnesium nitrate, 21% polyethylene glycol 2000, 0.8% 2---methyl---2,4---pentanediol in D 2 O) in sitting drops. Crystals grew to a final size of 0.1 0.15 0.5 mm 3 in a week, and were frozen in liquid N 2 before data collection. Cells expressing selenomethionine BtuCD protein were grown in M9 medium supplemented with seleno---D,L---methionine. Purification and crystallization of the selenomethionine protein was as described above for the native protein. Data sets were collected at the Advanced Photon Source (APS) or at the Stanford Synchrotron Radiation Laboratory (SSRL).

The motor domains of ABC-transporters

“…In 2002, Locher et al (2002) published the second complete ABC transporter structure. BtuCD is a bacterial importer, which together with its periplasmic binding protein BtuF, forms a functional vitamin B12 importer in E. coli.…”

“…In contrast, the obvious sequence and structural variation in the membrane domain of the Class 1 and 3 proteins is to be expected from the extreme variety of allocrites. As the recent crystal structures of full-length ABC-transporters have proved (Chang and Roth 2001;Locher et al 2002), the transmembrane spanning domain is built up from α-helices. Usually, there are 6 α-helices per transmembrane spanning domain (called the core domain), although some transporters exhibit more, e.g.…”

“…Usually, there are 6 α-helices per transmembrane spanning domain (called the core domain), although some transporters exhibit more, e.g. MRP1 or the BtuCD transporter (Locher et al 2002), and some less (Khwaja et al 2005).…”

“…Recently, in addition to the structures of the two full length ABC transporters (Chang 2003;Chang and Roth 2001;Locher et al 2002;Reyes and Chang 2005), several high resolution crystal structures of ATP-binding domains from all three classes of ABC proteins have been solved. Details of the origin, resolution and PDB access code for all these structures is shown in Table 1.…”

TAP and TAP-like — Brothers in arms?

“…However, there are also examples of 2×4 and 2×10 membrane helices as in the case of the macrolide transporter MacB (Kobayashi et al 2003b) and the vitamine B 12 uptake system BtuCD (Locher et al 2002), respectively. The TMDs form the translocation pathway and, in the case of exporters, the substrate-binding site.…”