ABC transporter architecture and regulatory roles of accessory domains

AbstractWe present an overview of the architecture of ATP-binding cassette (ABC) transporters and dissect the systems in core and accessory domains. The ABC transporter core is formed by the transmembrane domains (TMDs) and the nucleotide binding domains (NBDs) that constitute the actual translocator. The accessory domains include the substrate-binding proteins, that function as high affinity receptors in ABC type uptake systems, and regulatory or catalytic domains that can be fused to either the TMDs or NBDs. The regulatory domains add unique functions to the transporters allowing the systems to act as channel conductance regulators, osmosensors/regulators, and assemble into macromolecular complexes with specific properties

Ion Specificity and Ionic Strength Dependence of the Osmoregulatory ABC Transporter OpuA

The ATPase subunit of the osmoregulatory ATP-binding cassette transporter OpuA from Lactococcus lactis has a C-terminal extension, the tandem cystathionine β-synthase (CBS) domain, which constitutes the sensor that allows the transporter to sense and respond to osmotic stress. C-terminal of the tandem CBS domain is an 18-residue anionic tail (DIPDEDEVEEIEKEEENK). To investigate the ion specificity of the full transporter, we probed the activity of inside-out reconstituted wild-type OpuA and the anionic tail deletion mutant OpuAΔ12; these molecules have the tandem CBS domains facing the external medium. At a mole fraction of 40% of anionic lipids in the membrane, the threshold ionic strength for activation of OpuA was ~0.15, irrespective of the electrolyte composition of the medium. At equivalent concentrations, bivalent cations (Mg2+ and Ba2+) were more effective in activating OpuA than NH4+, K+, Na+, or Li+, consistent with an ionic strength-based sensing mechanism. Surprisingly, Rb+ and Cs+ were potent inhibitors of wild-type OpuA, and 0.1 mM RbCl was sufficient to completely inhibit the transporter even in the presence of 0.2 M KCl. Rb+ and Cs+ were no longer inhibitory in OpuAΔ12, indicating that the anionic C-terminal tail participates in the formation of a binding site for large alkali metal ions. Compared with OpuAΔ12, wild-type OpuA required substantially less potassium ions (the dominant ion under physiological conditions) for activation. Our data lend new support for the contention that the CBS module in OpuA constitutes the ionic strength sensor whose activity is modulated by the C-terminal anionic tail.

Efficient biosynthetic incorporation of tryptophan and indole analogs in an integral membrane protein

Biosynthetic incorporation of tryptophan (Trp) analogs such as 7-azatryptophan, 5-hydroxytryptophan, and fluorotryptophan into a protein can facilitate its structural analysis by spectroscopic techniques such as fluorescence, phosphorescence, nuclear magnetic resonance, and Fourier transform infrared. Until now, the approach has dealt primarily with soluble proteins. In this article, we demonstrate that four different Trp analogs can be very efficiently incorporated into a membrane protein as demonstrated for the mannitol transporter of Escherichia coli (EIImtl). EIImtl overexpression was under control of the λPR promoter, and the E. coli Trp auxotroph M5219 was used as host. This strain constitutively expresses the heat labile repressor protein of the λPR promoter. Together with the presence of the repressor gene on the EIImtl plasmid, this resulted in a tightly controlled promoter system, a prerequisite for high Trp analog incorporation. A new method for determining the analog incorporation efficiency is presented that is suitable for membrane proteins. The procedure involves fitting of the phosphorescence spectrum as a linear combination of the Trp and Trp analog contributions, taking into account the influence of the protein environment on the Trp analog spectrum. The data show that the analog content of EIImtl samples is very high (>95%). In addition, we report here that biosynthetic incorporation of Trp analogs can also be effected with less expensive indole analogs, which in vivo are converted to L-Trp analogs