Membrane Porters of ATP-Binding Cassette Transport Systems Are Polyphyletic

The ATP-binding cassette (ABC) superfamily consists of both importers and exporters. These transporters have, by tradition, been classified according to the ATP hydrolyzing constituents, which are monophyletic. The evolutionary origins of the transmembrane porter proteins/domains are not known. Using five distinct computer programs, we here provide convincing statistical data suggesting that the transmembrane domains of ABC exporters are polyphyletic, having arisen at least three times independently. ABC1 porters arose by intragenic triplication of a primordial two-transmembrane segment (TMS)-encoding genetic element, yielding six TMS proteins. ABC2 porters arose by intragenic duplication of a dissimilar primordial three-TMS-encoding genetic element, yielding a distinctive protein family, nonhomologous to the ABC1 proteins. ABC3 porters arose by duplication of a primordial four-TMS-encoding genetic element, yielding either eight- or 10-TMS proteins. We assign each of 48 of the 50 currently recognized families of ABC exporters to one of the three evolutionarily distinct ABC types. Currently available high-resolution structural data for ABC porters are fully consistent with our findings. These results provide guides for future structural and mechanistic studies of these important transport systems

Inter-domain Communication Mechanisms in an ABC Importer: A Molecular Dynamics Study of the MalFGK2E Complex

ATP-Binding Cassette transporters are ubiquitous membrane proteins that convert the energy from ATP-binding and hydrolysis into conformational changes of the transmembrane region to allow the translocation of substrates against their concentration gradient. Despite the large amount of structural and biochemical data available for this family, it is still not clear how the energy obtained from ATP hydrolysis in the ATPase domains is “transmitted” to the transmembrane domains. In this work, we focus our attention on the consequences of hydrolysis and inorganic phosphate exit in the maltose uptake system (MalFGK2E) from Escherichia coli. The prime goal is to identify and map the structural changes occurring during an ATP-hydrolytic cycle. For that, we use extensive molecular dynamics simulations to study three potential intermediate states (with 10 replicates each): an ATP-bound, an ADP plus inorganic phosphate-bound and an ADP-bound state. Our results show that the residues presenting major rearrangements are located in the A-loop, in the helical sub-domain, and in the “EAA motif” (especially in the “coupling helices” region). Additionally, in one of the simulations with ADP we were able to observe the opening of the NBD dimer accompanied by the dissociation of ADP from the ABC signature motif, but not from its corresponding P-loop motif. This work, together with several other MD studies, suggests a common communication mechanism both for importers and exporters, in which ATP-hydrolysis induces conformational changes in the helical sub-domain region, in turn transferred to the transmembrane domains via the “coupling helices”

In vitro functional characterization of BtuCD-F, the Escherichia coli ABC transporter for vitamin B12 uptake

BtuCD is an ATP binding cassette (ABC) transporter that facilitates uptake of vitamin B-12 into the cytoplasm of Escherichia coli. The crystal structures of BtuCD and its cognate periplasmic binding protein BtuF have been recently determined. We have now explored BtuCD-F function in vitro, both in proteoliposomes and in various detergents. BtuCD reconstituted into proteoliposomes has a significant basal ATP hydrolysis rate that is stimulated by addition of BtuF and inhibited by sodium ortho-vanadate. When using different detergents to solubilize BtuCD, the basal ATP hydrolysis rate, the ability of BtuF to stimulate hydrolysis, and the extent to which sodium ortho-vanadate inhibits ATP hycrolysis all vary significantly. Reconstituted BtuCD can mediate transport of vitamin B12 against a concentration gradient when coupled to ATP hydrolysis by BtuD in the liposome lumen and BtuF outside the liposomes. These in vitro studies establish the functional competence of the BtuCD and BtuF preparations used in the crystallographic analyses for both ATPase and transport activities. Furthermore, the tight binding of BtuF to BtuCD under the conditions studied suggests that the binding protein may not dissociate from the transporter during the catalytic cycle, which may be relevant to the mechanisms of other ABC transporter systems

Structure of AMP-PNP-bound BtuCD and mechanism of ATP-powered vitamin B12 transport by BtuCD-F

ISSN:1545-9993ISSN:1545-998

EPR Techniques to Probe Insertion and Conformation of Spin-Labeled Proteins in Lipid Bilayers

Electron paramagnetic resonance (EPR) spectroscopy of spin-labeled membrane proteins is a valuable biophysical technique to study structural details and conformational transitions of proteins close to their physiological environment, e.g., in liposomes, membrane bilayers, and nanodiscs. Unlike in nuclear magnetic resonance spectroscopy, having only one or few specific side chains labeled at a time with paramagnetic probes makes the size of the object under investigation irrelevant in terms of technique sensitivity. As a drawback, extensive site-directed mutagenesis is required in order to analyze the properties of the protein under investigation. EPR can provide detailed information on side chain dynamics of large membrane proteins or protein complexes embedded in membranes with an exquisite sensitivity for flexible regions and on water accessibility profiles across the membrane bilayer. Moreover, distances between the two spin-labeled side chains in membrane proteins can be detected with high precision in the 1.5–6 nm range at cryogenic temperatures. The application of EPR to membrane proteins still presents some challenges in terms of sample preparation, sensitivity, and data interpretation; thus no ready-to-go methodological recipes can be given. However this chapter describes the state of the art in the application of nitroxide-based site-directed spin labeling EPR to membrane proteins, with specific focus on the different types of information which can be obtained with continuous wave and pulsed techniques and on the challenges in sample preparation and data analysis for functional and structural membrane protein studies