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Structural Bioenergetics
Summary: Douglas Rees is interested in the structure and function of metalloproteins and membrane proteins, particularly those involved in cellular energy metabolism.
Our research emphasizes the area of structural bioenergetics, which seeks to describe the molecular basis of biological energy transduction processes. Coupling of cellular energy metabolism to environmental energy sources occurs primarily through consumption of nutrients (redox energy) and absorption of light. These energy sources must then be converted into biologically usable forms, such as ATP and concentration gradients, that are required to drive biosynthetic reactions, motility, electrical signaling, and other metabolic processes. Energy transduction processes mediate these interconversions of different energy forms. A major goal of our group is to characterize the structures and mechanisms of ATP-dependent transduction systems, including membrane proteins that catalyze energy transduction processes associated with transport, mechanosensation, and respiration-linked electron transfer reactions.
An Inward-Facing Conformation of the ABC Transporter HI1470/1
Transporters have the remarkable ability to pump molecules against concentration gradients across cell membranes through the coupling to a second, energetically favorable process. One of the most widespread families of transporters, the ATP-binding cassette (ABC) family, uses the binding and hydrolysis of ATP to power substrate translocation. ABC transporters are minimally composed of four domains, with two transmembrane domains (TMDs) and two ABCs or nucleotide-binding domains (NBDs) located in the cytoplasm. Although diverse in physiological function and TMD architecture, ABC transporters are characterized by two highly conserved NBDs that contain critical sequence motifs for ATP binding and hydrolysis, including the P loop present in many nucleotide-binding proteins and the ABC signature motif (LSGGQ) specific to ABC transporters. These similarities suggest a common mechanism by which ABC transporters orchestrate a sequence of nucleotide- and substrate-dependent conformational changes that translocate the substrate across the membrane through interconversion of outward- and inward-facing conformations; this "alternating-access" model provides a framework for the mechanistic characterization of transporters. For prokaryotic ABC transporters functioning as importers, substrate translocation also depends on high-affinity periplasmic binding proteins that deliver the ligand to the outward-facing state of the cognate transporter.
The HI1470/1 transporter from Haemophilus influenzae belongs to the family of binding protein–dependent bacterial ABC transporters that mediate the uptake of metal-chelate species, including heme and vitamin B12. Since iron is often an essential nutrient, members of this family are widely distributed throughout bacteria, including pathogenic organisms such as H. influenzae. The molecular architecture for this family of ABC transporters was established when we solved the structure of BtuCD, the vitamin B12 importer from Escherichia coli. The transporter encoded by genes HI1470 and HI1471 of H. influenzae exhibits 24 and 33 percent sequence identity to the ABC subunit BtuD and the membrane-spanning BtuC, respectively, and was identified as a candidate for structural study during the original screen of homologs explored in the BtuCD analysis. Following overexpression and purification in decylmaltoside of a histidine-tagged construct, we crystallized and solved the structure of the intact, nucleotide-free HI1470/1 transporter by isomorphous and multiwavelength anomalous diffraction methods at 2.4-Å resolution.
Although the overall architecture of the intact HI1470/1 transporter resembles that of BtuCD, more-detailed comparisons highlight differences in tertiary and quaternary arrangements that may be functionally relevant. An important distinction is that, although each transporter maintains a tapered pathway across the membrane-spanning region, the pathways open to opposite sides of the membrane: HI1470/1 and BtuCD adopt inward- and outward-facing conformations, respectively. Each of the membrane-spanning subunits HI1471 and BtuC contains 10 transmembrane helices that are organized into two sets suggestive of an internal duplication. In comparison to a structurally conserved core composed of seven of these helices, a twist of ~9˚ about an axis perpendicular to the translocation pathway is required to interconvert the TMDs of the two transporters. Together with the changes in the three remaining helices, these conformational rearrangements result in a translocation pathway that is closed to the periplasm and open to the cytoplasm in HI1470/1; the converse is observed in BtuCD.
The orientation of the permeation pathways of HI1470/1 and BtuCD in opposite directions allows us to identify structural elements underlying this transition. The conformational transformations between the polypeptide chains of HI1470/1 and BtuCD, while maintaining the overall twofold molecular symmetry, do not exclusively involve rigid body movements of individual subunits. Still, the rigid body description is a useful framework for comparison of the two structures; for example, when the catalytic domains of one NBD in the intact HI1470/1 and BtuCD transporters are superimposed, the relative positions of the partner NBDs in these structures are shifted by a translation of ~4.5 Å along an axis parallel to the interface between NBDs. The direction of this shift coincides with the direction of the twist motion observed between the membrane-spanning subunits of HI1470/1 and BtuCD. This translation component arises from the coupling of the local rotation axes relating individual NBDs in different structures to the molecular twofold rotation, which generates a displacement along the subunit-subunit interface as the separation between NBDs varies. The linkage between NBD positioning and the twist between TMDs supports a coupling mechanism connecting the permeation pathway and nucleotide state of the transporter where the ABCs can remain juxtaposed during the transport cycle.
The differences observed between the structures of the HI1470/1 and BtuCD ABC transporters involve relatively modest rearrangements and may serve as structural models for inward- and outward-facing conformations relevant to the alternating access mechanism of substrate translocation. As neither structure was crystallized in the presence of nucleotide, binding protein, or ligand, the energetic basis of the differential stabilization of alternate conformations is not obvious; one possibility we are studying is that replacement of the native bilayer with detergent has shifted the conformational equilibrium between inward- and outward-facing states. Intriguingly, the internal duplication evident in the helix-packing arrangements of HI1470/1 and BtuCD, as well as other channels and transporters, suggests that the internal symmetry in the membrane-spanning subunits is relevant to the mechanistic transition between inward- and outward-facing conformations. The roles of binding protein, ligand, and particularly nucleotide binding and hydrolysis in driving these conformational transitions are crucial mechanistic issues that we are exploring.
This work was supported in part by a grant from the National Institutes of Health.
Last updated: March 16, 2007
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