Our Scientists

Our research emphasizes the area of structural bioenergetics, which studies the molecular basis of biological energy transduction processes. The 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 involved in transport and oxidation-reduction processes.

Allosteric Regulation of ABC Transporters
Any object that enters or leaves a cell—whether a nutrient, a virus, or a waste product—must penetrate one or more enclosing membranes. Consequently, the movement of most molecules across membranes is mediated by specialized integral membrane proteins known as transporters. Currently, more than 500 families of transporters have been identified through biochemical and genomic analyses, highlighting the significance of transport processes in cellular metabolism. The proteins that mediate these transport processes may be divided into two general categories: channels that facilitate the movement of molecules across the membrane in their thermodynamically favorable direction, and active transporters that can move molecules across a membrane against a concentration gradient. Transporters are able to achieve this uphill process by coupling the unfavorable process to a second, energetically favorable process. The importance of transport activity to cellular energy metabolism can be appreciated from the nontrivial metabolic costs of maintaining the correct transmembrane concentrations of molecules, which is estimated to consume ~10–60 percent of the ATP requirements of bacteria and humans, depending on conditions.

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.

We have been interested in the structure and mechanism by which ABC transporters couple the binding and hydrolysis of ATP to movement of molecules across membranes. Our initial studies focused on two homologous transporters involved in the import of vitamin B12 and related compounds into bacteria to address the structural foundations of the alternating-access model. More recently, we have started focusing on potential regulatory aspects. In view of the metabolic costs associated with transporter activity, it is not surprising that the activity of ABC transporters can be regulated at the level of protein function, likely involving domains fused to the NBDs and/or TMDs. One example of such a regulatory process is the phenomenon of trans-inhibition, in which the uptake of an external ligand is inhibited by intracellular concentrations of the same ligand. Intriguingly, early studies by Robert Kadner established the phenomenon of trans-inhibition in the Escherichia coli methionine-uptake system, since uptake of external methionine is inhibited by intracellular methionine levels in a fashion consistent with the direct action of methionine on the transporter.

The high-affinity uptake of methionine in E. coli is mediated by the MetD system, a member of the methionine-uptake transporter family of ABC transporters. The MetD system was originally identified as an importer of both L- and D-methionine, either of which may be used as a source of methionine by E. coli. To further investigate this system, we determined the crystal structure at 3.7-Å resolution of the E. coli methionine ABC transporter MetNI solubilized in the detergent dodecylmaltoside. The overall architecture of MetNI consists of two copies of the ATPase MetN in complex with two copies of the transmembrane domain MetI, with the transporter adopting an inward-facing conformation and the NBDs widely separated. The ABC subunits contain a C-terminal extension unique to members of the methionine-uptake family that separate the NBDs in this inward-facing conformation; significantly, crystallographic studies established that selenomethionine, a transported substrate, binds to the isolated C-terminal domain.

Since ATP hydrolysis requires nucleotide binding to conserved sequence motifs at the interface between two closely juxtaposed NBDs, the crystallographically observed inward-facing conformation of the MetNI transporter, with the widely separated NBDs, corresponds to an ATPase-inactive state. Consistent with this assignment, kinetic studies demonstrated that the ATPase activity of the transporter decreased with increasing methionine concentrations. These results are consistent with a model in which an equilibrium exists between conformational states of MetNI differing in the extent of NBD separation. As methionine binds to the C-terminal domains of MetNI, the equilibrium shifts toward transporter conformations with separated NBDs, and as a consequence, the rate of ATP hydrolysis decreases. By sterically interfering with NBD association, the ATP-driven engine that powers transport will be disrupted, thereby providing a molecular mechanism for Kadner's observation that increasing levels of internal methionine inhibit transport. The structure of the MetNI ABC transporter provides a foundation for the characterization of regulatory mechanisms relevant to the uptake of methionine and provides an opportunity for establishing how these processes are more generally integrated with cellular metabolism.

This work was supported in part by a grant from the National Institutes of Health.

As of May 30, 2012