In the past year our work has focused on both GltPh, a model for the EAAT family of glutamate transporters and a new protein, vcINDY, a succinate transproter which has been implicated in longevity and obesity. It is critical to understand the fundamental mechanisms by which there transporters function because such knowledge could lead to the development of therapeutic agents active against these proteins. We seek to analyze the dynamic movements of the functioning transporter on the way to a detailed understanding of its mechanism. Our approach is to analyze the details of transport in model transporters obtained from bacteria. These can be expressed and purified in large quantities and are amenable to biophysical methods not available for their mammalian cousins. We have continued our work using EPR spectroscopy to monitor conformational changes in GltPh. This work has identified local changes in the protein that may be important for coupling between the driving ion, Na+, and the substrate, aspartate. We are continuing work to identify the nature of this change. We recently reported that a extracellular loop of gltPH must be intact for effective transport. Last year we probed the mechanism of this effect in detail and found that when the 34 loop is cut the proteins maintains substrate affinities but maximal transport is significantly reduced. We demonstrated that this effect relates to the activation energy of the substrate translocation step, implicating the loop in the piston like movement of the translocation domain. This year we also found that only the translocation of the substrate-bound form of the protein is affected--the apo, substrate-free transporter is unaffected by 34 loop cleavage. We have performed important controls eliminating alternative explanations for these effects and a paper describing this work is under review. In the past year we also made substantial progress in work on a vcINDY, a transporter which is important for longevity in flies and is involved in obesity and insulin resistance in mammals. We performed the first successful functional reconstitution of vcINDY and directly demonstrated that it is a Na+ coupled succinate transporter and completed a comprehensive analysis of its functional properties, which was published this spring. In the past year we shifted to more mechanistic analysis of the vcINDY mechanism. One structure of vcINDY is available (in an inward-facing state), but yields little information on the protein's strategy for transport. In collaboration with Lucy Forrest, we used computational methods to predict the outward facing state of the protein and found, surprisingly, that the state appears to require a major translocation of the substrate binding domain across the membrane, moving about 15 angstroms. We tested this prediction using biochemical and biophyiscal methods, introducing cysteine pairs at positions expected to be distant in the available inward-facing state but brought together by the predicted conformational change to outward-facing. We found that three different such pairs were able to form crosslinks, confirming the existence of our predicted outward-facing state. Furthermore, these crosslinks nearly completely inhibit vcINDY transport activity, demonstrating that the conformational change is required for transport. Similar exploration of the protein's dimer interface revealed little such movement. This work establishes vcINDY as only the second transporter to use an Elevator type mechanism with such a large conformational change required for transport and suggests that the very large family encompassing vcINDY also works this way. Further support for this idea came with the publication of structures of two distantly related members of the same broad family, the drug transporters MtrF and YdaH. We performed a computational analysis (again in collaboration with the Forrest lab) that reveals these proteins to be structurally related to INDY and therefore likely to operate via similar mechanisms. In another vein, we have been exploring methods for accurately determining transporter stoichiometry. In collaboration with Simon Newstead at Oxford, we determined that the PepT class of proton-coupled peptide transporters operate via a unique mechanism, with different coupling ratios of proton:peptide for different length peptides. In addition, we have developed a new and widely applicable method for determining transporter stoichiometry for Na-coupled transporters that will be especially useful for characterizing transporters amenable to structure determination.
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