In the past year our work has focused on the 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. In the past year we made substantial progress in work on a vcINDY, a representative of a transporter family which is important for longevity in flies and is involved in obesity and insulin resistance in mammals. 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. ALso in the last year we obtained preliminary results suggesting that the mammalian transporter, NaDC3, in the same family as vcINDY, operates via a similar elevator-type mechanism. We are now establishing new methods, including single molecule analysis and electron paramagnetic resonance techniques to examine these conformational changes more thoroughly. In another vein, we have been exploring methods for accurately determining transporter stoichiometry. 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. We applied this method to vcINDY and established that the transporter couples 3 Na ions to the transport of each succinate molecule, and shows that the transporter can concentrate succinate to high levels in the cell. We also confirmed the validity of the method using a sugar transporter of known stoichiometry, vSGLT. We are now applying the new method to other transporters for which accurate understanding of stoichiometry is important.
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