Computational studies of membrane transport proteins

National Institute Of Neurological Disorders And Stroke

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Abstract

Secondary active transporters are a class of membrane proteins that use pre-existing molecular concentration gradients as an energy source for translocating another substrate, such as a nutrient or a neurotransmitter, against its concentration gradient. This process requires the protein to change conformations so as to expose a pathway to the substrate binding site(s) on one or other side of the membrane, in a cycle known as alternating access. Every organism expresses dozens of different secondary transporter proteins, and these exhibit a diverse set of architectures, albeit always with some form of internal structural symmetry. Unprecedented, ground-breaking insights have been garnered from three-dimensional structures obtained in the last decade. Nevertheless, a detailed understanding of the mechanism of each membrane transport protein requires knowledge of its structure in many more conformational states, including identification of the binding regions for the substrate or substrates. Moreover, those structures need to be placed into a context of dynamic ensembles on a thermodynamic landscape separated by kinetic barriers. Studies from our group over the last year have continued to investigate these issues in many membrane proteins. A long-standing interest in our lab is transporters belonging to the families of neurotransmitter transporters, including NSS (neurotransmitter sodium symporter) or SLC6 family proteins. Although all NSS transporters use sodium ions for transport, a large subset also requires chloride ions. Sodium ions are known to act at two sites on these proteins to stabilize a conformation in which the pathway is open to the outside (i.e., outward-open), as well as to interact with substrate to facilitate transport, but the role of chloride ions has not previously been established. In collaboration with the Rudnick lab (Yale), we showed how a chloride ion can act independently from substrate to facilitate the conversion of an NSS transporter for glycine (SLC6A9) from outward- to inward-open states (Ref. 1). Using molecular dynamics simulations on a related transporter, LeuT, we showed how the presence of the negative charge influenced the behavior of specific amino acids in the pathway, so as to encourage or disfavor opening of the pathway. The second major family of neurotransmitter transporters are known as SLC1, or Excitatory Amino Acid Transporters (EAAT). This rather specialized family contains a unique structure with a highly-conserved NMDG motif responsible for binding of amino acids and for coupling to the required sodium ions. The second amino acid in the motif, methionine, plays a highly unusual role in EAATs. Despite the naturally hydrophobic character of methionine, in EAATs, it actually interacts with the charged sodium ions. In collaboration with the Faraldo-Gomez (NHLBI) and Slotboom (Univ. Groningen, Netherlands) labs, we investigated the role of this methionine and showed that it is crucial for defining the kinetics of the turnover mechanism, but not the ratio of three sodium ions required for every amino acid substrate (Ref. 2). In summary, our publications this year reflect ongoing efforts to utilize computational approaches in close collaboration with experimental laboratories, and drive understanding of the mechanism of biomedically-important proteins in neuronal processes.