|dc.description.abstract||Survival necessitates obtaining the right balance of nutrients from the environment. Most crucially, organisms must secure an adequate supply of each of the two dozen immutable chemical elements that are essential to life. Organisms have therefore evolved intricate cellular machinery—primarily in the form of membrane transport proteins—to import raw materials. The highly conserved natural resistance associated macrophage protein (Nramp) family facilitates the cross-membrane transport of transition metal ions, which all organisms use as co-factors in enzymes that catalyze myriad essential metabolic reactions. In mammals, Nramp homologs enable the uptake of dietary iron and contribute to the metal withdrawal innate immune defense strategy, while plants and bacteria use these proteins primarily for manganese acquisition. For my thesis I used biochemistry and structural biology to develop an atomic-resolution mechanistic model for Nramp transport, which sheds light on the fundamental principles of secondary transport.
To explain how Nramp distinguishes among various metal substrates, I demonstrate the role of a conserved metal-binding methionine residue in preferentially stabilize transition metals via its sulfur atom. Indeed, while Nramp mutants that lack this methionine can still transport some transition metals they lose the advantageous ability to reject calcium and magnesium, which are orders of magnitude more abundant environmentally. This work illustrates how evolution has exploited the chemical properties of its amino acid repertoire to enable organisms to sort through different metal ions and specifically take up the rare ones they need to survive.
Next, I demonstrate how Nramp cycles between two stable conformations – inward- and outward-facing – to provide a pathway for metal ion transport. Using a low-resolution inward-facing crystal structure determined by my colleagues, I use cysteine accessibility assays to animate this static snapshot of Nramp—illustrating how the protein’s transmembrane helices rearrange themselves in the alternate outward-facing conformation—and to show how anemia-causing mutations disrupt the conformational change process to impair metal transport.
I further exploit this structural knowledge to design an outward-locked Nramp mutant, whose high-resolution X-ray crystal structure I determined in the presence of metal substrate. This structure, along with an additional high-resolution inward-occluded structure determined by my colleague, provides the basis for understanding the complete metal transport cycle for the Nramp family, while also fully illustrating the conformational rearrangements that open and close the inner and outer gates. In addition, I show that Nramp uses separate pathways for metal ions and its proton co-substrates—such that proton shuttling does not require bulk conformational change—which may facilitate the co-transport of two like charges.
Lastly, I demonstrate that Nramp metal import is highly voltage dependent. In addition, in contrast to canonical secondary transporters, metal and proton co-substrates are not tightly coupled for Nramp, as metal-proton symport, metal uniport, and proton uniport all occur, with metal elemental identity affecting the co-transport stoichiometry. Mutagenesis studies to neutralize charged/protonatable residues demonstrate how an extensive conserved salt-bridge network allosterically imparts Nramp’s voltage-dependence and proton-metal coupling properties, which serve to prevent the deleterious back transport of acquired metals. More broadly, the demonstrated Nramp molecular mechanism highlights the underappreciated role of the physiological membrane potential to impact any membrane-associated biochemical process and illustrates the mechanistic fluidity between the realms of channels and transporters.
In summary, here I provide an atomic-level explanation of a conserved mechanism by which organisms acquire essential nutrients and developed a model system that provides opportunity for a deeper understanding of the general principles of membrane transport.||