Transport proteins embedded in the cellular membrane move chemical cargo into and out of cells, thus facilitating the acquisition of essential nutrients. Many transport proteins evolved from a common ancestor, retaining similar three-dimensional structures despite considerable divergence in their amino acid sequences. Over time these proteins specialized in how they harness the energy they may need and which chemical substances they transport. The Nramp family of transporters enables transition metal ions—including essential nutrients like iron and manganese—to cross cellular membranes. Dysfunction of one such Nramp transporter in humans leads to anemia, as insufficient dietary iron is absorbed in the intestines and delivered to red blood cells. In addition, bacterial pathogens express their own Nramp transporters to acquire essential metals during an infection.
In this study (PDF), we wanted to understand at the atomic level how an Nramp transporter works, which can ultimately help us better understand—and regulate—how cells acquire nutrients. A transporter can be conceptualized as “foyer” within the membrane, with a door to the outside and a door to the inside of cells. The doors are never open at the same time, but rather alternate which one is open and which is closed, a process referred to as “alternating access”, letting in one metal ion in each alternating cycle. To understand how the transporter rearranges itself to provide alternating access to transported metal ions, we need snapshots of the transporter in multiple states. To visualize our model Nramp transporter in distinct states, we designed complementary Nramp variants replacing a small amino acid with a large one in judiciously chosen locations we predicted would function as a doorstop, holding one or the other door open. We then used X-ray crystallography to determine atomic-resolution structures of each of these variants. One structure provides our first view of how an Nramp transporter binds its metal substrate from the outside of the cell, revealing the external water-filled cavity through which metal ions travel to reach the metal binding site used during the transport process. A second structure shows how the transporter releases its metal cargo through a separate water-filled cavity that opens to the inside of the cell after the external cavity has closed. A third structure reveals an intermediate conformation in which the protein has closed off access to the metal-binding site from both outside and inside. Interestingly, these structures show that Nramp’s conformational changes require different intramolecular rearrangements compared to previously studied transporters with similar structures.
It has long been known that Nramps also transport protons—perhaps to harness electrochemical energy—and we further aimed to discover how the protein binds both its metal ion and proton cargos. First, we measured transport activity to test the importance of the observed conformational changes. As expected, our engineered trapped Nramp variants did not transport metals, as these proteins could not rearrange to allow both metal entry and metal exit. Surprisingly, Nramp variants trapped in the outward-open state—but not the inward-open state—could still transport protons. This suggested that protons travel through the same external cavity as metal ions but can transit a separate pathway through the inside half of the protein to reach the inside of the cell. We identified a conserved network of protonatable amino acid residues that leads from the metal-binding site to the inside of the cell and form the proton transport pathway. Ultimately, the separate pathways we delineated for the metal ions and protons may allow Nramp to circumvent the expected electrostatic repulsion between the two positively charged ions—the metal ion and the proton—that it transports. When compared to structurally-related transporters, the parallel transport pathways appear to be a unique feature of the Nramp family. Our findings thus illustrate the flexibility of a common transporter structure to support a broad range of substrate transport and conformational change mechanisms.
Our new model for Nramp transport ultimately improves our understanding of how organisms have evolved to maintain metal homeostasis—the delicate balance between having enough essential metals while avoiding the toxicity of metal overaccumulation, which is an important component of human health. In addition, few transport proteins have mechanisms that are well understood at a structural level, so Nramp may help us understand more general principles about wider classes of transporters.
This study includes contributions from several current and former lab members. Aaron Bozzi (PhD’18) determined the “outward-open” structure with the help of Jack Nicoludis (PhD’18) and led the biochemical experiments. Former Postdoc Christina Zimanyi (currently a Research Scientist at the New York Structural Biology Center) determined the “occluded” structure. Brandon Lee (currently a Computer Science senior) engineered the “outward-open” variant. Casey Zhang (currently an Applied Math junior) performed sequence analyses and developed a new structure comparison tool that we used in our analyses of the structures.
Our new structures illustrate the three principal conformations, or states, of an Nramp transporter. From left to right: (Left) The transporter has a water-filled cavity (blue dots) open to the outside, enabling a metal ion (purple sphere) to come into the binding site. (Middle) The outside cavity closes, so the metal-binding site (star) is occluded from both sides of the membrane. (Right) The transporter has a water-filled cavity open to the inside of the cell, allowing the metal ion to enter the cell. The yellow sticks represent the amino acid residues that provide an alternate path for protons to enter the cells.
by Aaron Bozzi and Rachelle Gaudet
