Structures in multiple conformations reveal distinct transition metal and proton pathways in an Nramp transporter

Abstract

Nramp family transporters—expressed in organisms from bacteria to humans—enable uptake of essential divalent transition metals via an alternating-access mechanism that also involves proton transport. We present high-resolution structures of Deinococcus radiodurans (Dra)Nramp in multiple conformations to provide a thorough description of the Nramp transport cycle by identifying the key intramolecular rearrangements and changes to the metal coordination sphere. Strikingly, while metal transport requires cycling from outward- to inward-open states, efficient proton transport still occurs in outward-locked (but not inward-locked) DraNramp. We propose a model in which metal and proton enter the transporter via the same external pathway to the binding site, but follow separate routes to the cytoplasm, which could facilitate the co-transport of two cationic species. Our results illustrate the flexibility of the LeuT fold to support a broad range of substrate transport and conformational change mechanisms.

Unique structural features in an Nramp metal transporter impart substrate-specific proton cotransport and a kinetic bias to favor import

“…Recent studies using different Nramp homologues implicated E134 or H232 in proton-metal coupling (Ehrnstorfer et al, 2017;Pujol-Giménez et al, 2017), and we demonstrated the importance of D56 and D131 in addition to E134 and H232 to DraNramp H + transport (Bozzi et al, 2019). From structure-based pK a predictions, we therefore proposed D56 as the initial protonbinding site and D131 as the subsequent proton acceptor required for TM transport (Bozzi et al, 2019).…”

“…We previously showed that mutations to D56, H232, E134, and D131 eliminated DraNramp's voltage-driven proton uniport, thus leading us to propose those four residues as the core of a conserved proton-transport pathway (Bozzi et al, 2019). In Figure 7.…”

“…Like other Nramp homologues (Gunshin et al, 1997;Chen et al, 1999;Mackenzie et al, 2007;Ehrnstorfer et al, 2017), DraNramp also transports protons in addition to divalent metals (Bozzi et al, 2019). We therefore measured H + transport by DraNramp ( Fig.…”

“…6 A). Conserved residues D56, N59, and M230 coordinate metal substrate throughout the transport process (Ehrnstorfer et al, 2014;Bozzi et al, 2019; Fig. 6 B).…”

“…However, strict thermodynamic coupling, such that a gradient of one substrate clearly drives uphill transport of the other, between metal and proton cosubstrates has yet to be demonstrated. In addition, electrophysiological studies of Nramps showed variable proton-metal transport stoichiometries depending on pH and membrane potential (Chen et al, 1999;Nevo and Nelson, 2004), as well as significant proton uniport (Gunshin et al, 1997;Chen et al, 1999;Xu et al, 2004;Mackenzie et al, 2006Mackenzie et al, , 2007Shawki and Mackenzie, 2010;Bozzi et al, 2019), which suggests loose coupling (Nelson et al, 2002;Henderson et al, 2019). Furthermore, multiple Nramp homologues exhibit a pronounced voltage dependence of metal transport rate (Gunshin et al, 1997;Chen et al, 1999;Xu et al, 2004;Mackenzie et al, 2007).…”