Transmembrane helix 6b links proton and metal release pathways and drives conformational change in an Nramp-family transition metal transporter

Abstract

The natural resistance-associated macrophage protein (Nramp) family encompasses transition metal and proton cotransporters that are present in many organisms from bacteria to humans. Recent structures of Deinococcus radiodurans Nramp (DraNramp) in multiple conformations revealed the intramolecular rearrangements required for alternating access of the metal-binding site to the external or cytosolic environment. Here, using recombinant proteins and metal transport and cysteine accessibility assays, we demonstrate that two parallel cytoplasm-accessible networks of conserved hydrophilic residues in DraNramp, one lining the wide intracellular vestibule for metal release and the other forming a narrow proton transport pathway, are essential for metal transport. We further show that mutagenic or posttranslational modifications of transmembrane helix (TM) 6b, which structurally links these two pathways, impede normal conformational cycling and metal transport. TM6b contains two highly conserved histidines, His232 and His237. We found that different mutagenic perturbations of His232, just below the metal-binding site along the proton exit route, differentially affect DraNramp's conformational state, suggesting that His232 serves as a pivot point for conformational changes. In contrast, any replacement of His237, lining the metal exit route, locked the transporter in a transport-inactive outward-closed state. We conclude that these two histidines, and TM6b more broadly, help trigger the bulk rearrangement of DraNramp to the inward-open state upon metal binding and facilitate return of the empty transporter to an outward-open state upon metal release.

Structural and functional properties of a magnesium transporter of the SLC11/NRAMP family

“…Similar to the altered substrate preference, also the second functional hallmark of EleNRMT concerning the absent coupling of metal ion transport to protons, is manifested in its structure ( Figure 7D and E ). Proton transport in classical NRAMP transporters was proposed to be linked to structural features extending from the ion binding site ( Bozzi et al, 2019a ; Bozzi et al, 2020 ; Bozzi et al, 2019b ; Ehrnstorfer et al, 2017 ; Mackenzie et al, 2006 ; Pujol-Giménez et al, 2017 ). These include the conserved aspartate involved in the coordination of metal ions, two conserved histidines on α-helix 6b downstream of the binding site methionine and a continuous path of acidic and basic residues on α-helices 3 and 9 surrounding a narrow aqueous cavity, which together appear to constitute an intracellular H + release pathway.…”

“…Similar to the altered substrate preference, also the second functional hallmark of EleNRMT concerning the absent coupling of metal ion transport to protons, is manifested in its structure (Figure 7D, E). Proton transport in classical NRAMP transporters was proposed to be linked to structural features extending from the ion binding site (Bozzi et al, 2019a;Bozzi et al, 2020;Bozzi et al, 2019b;Ehrnstorfer et al, 2017;Mackenzie et al, 2006;Pujol-Gimenez et al, 2017).…”

Electrophysiology Measurements of Metal Transport by MntH2 from Enterococcus faecalis

“…Proton co-transport is known to vary between members of the Nramp family and does not always follow a strict stoichiometric ratio, but depends on the condition such as pH and even the metal substrate [ 28 , 29 , 30 ]. Functional studies on Nramp highlighted several conserved residues in close vicinity of the metal-binding pocket that are important for the proton-coupled aspect of the transport mechanism [ 15 , 17 , 30 , 31 ]. Notably, mutating a conserved histidines to an alanine in Nramp from S. capitis (His236) or human (His272) retains their ability to transport manganese, but proton transport was abolished [ 15 , 29 ].…”

“…This correlates with studies on E. coli MntH, in which the LeuT motifs in transmembrane (TM) helices 1 (Asp-Pro-Gly) and 6 (Met-Pro-His) were found to be key to the metal-proton symport activity (the histidine in the Met-Pro-His motif aligns with the S. capitis (His236) and human (His272) Nramp) [ 25 ]. In stark contrast, however, mutating the homologue histidine in D. radiodurans (His237) locks the protein in an outward closed state, rendering the transporter inactive [ 31 ]. Furthermore, other D. radiodurans MntH mutants, which lock the transporter into either an inside- or outside-open state, show proton transport in the locked outside-open state (without metal transport), but not the inside-open state.…”

Structural and functional properties of a magnesium transporter of the SLC11/NRAMP family

“…Similar to the altered substrate preference, also the second functional hallmark of EleNRMT concerning the absent coupling of metal ion transport to protons, is manifested in its structure (Figure 7D, E). Proton coupling in classical NRAMP transporters was assigned to structural features extending from the ion binding site (Bozzi et al, 2019a;Bozzi et al, 2020;Ehrnstorfer et al, 2017;Mackenzie et al, 2006;Pujol-Gimenez et al, 2017). These include a conserved histidine on α-helix 6b located two residues downstream of the binding site methionine and a continuous path of acidic and basic residues on α-helices 3 and 9 surrounding a narrow aqueous cavity, which together appear to constitute an intracellular H + release pathway.…”