Importance of Conserved Acidic Residues in MntH, the Nramp homolog of Escherichia coli

Abstract

A bioinformatic approach was used for the identification of residues that are conserved within the Nramp family of metal transporters. Site-directed mutagenesis was then carried out to change six conserved acidic residues (i.e., Asp-34, Glu-102, Asp-109, Glu-112, Glu-154, and Asp-238) in the E. coli Nramp homolog mntH. Of these six, five of them, Asp-34, Glu-102, Asp-109, Glu-112, and Asp-238 appear to be important for function since conservative substitutions at these sites result in a substantial loss of transport function. In addition, all of the residues within the signature sequence of the Nramp family, DPGN, were also mutated in this study. Each residue was changed to several different side chains, and of ten site-directed mutations made in this motif, only P35G showed any measurable level of (54)Mn(2+) uptake with a V(max) value of approximately 10% of wild-type and a slightly elevated K(m) value. Overall, the data are consistent with a model where helix breakers in the conserved DPGN motif in TMS-1 provide a binding pocket in which Asp-34, Asn-37, Asp-109, Glu-112 (and possibly other residues) are involved in the coordination of Mn(2+). Other residues such as Glu-102 and Asp238 may play a role in the release of Mn(2+) to the cytoplasm or may be involved in maintaining secondary structure.

Conserved methionine dictates substrate preference in Nramp-family divalent metal transporters

“…1C). Loss of a metal-coordinating residue that uses oxygen to bind the metal, D56 or N59, was detrimental to Co 2+ uptake, consistent with the previously demonstrated importance of these residues to the transport of Co 2+ , Fe 2+ , Mn 2+ , and Cd 2+ in bacterial and eukaryotic Nramp homologs (9,(17)(18)(19)(20).…”

Solute Carrier 11 Cation Symport Requires Distinct Residues in Transmembrane Helices 1 and 6

“…The TM location of the targeted sites is schematized (Fig. 1A) based on previous predictions and experimental determinations (11,14,15,39). The mutant proteins displayed membrane expression levels similar to MntH-WT, indicating that the substitutions were structurally well tolerated (Fig.…”

“…The TMS1 Asp residue is part of a conserved DPGN motif that has been subjected to mutagenesis in studies using MntH or Nramp2 homologs, which showed loss of Me 2ϩ uptake caused by Gly exchange (11,47). The carboxyl end of Nramp2 TMS1 and adjacent extra loop were implicated in Me 2ϩ binding and coupling of Me 2ϩ uptake to the proton-motive force ((C/S/T)P(C/H)) that is conserved in the TMS6 of P 1B -type ATPases, which pump heavy metal cations using energy provided by ATP hydrolysis (48,49).…”

“…Four type II rate shifts were identified at TM sites displaying polar or charged SLC11-specific amino acids matched by distinct outgroup-specific residues (10). Reciprocal residue exchange and additional mutations at each of these sites in TMS1, TMS6, and TMS11 were characterized, showing individual roles of each site in the Me 2ϩ and/or H ϩ symport (10); among them residue Asp 34 appeared crucial for transport activity but its functional role remains undefined (10,11).…”

Structural and Mechanistic Basis of Proton-Coupled Metal Ion Transport in the SLC11/NRAMP Family

“…This suggests that Glu129 is not the primary H þ acceptor during transport. Still, since at lower pH, the corresponding mutation of the E. coli homologue MntH strongly decreased the rate of Mn 2 þ uptake into cells 40 , this residue probably plays an important role for protein function.…”