Evidence that highly conserved residues of transmembrane segment 6 of escherichia coli mnth are important for transport activity

Heather A.H. Haemig, Patrick J. Moen, Robert J. Brooker

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

Nramp (natural resistance-associated macrophage protein) family members have been characterized in mammals, yeast, and bacteria as divalent metal ion/H+ symporters. In previous work, a bioinformatic approach was used for the identification of residues that are conserved within the Nramp family [Haemig, H. A., and Brooker, R. J. (2004) J. Membr. Biol. 201 (2), 97?107]. On the basis of site-directed mutagenesis of highly conserved negatively charged residues, a model was proposed for the metal binding site of the Escherichia coli homologue, MntH. In this study, we have focused on the highly conserved residues, including two histidines, of transmembrane segment 6 (TMS-6). Multiple mutants were made at the eight conserved sites (i.e., Gly-205, Ala-206, Met-209, Pro-210, His-211, Leu-215, His-216, and Ser-217) in TMS-6 of E. coli MntH. Double mutants involving His-211 and His-216 were also created. The results indicate the side chain volume of these residues is critically important for function. In most cases, only substitutions that are closest in side chain volume still permit transport. In addition, the Km for metal binding is largely unaffected by mutations in TMS-6, whereas V max values were decreased in all mutants characterized kinetically. Thus, these residues do not appear to play a role in metal binding. Instead, they may comprise an important face on TMS-6 that is critical for protein conformational changes during transport. Also, in contrast to other studies, our data do not strongly indicate that the conserved histidine residues play a role in the pH regulation of metal transport.

Original languageEnglish (US)
Pages (from-to)4662-4671
Number of pages10
JournalBiochemistry
Volume49
Issue number22
DOIs
StatePublished - Jun 8 2010

Fingerprint

Escherichia coli
Metals
Histidine
Symporters
Mutagenesis
Mammals
Bioinformatics
Yeast
Metal ions
Bacteria
Site-Directed Mutagenesis
Computational Biology
Substitution reactions
Binding Sites
Yeasts
Ions
Mutation
Proteins
natural resistance-associated macrophage protein 1

Cite this

Evidence that highly conserved residues of transmembrane segment 6 of escherichia coli mnth are important for transport activity. / Haemig, Heather A.H.; Moen, Patrick J.; Brooker, Robert J.

In: Biochemistry, Vol. 49, No. 22, 08.06.2010, p. 4662-4671.

Research output: Contribution to journalArticle

@article{128d9f4e89c54d42be7e1cdf306a5aa9,
title = "Evidence that highly conserved residues of transmembrane segment 6 of escherichia coli mnth are important for transport activity",
abstract = "Nramp (natural resistance-associated macrophage protein) family members have been characterized in mammals, yeast, and bacteria as divalent metal ion/H+ symporters. In previous work, a bioinformatic approach was used for the identification of residues that are conserved within the Nramp family [Haemig, H. A., and Brooker, R. J. (2004) J. Membr. Biol. 201 (2), 97?107]. On the basis of site-directed mutagenesis of highly conserved negatively charged residues, a model was proposed for the metal binding site of the Escherichia coli homologue, MntH. In this study, we have focused on the highly conserved residues, including two histidines, of transmembrane segment 6 (TMS-6). Multiple mutants were made at the eight conserved sites (i.e., Gly-205, Ala-206, Met-209, Pro-210, His-211, Leu-215, His-216, and Ser-217) in TMS-6 of E. coli MntH. Double mutants involving His-211 and His-216 were also created. The results indicate the side chain volume of these residues is critically important for function. In most cases, only substitutions that are closest in side chain volume still permit transport. In addition, the Km for metal binding is largely unaffected by mutations in TMS-6, whereas V max values were decreased in all mutants characterized kinetically. Thus, these residues do not appear to play a role in metal binding. Instead, they may comprise an important face on TMS-6 that is critical for protein conformational changes during transport. Also, in contrast to other studies, our data do not strongly indicate that the conserved histidine residues play a role in the pH regulation of metal transport.",
author = "Haemig, {Heather A.H.} and Moen, {Patrick J.} and Brooker, {Robert J.}",
year = "2010",
month = "6",
day = "8",
doi = "10.1021/bi100320y",
language = "English (US)",
volume = "49",
pages = "4662--4671",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "22",

}

TY - JOUR

T1 - Evidence that highly conserved residues of transmembrane segment 6 of escherichia coli mnth are important for transport activity

AU - Haemig, Heather A.H.

AU - Moen, Patrick J.

AU - Brooker, Robert J.

PY - 2010/6/8

Y1 - 2010/6/8

N2 - Nramp (natural resistance-associated macrophage protein) family members have been characterized in mammals, yeast, and bacteria as divalent metal ion/H+ symporters. In previous work, a bioinformatic approach was used for the identification of residues that are conserved within the Nramp family [Haemig, H. A., and Brooker, R. J. (2004) J. Membr. Biol. 201 (2), 97?107]. On the basis of site-directed mutagenesis of highly conserved negatively charged residues, a model was proposed for the metal binding site of the Escherichia coli homologue, MntH. In this study, we have focused on the highly conserved residues, including two histidines, of transmembrane segment 6 (TMS-6). Multiple mutants were made at the eight conserved sites (i.e., Gly-205, Ala-206, Met-209, Pro-210, His-211, Leu-215, His-216, and Ser-217) in TMS-6 of E. coli MntH. Double mutants involving His-211 and His-216 were also created. The results indicate the side chain volume of these residues is critically important for function. In most cases, only substitutions that are closest in side chain volume still permit transport. In addition, the Km for metal binding is largely unaffected by mutations in TMS-6, whereas V max values were decreased in all mutants characterized kinetically. Thus, these residues do not appear to play a role in metal binding. Instead, they may comprise an important face on TMS-6 that is critical for protein conformational changes during transport. Also, in contrast to other studies, our data do not strongly indicate that the conserved histidine residues play a role in the pH regulation of metal transport.

AB - Nramp (natural resistance-associated macrophage protein) family members have been characterized in mammals, yeast, and bacteria as divalent metal ion/H+ symporters. In previous work, a bioinformatic approach was used for the identification of residues that are conserved within the Nramp family [Haemig, H. A., and Brooker, R. J. (2004) J. Membr. Biol. 201 (2), 97?107]. On the basis of site-directed mutagenesis of highly conserved negatively charged residues, a model was proposed for the metal binding site of the Escherichia coli homologue, MntH. In this study, we have focused on the highly conserved residues, including two histidines, of transmembrane segment 6 (TMS-6). Multiple mutants were made at the eight conserved sites (i.e., Gly-205, Ala-206, Met-209, Pro-210, His-211, Leu-215, His-216, and Ser-217) in TMS-6 of E. coli MntH. Double mutants involving His-211 and His-216 were also created. The results indicate the side chain volume of these residues is critically important for function. In most cases, only substitutions that are closest in side chain volume still permit transport. In addition, the Km for metal binding is largely unaffected by mutations in TMS-6, whereas V max values were decreased in all mutants characterized kinetically. Thus, these residues do not appear to play a role in metal binding. Instead, they may comprise an important face on TMS-6 that is critical for protein conformational changes during transport. Also, in contrast to other studies, our data do not strongly indicate that the conserved histidine residues play a role in the pH regulation of metal transport.

UR - http://www.scopus.com/inward/record.url?scp=77953111415&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=77953111415&partnerID=8YFLogxK

U2 - 10.1021/bi100320y

DO - 10.1021/bi100320y

M3 - Article

C2 - 20441230

AN - SCOPUS:77953111415

VL - 49

SP - 4662

EP - 4671

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 22

ER -