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 K(m) 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.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Haemig HA,Moen PJ,Brooker RJdoi
10.1021/bi100320ysubject
Has Abstractpub_date
2010-06-08 00:00:00pages
4662-71issue
22eissn
0006-2960issn
1520-4995journal_volume
49pub_type
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