Identification of the dimer interface of a bacterial Ca2+/H + antiporter

Marc Ridilla, Anoop Narayanan, Jeffrey T. Bolin, Dinesh A. Yernool

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Members of the calcium/cation antiporter superfamily, including the cardiac sodium/calcium exchangers, are secondary active transporters that play an essential role in cellular Ca2+ homeostasis. A notable feature of this group of transporters is the high levels of sequence similarity in relatively short sequences constituting the functionally important α-1 and α-2 regions in contrast to relatively lower degrees of similarity in the extended adjoining sequences. This suggests a similar structure and function of core transport machinery but possible differences in topology and/or oligomerization, a topic that has not been adequately addressed. Here we present the first example of purification of a bacterial member of this superfamily (CAXCK31) and analyze its quaternary structure. Purification of CAXCK31 required the presence of a choline headgroup-containing detergent or lipid to yield stable preparations of the monomeric transporter. H+-driven Ca2+ transport was demonstrated by reconstituting purified CAXCK31 into liposomes. Dimeric CAX CK31 could be isolated by manipulation of detergent micelles. Dimer formation was shown to be dependent on micelle composition as well as protein concentration. Furthermore, we establish that CAXCK31 forms dimers in the membrane by analysis of cross-linked proteins. Using a dimeric homology model derived from the monomeric structure of the archaeal NCX homologue (Protein Data Bank entry 3V5U), we introduced cysteine residues and through cross-linking experiments established the role of transmembrane helices 2 and 6 in the putative dimer interface.

Original languageEnglish (US)
Pages (from-to)9603-9611
Number of pages9
JournalBiochemistry
Volume51
Issue number48
DOIs
StatePublished - Dec 4 2012

Fingerprint

Antiporters
Dimers
Micelles
Detergents
Purification
Sodium-Calcium Exchanger
Oligomerization
Proteins
Choline
Liposomes
Machinery
Cysteine
Cations
Homeostasis
Topology
Databases
Calcium
Membranes
Lipids
Chemical analysis

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Ridilla, Marc ; Narayanan, Anoop ; Bolin, Jeffrey T. ; Yernool, Dinesh A. / Identification of the dimer interface of a bacterial Ca2+/H + antiporter. In: Biochemistry. 2012 ; Vol. 51, No. 48. pp. 9603-9611.
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Identification of the dimer interface of a bacterial Ca2+/H + antiporter. / Ridilla, Marc; Narayanan, Anoop; Bolin, Jeffrey T.; Yernool, Dinesh A.

In: Biochemistry, Vol. 51, No. 48, 04.12.2012, p. 9603-9611.

Research output: Contribution to journalArticle

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