title: Identification of the dimer interface of a bacterial Ca(2+)/H(+) antiporter.
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 Ca(2+) 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 (CAX(CK31)) and analyze its quaternary structure. Purification of CAX(CK31) required the presence of a choline headgroup-containing detergent or lipid to yield stable preparations of the monomeric transporter. H(+)-driven Ca(2+) transport was demonstrated by reconstituting purified CAX(CK31) 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 CAX(CK31) 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.
authors.affiliation: Department of Biological Sciences, Purdue University, 240 South Martin Jischke Drive, West Lafayette, IN 47906, USA.
authors.forename: Marc
authors.initials: M
authors.lastname: Ridilla
authors.forename: Anoop
authors.initials: A
authors.lastname: Narayanan
authors.forename: Jeffrey T
authors.initials: JT
authors.lastname: Bolin
authors.forename: Dinesh A
authors.initials: DA
authors.lastname: Yernool
mesh_terms: D017920:Antiporters / Q000737:chemistry*; D027682:Cation Transport Proteins / Q000737:chemistry*; D002942:Circular Dichroism; D003902:Detergents / Q000737:chemistry; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins / Q000737:chemistry*; D008823:Micelles; D008958:Models, Molecular
chemical_list: D017920:Antiporters; D027682:Cation Transport Proteins; D003902:Detergents; D029968:Escherichia coli Proteins; D008823:Micelles; C027614:calcium-hydrogen antiporters