title: Ca2+ binding alters the interdomain flexibility between the two cytoplasmic calcium-binding domains in the Na+/Ca2+ exchanger.
abstract: The Na(+)/Ca(2+) exchanger (NCX) is a membrane protein, which catalyzes the counter transport of Na(+) and Ca(2+) ions across the plasma membrane, playing a key role in the maintenance of the intracellular Ca(2+) homeostasis in various cell types. NCX consists of a transmembrane part and a large intracellular loop. The activation of the NCX transport function requires the binding of Ca(2+) to two tandem C2 domains, CBD1 and CBD2, which are an integral part of the exchanger's intracellular loop. Although high-resolution structures of individual CBD1 and CBD2 are available, their interdomain structure and dynamics and the atomic level mechanism of allosteric Ca(2+)-regulation remains unknown. Here, we use solution NMR spectroscopy to study the interdomain dynamics of CBD12, a 32 kDa construct that contains both the CBD1 and CBD2 domains connected by a short linker. Analysis of NMR residual dipolar couplings shows that CBD12 assumes on average an elongated shape both in the absence and in the presence of Ca(2+). NMR (15)N relaxation data of the Apo state indicate that the two domains sample a wide range of relative arrangements on the nanosecond time scale. These arrangements comprise significantly non-linear interdomain orientations. Binding of Ca(2+) to CBD1 significantly restricts the interdomain flexibility, stabilizing a more rigid elongated conformation. These findings suggest a molecular mechanism for the role of CBD12 in the function of NCX.
authors.affiliation: Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32306-4390, USA.
authors.forename: Roberto K
authors.initials: RK
authors.lastname: Salinas
authors.forename: Lei
authors.initials: L
authors.lastname: Bruschweiler-Li
authors.forename: Eric
authors.initials: E
authors.lastname: Johnson
authors.forename: Rafael
authors.initials: R
authors.lastname: Brüschweiler
mesh_terms: D000494:Allosteric Regulation / Q000502:physiology; D000818:Animals; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D004285:Dogs; D019906:Nuclear Magnetic Resonance, Biomolecular; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D019831:Sodium-Calcium Exchanger / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D019831:Sodium-Calcium Exchanger; C110374:sodium-calcium exchanger 1; D002118:Calcium