Sodium recognition by the Na+/Ca2+ exchanger in the outward-facing conformation. Proc Natl Acad Sci U S A 2014 Dec 16;111(50):E5354-62
Date
12/04/2014Pubmed ID
25468964Pubmed Central ID
PMC4273333DOI
10.1073/pnas.1415751111Scopus ID
2-s2.0-84919343757 (requires institutional sign-in at Scopus site) 65 CitationsAbstract
Na(+)/Ca(2+) exchangers (NCXs) are ubiquitous membrane transporters with a key role in Ca(2+) homeostasis and signaling. NCXs mediate the bidirectional translocation of either Na(+) or Ca(2+), and thus can catalyze uphill Ca(2+) transport driven by a Na(+) gradient, or vice versa. In a major breakthrough, a prokaryotic NCX homolog (NCX_Mj) was recently isolated and its crystal structure determined at atomic resolution. The structure revealed an intriguing architecture consisting of two inverted-topology repeats, each comprising five transmembrane helices. These repeats adopt asymmetric conformations, yielding an outward-facing occluded state. The crystal structure also revealed four putative ion-binding sites, but the occupancy and specificity thereof could not be conclusively established. Here, we use molecular-dynamics simulations and free-energy calculations to identify the ion configuration that best corresponds to the crystallographic data and that is also thermodynamically optimal. In this most probable configuration, three Na(+) ions occupy the so-called Sext, SCa, and Sint sites, whereas the Smid site is occupied by one water molecule and one H(+), which protonates an adjacent aspartate side chain (D240). Experimental measurements of Na(+)/Ca(2+) and Ca(2+)/Ca(2+) exchange by wild-type and mutagenized NCX_Mj confirm that transport of both Na(+) and Ca(2+) requires protonation of D240, and that this side chain does not coordinate either ion at Smid. These results imply that the ion exchange stoichiometry of NCX_Mj is 3:1 and that translocation of Na(+) across the membrane is electrogenic, whereas transport of Ca(2+) is not. Altogether, these findings provide the basis for further experimental and computational studies of the conformational mechanism of this exchanger.
Author List
Marinelli F, Almagor L, Hiller R, Giladi M, Khananshvili D, Faraldo-Gómez JDAuthor
Fabrizio Marinelli PhD Associate Professor in the Biophysics department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
Escherichia coliGenetic Vectors
Hydrogen-Ion Concentration
Methanocaldococcus
Models, Molecular
Molecular Dynamics Simulation
Protein Conformation
Sodium
Sodium-Calcium Exchanger
Thermodynamics
