Structure-function relationships underlying allosteric regulation of Na+/Ca2+ exchanger (NCX) proteins

Orateur : Moshe GILADI (Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Israel)

Na+/Ca2+ exchanger (NCX) proteins move one Ca2+ ion across the membrane against its electrochemical gradient in exchange for three Na+ ions moving along their electrochemical gradient. NCX proteins are allosterically regulated by intracellular [Ca2+] via its interactions with two Ca2+ binding domains, CBD1 and CBD2, forming a two-domain tandem (CBD12) through a short linker. Despite striking similarities between CBDs from different splice-variants, isoforms and orthologs, NCX proteins exhibit different responses to regulatory Ca2+ - activation, inhibition or no response. To resolve the structural basis of allosteric regulation by Ca2+, we examined CBD12 mutants, splice variants and orthologs using X-ray crystallography, small-angle X-ray scattering (SAXS) and hydrogendeuterium exchange mass spectrometry (HDX-MS). The 2.7Å crystal structure revealed a Ca2+-driven interdomain switch, which drives a population-shift of the conformational distributions towards an elongated orientation of CBD12 as revealed by our SAXS analysis. These mechanisms are common among different NCX proteins, even in cases where the regulatory responses to Ca2+ are completely different. Using HDX-MS we show that the differential response stems from differences in Ca2+-dependent rigidification of the main-chain within the CBD2 domain where the extent of the dynamic response correlates with positive, negative or no response to regulatory Ca2+ in a given ortholog/splice variant.

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