Dynamic distinctions in the Na+/Ca2+ exchanger adopting the inward- and outward-facing conformational states

Abstract

Na/Ca exchanger (NCX) proteins operate through the alternating access mechanism, where the ion-binding pocket is exposed in succession either to the extracellular or the intracellular face of the membrane. The archaeal NCX_Mj ( NCX) system was used to resolve the backbone dynamics in the inward-facing (IF) and outward-facing (OF) states by analyzing purified preparations of apo- and ion-bound forms of NCX_Mj-WT and its mutant, NCX_Mj-5L6-8. First, the exposure of extracellular and cytosolic vestibules to the bulk phase was evaluated as the reactivity of single cysteine mutants to a fluorescent probe, verifying that NCX_Mj-WT and NCX_Mj-5L6-8 preferentially adopt the OF and IF states, respectively. Next, hydrogen-deuterium exchange-mass spectrometry (HDX-MS) was employed to analyze the backbone dynamics profiles in proteins, preferentially adopting the OF (WT) and IF (5L6-8) states either in the presence or absence of ions. Characteristic differences in the backbone dynamics were identified between apo NCX_Mj-WT and NCX_Mj-5L6-8, thereby underscoring specific conformational patterns owned by the OF and IF states. Saturating concentrations of Na or Ca specifically modify HDX patterns, revealing that the ion-bound/occluded states are much more stable (rigid) in the OF than in the IF state. Conformational differences observed in the ion-occluded OF and IF states can account for diversifying the ion-release dynamics and apparent affinity ( ) at opposite sides of the membrane, where specific structure-dynamic elements can effectively match the rates of bidirectional ion movements at physiological ion concentrations.

Hydrogen-Deuterium Exchange Mass-Spectrometry of Secondary Active Transporters: From Structural Dynamics to Molecular Mechanisms

“…We detected a Na + -dependent decrease in deuterium uptake at S int , S Ca , and S ext , but not S mid , whereas in the presence of Ca 2+ the decrease in deuterium uptake was mainly observed at S Ca . This is consistent with the predictions made by the MD simulations and mutational analyses foreseeing the occupation of S mid by a water molecule but not by Na + or Ca 2+ in the ground state (Marinelli et al, 2014;Giladi et al, 2016a;Giladi et al, 2017;van Dijk et al, 2018). Notably, subsequent crystallographic studies have validated our binding sites' assignment (Liao et al, 2016).…”

“…Surprisingly, the crystal structure of NCX from Methanocaldococcus jannaschii (NCX_Mj) revealed four ion binding sites, simultaneously occupied by three Na + ions at sites termed S int , S mid , S ext , and one Ca 2+ ion at a site termed S Ca (Liao et al, 2012). Since this binding mode was inconsistent with previous studies, MD simulations and ion flux analyses of mutants were performed, suggesting that Na + ions occupy S int , S Ca , and S ext , whereas Ca 2+ occupies S Ca (Marinelli et al, 2014;Giladi et al, 2016b;Giladi et al, 2017;van Dijk et al, 2018). Thus, the Na + and Ca 2+ ions are bound in a mutually exclusive manner along the transport cycle.…”

“…To experimentally establish the assignment of ion binding sites, we compared the apo-state with the ion-bound states of NCX_Mj using HDX-MS (Giladi et al, 2016a;Giladi et al, 2017). Despite low sequence coverage, our analysis included 10 out of 12 ion-coordinating residues.…”

A glimpse into the molecular mechanism of integral membrane proteins through hydrogen–deuterium exchange mass spectrometry

“…A study on the exchanger NCX_Mj responsible for Ca 2+ homeostasis is an elegant demonstration that obtaining a lot of sequence coverage is not always necessary to answer specific mechanistic questions . NCX transporters exchange three Na + ions for one Ca 2+ but the binding sequence of events is difficult to understand.…”

MS-Based Approaches Enable the Structural Characterization of Transcription Factor / DNA Response Element Complex

“…In HDX experiments, the exchange rate of backbone amide hydrogens, which is affected by solvent accessibility and possible involvement in hydrogen bonding, can be directly determined by MS analysis. This technique has been broadly employed to study conformational changes [19,20], protein folding [21,22] and protein-protein interactions [23,24], as well as nucleic acid-protein complexes [25][26][27][28][29][30]. Among the MS3D techniques, chemical and photo-activated cross-linking (XL) are employed to generate stable covalent bridges between contiguous functional groups, which can reveal their mutual placement in the targeted assembly [13,16].…”