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

Moshe Giladi,Liat Van Dijk,Bosmat Refaeli,Lior Almagor,Reuben Hiller,Petr Man,Eric Forest,Daniel Khananshvili

doi:10.1074/jbc.m117.787168

Abstract

Na+/Ca2+ 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 (Methanococcus jannaschii 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 Ca2+ 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 (Km ) at opposite sides of the membrane, where specific structure-dynamic elements can effectively match the rates of bidirectional ion movements at physiological ion concentrations.

Highlights

IntroductionAssigned as the inward-facing (IF) and outward-facing (OF) states [1,2,3]
To verify that NCX from Methanococcus jannaschii (NCX_Mj)-WT and NCX_Mj-5L6 – 8 represent the OF and IF stabilized states and are suitable for hydrogen-deuterium exchange–mass spectrometry (HDX-MS) analysis, we sought to find an independent method to determine the exposure of cytosolic and extracellular vestibules to bulk phase
According to the crystal structure of NCX_Mj and the model of IF NCX_Mj, Gly-42 should be accessible to the bulk phase solution in the IF state and not in the OF state, whereas the opposite is true for Gly-201 (Fig. 2)

Summary

Introduction

Assigned as the inward-facing (IF) and outward-facing (OF) states [1,2,3] In agreement with this dogma, extensive crystallographic studies have identified the OF, IF, and occluded states for many transporters [3,4,5], but the dynamic features of alternative OF/IF shuttling remain unclear even for proteins with a known crystal structure. Conformational dynamics of the NCX transporter ally exclusive manner, where 3Naϩ ions occupy Sext, SCa, and Sint and Ca2ϩ occupies SCa (Fig. 1, D and E) [27]. This assignment of ion-binding sites was further confirmed by crystallographic studies [26]. In the semi-open OF state, two Naϩ ions occupy Sint and SCa where the binding of the third Naϩ to Sext results in 3Naϩ occlusion with associated backbone bending between TM7A and TM7B (Fig. 1F) [26]

Methods

Results

Discussion

Conclusion