Substrate-modulated ADP/ATP-transporter dynamics revealed by NMR relaxation dispersion

Abstract

The ADP/ATP carrier (AAC) transports ADP and ATP across the inner mitochondrial membrane. Unlike most transporters that have 2-fold direct or inverted quasi-symmetry, AAC has the apparent 3-fold rotational symmetry. Further, its transport rate is fast for transporters that carry large solutes. Here, we perform comprehensive NMR relaxation dispersion measurements for the yeast AAC carrier 3, which provide residue-specific information on the protein conformational exchange. Our data indicate that AAC is predominantly in the cytosol-facing open state and converts to a lowly populated state in an asymmetric manner despite its three-fold structural symmetry. Binding of the substrate ADP significantly increases the rate of conformational exchange, whereas the inhibitor CATR slows the exchange. These results suggest that while the transporter catalyzes the translocation of substrate, the substrate also facilitates interconversion between alternating states that may be relevant to the transport function.

Dynamics and interactions of ADP/ATP transporter AAC3 in DPC detergent are not functionally relevant

“…2 The thermal stability of AACs in mild detergent increases by more than 30°C upon binding of CATR, 5,6 revealing a considerable change in the properties induced by inhibitor-binding, at variance with the very modest CATR-induced effects reported in ref. 1. The lack of binding specificity, low affinity and hardly detectable effects on dynamics all point to a non-functional state of yAAC3 in DPC, in line with other reports on mitochondrial carriers.…”

“…We used the exchange-rate constants (kex), population of "excited" state (pe), as well as chemical-shift differences (Δν) reported in ref. 1, to generate theoretical CPMG dispersion curves, and superimposed them onto the original CPMG data ( Figure 1C, grey/black). The strong disagreement with the experimental data shows that the fit parameters by Brüschweiler et al do not describe their experimental data, thus questioning an interpretation that the authors put forward: they used the large values of Δν, particularly in the proline kink regions, as an argument to propose that the excited state may resemble the m-state.…”

In vitro reconstitution, functional dissection, and mutational analysis of metal ion transport by mitoferrin-1

“…Moreover, eukaryotic membrane proteins are fragile molecules once extracted from the membrane and the harsh detergents used for solubilization and purification of the carriers using this method cast doubt about the integrity of the proteins throughout such preparations. Accordingly, biophysical data on interactions between the carriers and their respective substrates exist for only a few MCs (24)(25)(26)(27).…”

NMR as a tool to investigate the structure, dynamics and function of membrane proteins

“…39 A comprehensive CPMG relaxation dispersion study revealed that slow conformational exchanges in the ADP/ATP carrier (AAC) were asymmetrically distributed, with large exchanges concentrated in domain I despite its unique three-fold quasi-symmetry. 42 The exchange rate of the TM region was increased or decreased substantially when a substrate or inhibitor bound, respectively. Surprisingly, the relative populations of the bound and unbound states did not change significantly, suggesting that ligand binding changed the energy barrier of conversion by stabilizing (substrate) or destabilizing (inhibitor) the transition state.…”