Solid-state NMR analysis of the sodium pump Krokinobacter rhodopsin 2 and its H30A mutant

Krokinobacter eikastus rhodopsin 2 (KR2) is a pentameric, light-driven ion pump, which selectively transports sodium or protons. The mechanism of ion selectivity and transfer is unknown. By using conventional as well as dynamic nuclear polarization (DNP)-enhanced solid-state NMR, we were able to analyse the retinal polyene chain between positions C10 and C15 as well as the Schiff base nitrogen in the KR2 resting state. In addition, 50% of the KR2 13C and 15N resonances could be assigned by multidimensional high-field solid-state NMR experiments. Assigned residues include part of the NDQ motif as well as sodium binding sites. Based on these data, the structural effects of the H30A mutation, which seems to shift the ion selectivity of KR2 primarily to Na+, could be analysed. Our data show that it causes long-range effects within the retinal binding pocket and at the extracellular Na+ binding site, which can be explained by perturbations of interactions across the protomer interfaces within the KR2 complex. This study is complemented by data from time-resolved optical spectroscopy

Probing the photointermediates of light-driven sodium ion pump KR2 by DNP-enhanced solid-state NMR

The functional mechanism of the light-driven sodium pump Krokinobacter eikastus rhodopsin 2 (KR2) raises fundamental questions since the transfer of cations must differ from the better-known principles of rhodopsin-based proton pumps. Addressing these questions must involve a better understanding of its photointermediates. Here, dynamic nuclear polarization-enhanced solid-state nuclear magnetic resonance spectroscopy on cryo-trapped photointermediates shows that the K-state with 13-cis retinal directly interconverts into the subsequent L-state with distinct retinal carbon chemical shift differences and an increased out-of-plane twist around the C14-C15 bond. The retinal converts back into an all-trans conformation in the O-intermediate, which is the key state for sodium transport. However, retinal carbon and Schiff base nitrogen chemical shifts differ from those observed in the KR2 dark state all-trans conformation, indicating a perturbation through the nearby bound sodium ion. Our findings are supplemented by optical and infrared spectroscopy and are discussed in the context of known three-dimensional structures

High-Field Solid-State NMR with Dynamic Nuclear Polarization

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