Structural changes of Natronomonas pharaonis halorhodopsin in its late photocycle revealed by solid-state NMR spectroscopy

Natronomonas pharaonis halorhodopsin (NpHR) is a light-driven Cl⁻ inward pump that is widely used as an optogenetic tool. Although NpHR is previously extensively studied, its Cl⁻ uptake process is not well understood from the protein structure perspective, mainly because in crystalline lattice, it has been difficult to analyze the structural changes associated with the Cl⁻ uptake process. In this study, we used solid-state NMR to analyze NpHR both in the Cl⁻-bound and -free states under near-physiological transmembrane condition. Chemical shift perturbation analysis suggested that while the structural change caused by the Cl⁻ depletion is widespread over the NpHR molecule, residues in the extracellular (EC) part of helix D exhibited significant conformational changes that may be related to the Cl⁻ uptake process. By combining photochemical analysis and dynamic nuclear polarization (DNP)-enhanced solid-state NMR measurement on NpHR point mutants for the suggested residues, we confirmed their importance in the Cl⁻ uptake process. In particular, we found the mutation at Ala165 position, located at the trimer interface, to an amino acid with bulky sidechain (A165V) significantly perturbs the late photocycle and disrupts its trimeric assembly in the Cl⁻-free state as well as during the ion-pumping cycle under the photo-irradiated condition. This strongly suggested an outward movement of helix D at EC part, disrupting the trimer integrity. Together with the spectroscopic data and known NpHR crystal structures, we proposed a model that this helix movement is required for creating the Cl⁻ entrance path on the extracellular surface of the protein and is crucial to the Cl⁻ uptake process.