title: Solid-State Nuclear Magnetic Resonance Structural Study of the Retinal-Binding Pocket in Sodium Ion Pump Rhodopsin.
abstract: The recently identified Krokinobacter rhodopsin 2 (KR2) functions as a light-driven sodium ion pump. The structure of the retinal-binding pocket of KR2 offers important insights into the mechanisms of KR2, which has motif of Asn112, Asp116, and Gln123 (NDQ) that is common among sodium ion pump rhodopsins but is unique among other microbial rhodopsins. Here we present solid-state nuclear magnetic resonance (NMR) characterization of retinal and functionally important residues in the vicinity of retinal in the ground state. We assigned chemical shifts of retinal C14 and C20 atoms, and Tyr218Cζ, Lys255Cε, and the protonated Schiff base of KR2 in lipid environments at acidic and neutral pH.15N NMR signals of the protonated Schiff base showed a twist around the N-Cε bond under neutral conditions, compared with other microbial rhodopsins. These data indicated that the location of the counterion Asp116 is one helical pitch toward the cytoplasmic side. In acidic environments, the15N Schiff base signal was shifted to a lower field, indicating that protonation of Asp116 induces reorientation during interactions between the Schiff base and Asp116. In addition, the Tyr218 residue in the vicinity of retinal formed a weak hydrogen bond with Asp251, a temporary Na+-binding site during the photocycle. These features may indicate unique mechanisms of sodium ion pumps.
authors.affiliation: Graduate School of Engineering, Yokohama National University , Hodogaya-ku, Yokohama 240-8501, Japan.
authors.forename: Arisu
authors.initials: A
authors.lastname: Shigeta
authors.affiliation: Department of Frontier Materials, Nagoya Institute of Technology , Showa-ku, Nagoya 466-8555, Japan.
authors.forename: Shota
authors.initials: S
authors.lastname: Ito
authors.affiliation: Department of Frontier Materials, Nagoya Institute of Technology , Showa-ku, Nagoya 466-8555, Japan.|OptoBioTechnology Research Center, Nagoya Institute of Technology , Showa-ku, Nagoya 466-8555, Japan.|PRESTO, Japan Science and Technology Agency (JST) , 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan.
authors.forename: Keiichi
authors.initials: K
authors.lastname: Inoue
authors.affiliation: Department of Organic Chemistry for Life Science, Kobe Pharmaceutical University , Higashinada-ku, Kobe 658-8558, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Okitsu
authors.affiliation: Department of Organic Chemistry for Life Science, Kobe Pharmaceutical University , Higashinada-ku, Kobe 658-8558, Japan.
authors.forename: Akimori
authors.initials: A
authors.lastname: Wada
authors.affiliation: Department of Frontier Materials, Nagoya Institute of Technology , Showa-ku, Nagoya 466-8555, Japan.|OptoBioTechnology Research Center, Nagoya Institute of Technology , Showa-ku, Nagoya 466-8555, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Kandori
authors.affiliation: Graduate School of Engineering, Yokohama National University , Hodogaya-ku, Yokohama 240-8501, Japan.
authors.forename: Izuru
authors.identifier: 0000-0002-8163-9695
authors.initials: I
authors.lastname: Kawamura
mesh_terms: D020816:Amino Acid Motifs; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D041965:Flavobacteriaceae / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D017136:Ion Transport; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011485:Protein Binding; D000072417:Protein Domains; D017433:Protein Structure, Secondary; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012172:Retinaldehyde / Q000737:chemistry* / Q000378:metabolism; D025604:Rhodopsins, Microbial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012545:Schiff Bases / Q000737:chemistry / Q000378:metabolism; D012964:Sodium / Q000737:chemistry* / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D011522:Protons; D011994:Recombinant Proteins; D025604:Rhodopsins, Microbial; D012545:Schiff Bases; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D012172:Retinaldehyde