Poster Presentation 23rd International Society of Magnetic Resonance Conference 2023

Solid-state NMR study of zinc-bound Thermoplasmatales archaeon heliorhodopsin (#221)

Sari Kumagai 1 , Shibuki Suzuki 1 , Toshio Nagashima 2 , Toshio Yamazaki 2 , Kota Katayama 3 , Hideki Kandori 3 , Izuru Kawamra 1
  1. Graduate School of Engineering Science, Yokohama National University, Yokohama, Japan
  2. RIKEN RSC, Hyogo, Japan
  3. Nagoya Institute of Technology, Nagoya, Japan

Heliorhodopsin (HeR) is a transmembrane protein with a retinal chromophore that constitutes a new rhodopsin family [1]. The unique sequence homology, inverted membrane topology, and the long photocycle distinguishes HeR from microbial and animal rhodopsin families [1]. The crystal structure of Thermoplasmatales archaeon heliorhodopsin (TaHeR) revealed that it forms a dimer in the membrane [2]. In addition, 15N protonated Schiff base (RPSB)/λmax of TaHeR with the all-trans form (178.9 ppm / 542 nm) and 15N chemical shift anisotropy (CSA) deviated more strongly from the linear relationship of the model retinylidene PSB compounds compared to other microbial rhodopsins [3]. Thus, it is suggested that the RPSB-counterion interaction is clearly different from those of other kinds. Ser112 and Ser234, which are distinctive residues around the RPSB, may have a role in stabilizing the excited state of all-trans retinal. Furthermore, only helical structural perturbations were observed for zinc-bound TaHeR by FTIR [4]. Here, using solid-state NMR spectroscopy, we investigated whether the structure of membrane embedded TaHeR in POPE/POPG is affected by zinc-binding. We exhibited zinc concentration dependency in changes of 15N NMR signals of some His imidazole rings using 15N3 His-labeled sample. A new peak at 210 ppm was detected due to the increase of electron density of Nδ1. The dissociation constant was estimated as 0.24 mM from the peak growth. Moreover, improvement of acquisition efficiency due to restriction of side chains and existence of hydration water contributed to the intensity increase of Nε2 region. Despite the drastic signal changes of some His, the peak of RPSB did not shift using 15N Lys-labeled sample under ZnCl2 condition. From our results, we assume that the binding site of zinc is located at His residues in the cytoplasmic region, distant from the retinal. Now, mutations are measured to assign and specify the His residue which is responsible for zinc-binding. These new structural insights will be helpful to discover the binding site of zinc and the mechanism of zinc-binding. This binding may contribute to the unknown function of HeR, which is thought to be signal transduction.

  1. A. Pushkarev et al. (2018) Nature, 558, 595.
  2. W. Shihoya et al. (2019) Nature, 574, 132.
  3. S. Suzuki et al. (2023) Biophys. Chem. 296, 106991.
  4. M. Hashimoto et al. (2020) JPC Lett. 11, 8604.