Homotrimer formation and dissociation of pharaonis halorhodopsin in detergent system

Abstract

Halorhodopsin from NpHR is a light-driven Cl(-) pump that forms a trimeric NpHR-bacterioruberin complex in the native membrane. In the case of NpHR expressed in Escherichia coli cell, NpHR forms a robust homotrimer in a detergent DDM solution. To identify the important residue for the homotrimer formation, we carried out mutation experiments on the aromatic amino acids expected to be located at the molecular interface. The results revealed that Phe(150) was essential to form and stabilize the NpHR trimer in the DDM solution. Further analyses for examining the structural significance of Phe(150) showed the dissociation of the trimer in F150A (dimer) and F150W (monomer) mutants. Only the F150Y mutant exhibited dissociation into monomers in an ionic strength-dependent manner. These results indicated that spatial positions and interactions between F150-aromatic side chains were crucial to homotrimer stabilization. This finding was supported by QM calculations. In a functional respect, differences in the reaction property in the ground and photoexcited states were revealed. The analysis of photointermediates revealed a decrease in the accumulation of O, which is important for Cl(-) release, and the acceleration of the decay rate in L1 and L2, which are involved in Cl(-) transfer inside the molecule, in the trimer-dissociated mutants. Interestingly, the affinity of them to Cl(-) in the photoexcited state increased rather than the trimer, whereas that in the ground state was almost the same without relation to the oligomeric state. It was also observed that the efficient recovery of the photocycle to the ground state was inhibited in the mutants. In addition, a branched pathway that was not included in Cl(-) transportation was predicted. These results suggest that the trimer assembly may contribute to the regulation of the dynamics in the excited state of NpHR.

Figure 1

(A) Trimeric assembly of HsHR (PDB code; 1E12) and (B) NpHR (PDB code; 3A7K) viewed from the cytoplasmic side. The extended figures around the center of the trimer are shown below. Homologous three aromatic side chains of HsHR-F135 and NpHR-F150 are shown in red. The crystal structure of NpHR contains the secondary pigment bacterioruberin shown as an orange sphere. (C) Mapping the aromatic amino acids located at the protein interface on the x-ray crystal structure of HsHR. The molecular surface corresponding to the contact region was removed. The helices in this region are shown in brown. Four aromatic amino acids, Y39, W121, F150, and W179 of NpHR, were mutated into alanine. (D) Sequence alignment of HRs and other archaeal rhodopsins, BR, SRI, and SRII. Amino acid sequences were derived from DDBJ. The conserved aromatic residues are highlighted in gray.

Figure 2

(A and B) Visible CD spectra of NpHR-WT and mutants. Proteins were solubilized in 10 mM MOPS buffer (pH 7, 0.3 M NaCl, 0.1% DDM) containing 0.3 M Na₂SO₄. Spectra were recorded at 25°C. (A) Representative amino acids correspond to those shown in Fig. 1. (B) Phenylalanine 150 of NpHR was additionally mutated into other aromatic residues, Tyr and Trp. (C and D) Molecular size analysis of NpHR-WT and F150 mutants. (C) Size-exclusion chromatograms of NpHRs. The samples containing 20 μM protein in 10 mM MOPS buffer (pH 7, 0.3 M NaCl, 0.1% DDM) were applied to the column. The program was run at a flow rate of 0.4 mL/min, and the eluting proteins were detected by the absorption at 580 nm. (D) SDS-PAGE analysis on cross-linked NpHRs. Cross-link was induced by the addition of 1% GA (9). Molecular mass of monomeric NpHR is ∼32 kDa. Symbols − and + denote the absence and presence of GA; t, d, and m denote the trimer, dimer, and monomer, respectively.

Figure 3

Flash-induced transient absorption changes of NpHR-WT and F150 mutants at typical wavelengths. (A–D) The absorption changes in the presence of 1 M NaCl. Proteins were solubilized in 10 mM MOPS buffer (pH 7, 1 M NaCl, 0.1% DDM). All measurements were performed at 20°C. (E) Time constants in the photocycle in the presence of 1 M NaCl. The values were obtained from the global fitting analysis on the data shown in the spectra A–D.

Figure 4

(A–C) Multi-Gaussian fit of the P3 state of NpHR mutants. The absolute spectra of this state at various Cl⁻ concentrations (open circles) were fitted in the same way as previously reported (19). Chloride-dependent absorption changes in the photo-excited N and O observed in the P3 spectra of F150 mutants. The samples were titrated with NaCl from 0.04 to 4 M. The arrows indicated the changes in the accumulation of N (520 nm) and of O (600 nm). (D) Chloride-dependent changes in the absorbance at 520 nm, which represents the fraction of N, of F150 mutants shown in spectra A–C. (E and F) Chloride-dependent absorption changes in the photoexcited NpHR′ observed in P4 spectra of (E) NpHR-F150A and (F) F150W. The arrows indicate the spectral shift resulting from the addition of Cl⁻.

Figure 5

Electrostatic potential maps of QM/MM optimized NpHR model structures of (A) phenylalanines in WT, (B) alanines in F150A, (C) tryptophans in F150W, and (D) tyrosines in F150Y. The green and red areas represent positively and negatively charged potential energy, respectively. Computational details are described in the Supporting Material.

Figure 6

Possible schemes of the photocycle estimated from the data on NpHR-F150A and F150W. (I) Equilibrium among three components, (II) Branched pathway.