Structural and Functional Studies of a Newly Grouped Haloquadratum walsbyi Bacteriorhodopsin Reveal the Acid-resistant Light-driven Proton Pumping Activity

Background: Most bacteriorhodopsins demonstrate red-shifted spectrum in acidic condition. Results: Structures of Haloquadratum walsbyi bacteriorhodopsin explain stable action spectra from pH 2 to 8. Conclusion: The extracellular hydrogen-bonding network assists in the maintenance of protonation status in the Haloquadratum walsbyi bacteriorhodopsin retinal-binding pocket. Significance: A bacteriorhodopsin subfamily has a stable optical property, and its structure is useful for protein engineering in optogenetic tools.

Retinal bound light-driven proton pumps are widespread in eukaryotic and prokaryotic organisms. Among these pumps, bacteriorhodopsin (BR) proteins cooperate with ATP synthase to convert captured solar energy into a biologically consumable form, ATP. In an acidic environment or when pumped-out protons accumulate in the extracellular region, the maximum absorbance of BR proteins shifts markedly to the longer wavelengths. These conditions affect the light-driven proton pumping functional exertion as well. In this study, wild-type crystal structure of a BR with optical stability under wide pH range from a square halophilic archaeon, Haloquadratum walsbyi (HwBR), was solved in two crystal forms. One crystal form, refined to 1.85 Å resolution, contains a trimer in the asymmetric unit, whereas another contains an antiparallel dimer was refined at 2.58 Å. HwBR could not be classified into any existing subgroup of archaeal BR proteins based on the protein sequence phylogenetic tree, and it showed unique absorption spectral stability when exposed to low pH values. All structures showed a unique hydrogen-bonding network between Arg 82 and Thr 201 , linking the BC and FG loops to shield the retinal-binding pocket in the interior from the extracellular environment. This result was supported by R82E mutation that attenuated the optical stability. The negatively charged cytoplasmic side and the Arg 82 -Thr 201 hydrogen bond may play an important role in the proton translocation trend in HwBR under acidic conditions. Our findings have unveiled a strategy adopted by BR proteins to solidify their defenses against unfavorable environments and maintain their optical properties associated with proton pumping.
The light-driven proton pumps feature a seven-transmembrane ␣-helical region with a lysine-bound retinal that serves as a chromophore responsive to light. These BR proteins respond to ϳ550 nm light and exert outward proton pumping, resulting in a proton gradient in the extracellular region (10 -13). These proteins consequently facilitate the inflow of protons back into the cell through ATP synthase to generate ATP (14). The first and most well studied BR from Halobacterium salinarum, HsBR (10), was shown to be optically and functionally durable under heat and high salinity conditions (15), making it one of the most stable membrane proteins. However, BR proteins have a well established and significant property, a red-shifted activity spectrum at acidic pH, wherein a maximum red-shift of ϳ55 nm in max for HsBR is from protonation of an aspartate residue at the retinal Schiff base (Asp 85 in HsBR) (16 -18). At acidic pH, the lack of proton transport is due to the protonated aspartate, which should be the proton acceptor for the Schiff base during the photocycle; in the absence of an acceptor, the proton transfer cannot take place, and a critical step in the transport does not occur. Those results are expected because releasing a proton from a protein into an environment of low pH is not chemically favored, and a redshifted action spectrum is a conventional indicator for the protonated aspartate in the Schiff base binding pocket.
Most BR proteins are not functional under acidic conditions. After we reported a BR from Haloarcula marismortui, HmBRII, BR proteins started to surface in the past few years. HmBRII showed high optical stability in acidic conditions even down to pH of 1.6 and maintained its light-driven proton pumping activity at pH of 4.0 (19). This observation was extended when we identified another BR from Haloquadratum walsbyi, HwBR, which showed optical durability in acidic conditions. 6 After the structures of HsBR were solved by electron microscopy in 1996 (20) and by x-ray in 1997 (21), more than 70 HsBR structures of different length proteins, intermediates, mutants, and binding statuses have been reported, and the molecular mechanism of light-driven proton transportation was described in detail (11,13,22). Furthermore, six BR-like crystal structures were determined in the last decade, including bacteriorhodopsin (bR) from H. salinarum (21,23,24), archaerhodopsin-1 and -2 (aR-1 and aR-2) from Halorubrum sp. aus-1 and -2 (25, 26), deltarhodopsin-3 (dR3) from Haloterrigena thermotolerans (27), and cruxrhodopsin-3 (cR-3) from Haloarcula vallismortis (28). Moreover, structural information also became available for seven light-driven proton translocators identified from bacteria and eukaryota, xanthorhodopsin (XR) from S. ruber (29), Acetabularia rhodopsin (ARII) from the marine plant Acetabularia acetabulum (30), channelrhodopsin (ChR) chimera from Chlamydomonas reinhardtii (31), and both blue and green proteorhodopsin (BPR and GPR) (32)(33)(34). However, none of them has a relatively consistent activity spectrum in broad pH conditions.
Here, we report the atomic structure, sequence analysis, and photochemical properties of a BR protein, HwBR, and we propose that HwBR belongs to a new subfamily of BRs that we have named qR. The crystal structures of HwBR revealed that a unique arginine residue stabilizes the extracellular loop region by forming hydrogen bonds with a threonine residue located in the membrane edge of extracellular region. The importance of this local structure, which shields the interior environment of HwBR from the low pH extracellular area, was further validated by the mutagenesis approach.

Experimental Procedures
Phylogenomic Analysis-Thirteen BR-like amino acid sequences were used for phylogenomic analysis 7 . The unweighted pair group method with arithmetic mean (UPGMA) algorithm was employed in this work, with the Kimura protein distance measure from CLC sequence Viewer 6.9. The bootstrap is based on 100 replicates. Bacterial Strains and Expression of BR from H. walsbyi-Routine DNA manipulations were carried out according to standard molecular cloning procedures. The HwBR gene was cloned into a pUC57 vector using directed synthesis. The final sequence was CC 1 ATGgCTXXXX 753 GACCTCGAG (underlining indicates the restriction sites for NcoI and XhoI, respectively, and g indicates a modified base.) The DNA fragments were obtained by NcoI and XhoI and were then inserted into the NcoI and XhoI sites of the pET-21d vector (Novagen). Consequently, a plasmid encoding hexahistidines at the C terminus was constructed. HwBR gene mutants were generated using the QuikChange site-directed mutagenesis method (Stratagene). The constructed plasmids were confirmed to have the expected nucleotide sequence using an automated sequencer.
Protein Expression in Escherichia coli and Purification-HwBR protein with hexahistidines at the C terminus was expressed in E. coli C43(DE3). The protein was purified using nickel-nitrilotriacetic acid resin chromatography (GE Healthcare) as described previously (35) and was solubilized in 0.05% n-dodecyl-␤-D-maltoside (DDM).
UV-visible Spectroscopy-The purified sample was concentrated and exchanged to a buffer containing 4 M NaCl, 50 mM Tris-HCl, and 0.05% DDM using an Amicon apparatus (Millipore). UV-visible spectra were recorded using a U-1900 spectrophotometer (Hitachi). pH-dependent spectra and a titration curve were conducted as described in previous research (36). The temperature was maintained at 298 K.
Photocurrent Measurement-The electrochemical cell was designed by Chu et al. (37) and modified in our previous work (19). Photocurrent measurements of purified proteins were carried out by using a modulated continuous wave (CW) 532-nm green laser as the excitation light source and controlled by a data acquisition card. Light-driven Proton Transport Activity-Light-driven proton transport activity was measured by monitoring light-induced pH changes using a glass electrode in real time. E. coli cells expressing the target rhodopsin were harvested by centrifugation (4,800 ϫ g for 10 min). They were then washed three times and resuspended in measurement buffer (10 mM NaCl, 10 mM The key residues are annotated with different symbols. Circle, retinal-binding pocket; diamond, proton reuptake residue; square, proton releasing group. The key residue, Arg 82 (HwBR), in this study is marked by the red box. The secondary-structural information of HwBR is shown above the alignment. B, phylogenomics analysis of the amino acid sequences of the light-driven proton pumps in halobacteria. The analysis classified HwBR from a quadrate-shaped bacteria into a new separate superfamily, qR. MgSO 4 , and 100 M CaCl 2 ). The concentration of the cell suspension was adjusted to obtain an A 600 ϳ2.0; the suspension was maintained in the dark and then illuminated with a green CW laser at 1 watt (532 nm). A parallel experiment with 10 M carbonyl cyanide m-chlorophenyl hydrazone (CCCP) was conducted to confirm the proton-specificity of the assay.
Protein Preparation for Crystallography-To screen for the optimal HwBR crystallization conditions, purified HwBR was analyzed for monodispersity using a size exclusion column, and its absorption was monitored at 280 and 552 nm. The protein was loaded onto a size exclusion column (Superdex 200 10/30 GL; GE Health Sciences) using Buffer A (50 mM CH 3 COONa, pH 4.5, 200 mM NaCl) and Buffer B (20 mM Tris, pH 7.0, 150 mM NaCl) as the elution buffer in the presence of 0.05-0.15% DDM, n-octyl-␤-D-glucopyranoside (OG), or n-decyl-␤-maltoside (DM). After dialysis in buffer with 0.15% DM, the elution pattern showed a monodispersed peak as well. In our detergent screening experiment, the HwBR protein showed monodis-perse peaks in both buffers in 0.15% DM. Therefore, we used HwBR protein in Buffer A with 0.15% DM as the sample for crystallization.
Crystallization and X-ray Diffraction Data Collection-The purified HwBR protein was concentrated to ϳ17 mg/ml, as estimated by ultraviolet absorbance, and it was mixed with 1-oleoyl-rac-glycerol (monoolein; Sigma-Aldrich) at a 2:3 (w/w) protein-to-lipid ratio using the twin-syringe mixing method. The volume of each drop was 0.2 l of protein-lipid mixture plus 1 l. HwBR crystals of the trimeric form were grown in 0.05 M sodium citrate, pH 5.5, 0.05 M NaCl, and 15% (v/v) PEG 400, and antiparallel dimeric crystals were grown in 0.1 M ammonium sulfate, 0.1 M sodium chloride, 0.01 M sodium acetate, pH 4.0, and 16.5% (v/v) PEG 200. The size of the crystals reached about 50 ϫ 50 ϫ 5 m within 2-30 days at 20°C. The HwBR trimeric form crystals were soaked in 30% (v/v) glycerol as a cryoprotectant before harvest.
X-ray diffraction data were collected at BL15A1 of the National Synchrotron Radiation Research Center (NSRRC), Hsinchu, Taiwan and at 44XU of SPring-8, Sayo, Japan. The data were processed using HKL2000 (53). We obtained the phases by molecular replacement using archaerhodopsin-2 as a template (1VGO) (25). The PHENIX (38), refmac5 (39), and COOT (40) programs were used for molecular replacement, structural refinement, and structural viewing, respectively. All structure figures were prepared in PyMOL (Schrödinger, LLC).

Results
Sequence Analysis of BR-like Proteins-A previous study (41) compared the BR protein sequences of several species living in different environments, including salterns (42), spring areas (43), and others (44 -46), and reported BR subgroups designated aR to dR (47). Multiple alignment of BR amino acid sequences (Fig. 1A) shows that the BRs share 50 -80% identity. However, the amino acid sequence of HwBR from a quadrateshaped bacterium, H. walsbyi, constitutes a novel group with HmBRII according to the phylogenetic tree analysis (Fig. 1B). We named this unidentified and distinct superfamily qR.
Purification of Monodispersed HwBR Proteins-The HwBR gene was constructed with a C-terminal hexahistidine tag and expressed in E. coli C43 (DE3) as described previously (35,48). The purified HwBR protein in buffer with detergent had a max at 552 nm, which is almost consistent with the absorbance value of HsBR monomer, and as a reference, the absorption peak of HsBR trimer, known as purple membrane, showed a peak value at 568 nm, which was red-shifted 13 nm when compared with monomer BR (Fig. 2A) (49). Three commonly used detergents, DDM, DM, and OG, in different buffer conditions under pH 4.5 and 7.0, were tested for monodispersity properties of HwBR. The HwBR protein showed two very close peaks in both buffers with 0.02% DDM. In buffers with 0.2% OG, the HwBR protein formed broad peaks. In our detergent screening experiment, HwBR protein showed monodispersed peaks in both buffers with 0.15% DM. After the protein in OG was dialyzed against buffer A with 0.15% DM and loaded onto the column, the elution pattern was restored from the broad peaks to a monodispersed peak in DM (Fig. 3). Therefore, we selected HwBR pro- tein in Buffer A containing 0.15% DM as the sample for crystallization, which yielded purple crystals.
Overall Structures and Proton Translocation Path of HwBR-Previously, we crystallized the protein using the vapor diffusion method, and the crystals diffracted to around 7 Å (48). In this work, HwBR proteins were crystallized using the in meso method (50), and the proteins packed into parallel trimeric and antiparallel dimeric crystals diffracted to 1.85 and 2.58 Å, respectively ( Table 1). The structures of the monomers were almost identical and consisted of seven transmembrane ␣-helices, two ␤-strands in the BC loops, and a prosthetic group alltrans retinal bound to Lys 224 via a Schiff base (Fig. 2B). The values of root mean square deviation (r.m.s.d.) between monomers of the antiparallel dimer and of the trimer are ϳ0.36 Å.   The trimeric structure showed that lipids (1-monooleoyl-racglycerol, MPG) surrounded each monomer to induce the formation of a self-assembled trimeric structure (Fig. 2C). In previous studies, it was shown that lipids control the trimeric structure, conformational flexibility, and photocycle activity of BR (51).
We used 30% glycerol as the cryoprotectant when harvesting the crystals. The presence of glycerol improved the resolution from 4 to 1.85 Å. In the structure, one glycerol molecule was found to be bound with chain B (Fig. 2C). The antiparallel dimeric structure had one MPG and two acetate molecules bound around the dimeric protein. A top view of the dimer shows that helices A and B from both monomers form a fourhelix bundle for antiparallel dimer formation (Fig. 2D). In the overall structures, the BC and FG loops (Fig. 2E) have some variations that might control the differences between the superfamilies of BR-like proteins (r.m.s.d. ϳ0.3-0.4 Å).
The proton translocation path could be divided into three areas (Fig. 4). On the cytoplasmic side, Asp 104 (Fig. 4C) is the proton uptake accelerator, as seen for all other BR proteins, known as Asp 96 in HsBR. The D104N/HwBR mutant constructed in this work showed retarded proton uptake during the light-driven proton pumping cycle when compared with the wild-type HwBR (Fig. 5). In the photocycle process, once the retinal binding site was fully protonated, a proton was translocated from Asp 93 to the proton releasing group, which is composed of Arg 90 , Glu 202 , and Glu 212 , through the hydrogen bond rearrangement matching those observed in other BRs (Fig. 4B) (11,18). In our 1.85 Å resolution structure, all structural waters for the proton translocation path are conserved in HwBR when  compared with the 1.55 Å HsBR (1C3W). The proton outward cap (BC loop) at the extracellular site composed of a ␤ hairpin motif was partially sealed by a pair of amide-carbonyl hydrogen bonds formed between Arg 82 and Thr 201 (Fig. 4A).
In both structures of HwBR solved in this study, two guanidinium nitrogen atoms of Arg 82 located at the BC loop formed hydrogen bonds with the carboxyl group of the main chain of Thr 201 in the FG loop in the extracellular region. This ␤ hairpin of the BC loop forms a cap covering the proton translocation channel exit site (Fig. 4A). These hydrogen bond connections at this position have never been observed in any other known BR protein (Fig. 1A). In our HwBR structures from different crystal packing forms, Arg 82 was located at the center of the ␤ hairpin in both structures, forming hydrogen bond connections with the main chain atoms of Thr 201 at the C terminus of helix F.
To further investigate the structural residue corresponding to Arg 82 in other BR proteins, all resolved BR structures were aligned (Fig. 4E). Alignment of the BC loop region in HwBR with other structures of aR-1, aR-2, bR, cR-3, and dR3 (23,(25)(26)(27) showed that the residues corresponding to Arg 82 are glutamic acid in all other BR proteins, except in the aR-2, where threonine is the corresponding residue, and no hydrogen bond formation in the corresponding position was observed (Fig. 6). However, there is a hydrogen bond network formed between Arg 74 -Thr 197 in cR-3 structure. One of the hydrogen bonds in cR-3 linked to the hydrogen atom of the ⑀ nitrogen, but two hydrogen bonds in HwBR were formed on the hydrogen atom on nitrogen. The nitrogen is sensitive to the micro-chemical environment, and a flexible cap could be formed during proton translocation process.
HwBR Is an Optically Stable BR under Wide pH Range and Mutagenesis of Arg 82 Impairs Optical Stability of HwBR-An important feature of rhodopsin is the pH dependence of the maximum absorbance, max , or activation spectrum, which reflects the micro-environment of the retinal protonation state. Here, we found that HwBR has high optical durability under acidic conditions. E. coli-expressed HsBR and HwBR were preequilibrated with buffered solution at a pH of 2.0 (Fig. 7A, red) or 8.0 (Fig. 7A, blue) for spectral scanning over 250 -750 nm. A mere ϳ9-nm red-shift in max was recorded for HwBR (Fig. 7A), significantly less than the ϳ55-nm red-shift observed in HsBR (19) under the same conditions. The much smaller red-shift at a pH of 2.0 represents an unusual level of optical stability that has not been observed in BR proteins other than HmBRII (19). To further obtain spectra of the fully protonated counterion states for HwBR, D93N/HwBR, the D85N/HsBR-corresponding mutant proteins, were prepared for comparison (Fig. 7A,  brown). D93N/HwBR showed a red-shifted spectrum with peak at 581 nm similar to that of D85N/HsBR, being red-shifted 20 nm farther than wild-type HwBR in acidic conditions. The result hinted that the micro-environment of the fully protonated retinal-binding pocket was similar between HwBR and HsBR.
To directly corroborate the importance of the Arg 82 -Thr 201 hydrogen-bonding network in the BC loop of HwBR, a pair of mutants (R82E/HwBR and R82E/D93N/HwBR) was constructed to examine their optical stability under different pH conditions when the hydrogen-bonding network would be disrupted. A 21-nm red-shift was observed for R82E/HwBR, with a max value of 568 nm under a pH of 2.0 and a value of 547 nm under a pH of 8.0 (Fig. 7B). The fully protonated counterion state of R82E/HwBR was represented by R82E/D93N/HwBR, and its value remained at 581 nm (Fig. 7B, magenta). To evaluate the spectral shift upon protonation of the Schiff base proton acceptor, titration curves for both HwBR and R82E/HwBR were determined via pH-dependent spectra. HwBR showed a single titration curve with a pK a of 1.97 (Fig. 7C, solid circle), which is lower than HsBR and similar to HmBRII (19). Replacement of the arginine with glutamate caused the pK a to increase to 2.24 ( Fig. 7C, open circle). This result supports our hypothesis that the Arg 82 -Thr 201 hydrogen-bonding network might have some effect on the retinal Schiff base counterion.
A light-driven proton pump activity assay was also conducted on R82E/HwBR to confirm that the mutant does not interfere with the light-driven proton pumping activity. E. coli cells transformed with rhodopsins of interest were measured for their light-driven pH change property. The proton pump activity was directly monitored by a pH electrode during light and dark periods. Both wild type and R82E/HwBR showed a pH decrease upon illumination, which was eliminated by the protonophore CCCP, an inhibitor of proton motive force. This result indicated that R82E/HwBR still retains its overall proton translocation ability (Fig. 8).
R82E Mutation Slightly Changes the pH-dependent Thermal Stability of HwBR-The diffusion according to the extracellular proton concentration influences the protonation state of the Schiff base in the ground state. Arg 82 in HwBR locates on the BC loop and forms a cap above the proton translocation path so that the cap may shield the retinal-binding pocket from outside environment. To investigate this hypothesis, a time-dependent denaturation assay was conducted with slight modifications (43), and the experiments were monitored via spectroscopy at the corresponding max of wild type and R82E/HwBR under pH 4 and pH 8 at 75°C. Both wild type and R82E/HwBR showed a similar time-dependent decrease at pH 4 in 30 min, but R82E/ HwBR exhibited a faster denaturation pattern than wild-type HwBR at pH 8 within 15 min (Fig. 9). The results also suggested that the cap together with residue Arg 82 had a slight effect on the protonation state in the retinal binding pocket.

Discussion
H. walsbyi, a square halophilic archaeon, was first discovered by A. E. Walsby in 1980 (54). Because this unique square morphology halobacterium is abundant in salt lakes around the world, it plays an important role in ecology. Based on the phylogenetic tree from BR protein sequences, new subgroup qR  was firstly named as part of HwBR and HmBRII, which were studied by our research team (Fig. 1B) (35). Although the overall structure of HwBR is similar to most solved BR structures, a special hydrogen-bonding network located at the extracellular region of the proton pumping path was first found.
The significance of the Arg 82 -Thr 201 hydrogen-bonding network within the overall protein surface was summarized by an electrostatic state analysis between HwBR and other BR proteins (Fig. 10A). The key Arg 82 -Thr 201 hydrogen-bonding net-work sits in the center from the top view. The location of the Arg 82 -Thr 201 hydrogen-bonding network might shield the retinal-binding pocket from the outside proton equilibrium and protect the protonation condition of both the interior channel and the retinal-binding pocket. In other words, this hydrogen network might prevent the proton acceptor of the Schiff base from outside influences in the ground/resting state, thereby leading to the pH-independent activity spectrum. Worth et al. (52) reported that polar and certain charged side chains form hydrogen bonds to main chain atoms in the core of proteins, which is conserved in evolution. For instance, arginine exhibited the highest propensity to form capping interactions that are both conserved and buried at the C termini of ␣-helices.
When compared with the extracellular side, the cytoplasmic side of HwBR shows a negatively charged region (Fig. 10A, bottom view, area colored in red) with a significantly enlarged surface area among all BRs. Driving the re-uptake of proton from the cytoplasm by the negatively charged region could potentially increase the proton uptake efficiency. Taken together, HwBR has adopted a straightforward approach to achieve a negatively charged region with an enlarged surface area on the cytoplasmic side and a minimized region regulated by the Arg 82 -Thr 201 hydrogen-bonding network on the extracellular side protecting the retinal-binding pocket micro-environment from the extracellular proton concentration direct influence (Fig. 10, B and C). Together with these two properties, HwBR seems like a highly efficient machine for proton pumping in acidic condition.
In summary, we characterized the overall important structural and photochemical features in HwBR and in comparison with other known BRs. This study demonstrated how the Arg 82 -Thr 201 hydrogen-bonding network cap gives HwBR a stable optical property in a wide pH range. The stable optical property might lead to broaden functional pH range of lightdriven proton pump activity. This protein property might play an important role in the abundance of H. walsbyi cells in salt lakes around the world.