Comparison between Bacteriorhodopsin and Halorhodopsin. Halorhodopsin (HR) and Bacteriorhodopsin (BR) belong to a subfamily of

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1 Comparison between Bacteriorhodopsin and Halorhodopsin Halorhodopsin (HR) and Bacteriorhodopsin (BR) belong to a subfamily of heptahelical membrane proteins, the archaeal rhodopsins. They are found in the purple membrane, a part of the Halobacterium salinarum cell membrane, but BR is much more abundant than HR. Bacteriorrhodopsin is known as a light-driven proton pump (from cytoplasmic to extracellular side) while Halorhodopsin is a light-driven chloride pump (from extracellular to cytoplasmic side). They are called light-driven ion pumps because both of them have chromophores, retinal, bound covalently to their residues Lys as protonated Schiff bases. Absorption of light leads to the photoisomerization of retinal from all-trans to 13-cis configuration. Upon photoisomerization, the slower thermal reactions involving conformational changes of protein, ion transfer steps, and reisomerization lead to the net transportation of one ion per photon. Apparently, chloride transport through membrane proteins should impose different structural constraints on appropriate translocation pathway than proton transport (Kolbe, et al., 2000). Since proton transport is achieved by the internal chain of H-bonds through water molecules and protonable side chains where the structural changes are not necessary for the transport itself but only for energy conservation and ion transport unidirectionality (Kolbe, et al., 2000). On the contrary, chloride ions (Pauling radius = 1.81 Å) demand suitable sterically conditions and have high energetic penalties on desolvation (Kolbe, et al., 2000). However, the architecture of archaeal rhodopsins makes them compatible with both proton and chloride transports as investigated by single site mutants Asp to Thr or Ser in BR (Tittor et al., 1997) including the experiment in HR (Bamberg et al., 1993). To answer this confounding issue, the structures of BR and HR are examined.

2 BR and HR share 31% sequence identity. As shown by superposition of HR on BR in Fig.1, the transmembrane part of HR is structurally well conserved although BR and HR are different in ion specificities and transport directions (Kolbe, et al., 2000). Retinal is slightly different while the most obvious difference is found among helices A and B. CP D C BR B HR A C-terminus E F G EC N-terminus Fig.1 Structure comparison between HR (red, Protein Data Bank code 1E12) and BR (cyan, PDB code 1QHJ) showing retinals bound to helices G. EC is extracellular side and CP is cytoplasmic side. Picture created by VMD. The extracellular regions of BR and HR are dominated by BC loop which form two twisted antipararelled β-strands as shown in Fig.2. The BC loop of HR occupied by residues 83 to 105 is a little bit longer than of BR occupied by residues 63 to 79. Besides, there are kinks in helix E and in helix G near residues Lys linked to retinals due to the adopting of π bulge conformation (Luecke et al., 1999, Kolbe et al., 2000). Fig.2 Cartoon presentation of BR (cyan) and HR (red) showing helices and β-strands. BC loop of HR is more extended than of BR. A B C D E F G

3 As shown in Fig.3, HR and BR have hydrophobic regions favorable to the contact with lipid phase of membrane and expose hydrophilic regions mostly composed of polar residues at the cytoplasmic and extracellular sides where they have favorable interactions with solvent. Nevertheless, HR is titled by 11º compared with BR. As a result, the trimeric structure of HR containing intratrimer contacts between BC and CD helix pairs occlude 1057 Å 2 surface area from solvent access per monomer whereas BR trimers make a contact between each other only through B and D helices and occlude much smaller surface area of 659 Å 2 (Kolbe et al., 2000) BR CP HR Membrane 11º EC Figure 3 The surface of BR (left) and HR (right) colored as residue types; nonpolar (white), polar (green), basic (blue), acidic (red), and unassigned (cyan). BR HR Fig. 4 Charged residues of BR and HR. Positive charged residues (LYS ARG) are blue and Negative charged residues (ASP GLU) are red. Most of them are at cytoplasmic and extracellular sides outside membrane regions while some residues are occupied in protein interiors.

4 Despite the hydrophobic regions outside the membrane protein HR and BR, there are some charged residues at protein interiors as illustrated in Fig.4. Retinal binding pocket and the active site The retinal chromophores in ground state of both BR and HR are found in alltrans, 15-anti configuration. In BR, within 3.5 Å, the polyene chain and β-ionone ring are surrounded by the side chains of Tyr83, Asp85, Trp86, Thr89, Thr90, Leu93, Met118, Gly122, Trp138, Ser141, Thr142, Met145, Trp182, Try185, Pro186, Trp189, and Asp212. In HR, there are residues Trp112, Ser115, Thr116, Ile119, Met144, Gly148, Trp165, Ser168, Cys169, Phe172, Trp207, Tyr210, Pro211, Trp214, Asp238 surrounding polyene chain and β-ionone ring within 3.5 Å as shown in Fig.5. These residues surrounding chromophores are highly conserved among BR and HR. There are negative charged residues near protinated Schiff base and some polar side chains along the polyene chain and β-ionone ring. Many of these residues lead to the changes of absorption maxima or rate of thermal isomerization as investigated by site-specific mutagenesis in BR (Luecke et al., 1999) and the effect in Chloride binding in HR (Sato et al, 2003). BR HR Fig.5 Parts of residues side chains within 3.5 Å of chromophore, retinal linked to Lys in BR and HR. Retinals are colored as atom types, i.e., Carbon (Cyan), Hydrogen (white), and Nitrogen (blue). Side chains are colored as residue types, i.e., nonpolar (white), polar (green), basic (blue), acidic (red), and unassigned (cyan).

5 The active site for photoreaction of BR is stabilized by hydrogen-bonded network comprising the protonated Schiff base, three water molecules and side chains of Asp85, Arg82 and Asp212 as indicated in Fig.6. In contrast, a single chloride ion was found near protonated Schiff base in HR indicating the probable primary ion transport site (Kolbe et al., 2000). The chloride ion occupies almost the same position of OD1 atom of Asp85 in BR. With three water molecules and residues Arg108 and Asp202, chloride ion forms hydrogen-bonded network in HR similar to which in BR. In the other way, the complex counterion of protonated Schiff base comprising chloride ion, Arg108, and Asp238 in HR replaces the group of Asp85, Arg82, and Asp212 in BR. (Kolbe et al., 2000). Thr89 Ser115 Asp85 OD1 PSB Trp112 Cl - PSB Asp212 Asp238 Arg82 BR Arg108 HR Fig.6 Complex couterions of protonated Schiff base (PSB) in BR (left panel) and HR (right panel). Water molecules are illustrated as red spheres. Purple Sphere is Chloride ion in HR. PSB and Side chains are colored as atom types. Putative hydrogen-bonds including the bond lengths are shown in orange. Note that HR has more putative hydrogen-bonds. In BR, the anionic form of Asp85 is stabilized by further hydrogen-bonding with Thr89, while there are two additional hydrogen-bonds with Thr57 and Thr185 in the case of Asp212. After the all-trans to 13-cis photoisomeriztion of retinal, the disruption of complex counterion of protonated Schiff base will destabilizes the active site leading to the dissociation of the protonated Schiff base (Luecke et al., 1999).

6 Since there are more hydrogen-bonds to Asp212 than to Asp85, it can be inferred why Asp85 is the proton acceptor in the first event in proton transport (Luecke et al., 1999). The water molecule nearest to the protonated Schiff base is centered between three formally charged moieties as shown in Fig.6 for BR. This water molecule may participate in the early step of the protonation/deprotonation of proton transport as it is in the position where its dissociation to H + and OH - could directly involved in this event (Luecke et al., 1999). In contrast, there is no deprotonation step for the chloride transport in HR due to the missing of proton receptor (Asp85 in BR). However, the similarity in the structures of HR and BR in ground state indicated several evidences that support a model of mechanistic equivalence for halide and proton transport (Kolbe et al., 2000). Initially, the ion to be transported is covalently linked (BR) or ion paired (HR) to the protonated Schiff base. Second, when considering the ground state of archaeal rhodopsins, the cytoplasmic pathway is closed for ion condution (Kolbe et al., 2000). Third, the charge distribution of the complex counterion of protonated Schiff base in BR and HR are almost the same as chloride replaces the OD1 atom of Asp85 in BR. And lastly, the single site mutagenesis of BR, especially Asp85 to neutral residues such as Thr or Ser, makes BR capable of inward chloride transport like HR (Sasaki et al., 1995). The structure of K-like intermediate of BR indicates the flipping of N-H dipole moment of the Schiff base relative to Asp85 upon photoisomerization without major alteration of protein environment (Edman et al., 1999). If consider in HR, this flipping of N-H dipole should shift the unfavorable energy to the bound chloride due to the increased repulsion between the N-H dipole and the carboxylate of Asp238 (Kolbe et al., 2000). From the FTIR spectroscopy, the next step of interaction is found to be the loss of Arg108-chloride interaction and the stronger PSB-chloride binding

7 (Kolbe et al., 2000). It can be inferred in structural term that there is a chloride passage along the N-H dipole of protonated Schiff base toward the cytoplasmic side of membrane (Kolbe et al., 2000). The single Serine, Ser115, which is initially hydrogen-bound to chloride, might be involved in remaining chloride solvated; its hydroxyl is properly positioned right above the protonated Schiff base to keep a polar interaction with chloride mostly throughout the chloride passage (Kolbe et al., 2000). In addition, Arg108 plays a significant role in chloride-pumping as the replacement of Arg108 by neutral residue decreases the activity of chloride transport (Váró, 2000). The schematic mechanisms of chloride and proton transports are illustrated in Fig.7. Following the flipping of N-H dipole triggered by photoisomerization, there are three scenarios than can develop (Kolbe et al., 2000). In HR, the chloride ion interaction with N-H dipole elicits electrostatical dragging of itself along protonated Schiff base to the releasing toward the cytoplasm and followed with the entering of the new chloride at the transport site (Kolbe et al., 2000). In BR, for the same energetic basis that chloride is translocated in HR, the fixed negative charge of Asp85 attracts the proton from the protonated Schiff base; the deprotonation of PSB. Then the Schiff base obtains a new proton from the cytoplasm, and Asp85 eventually discharges its proton toward the extracellular side (Kolbe et al., 2000). If Asp85 is replaced by neutral residue, the HR mechanism comes into action along with the presence of chloride for BR (Kolbe et al., 2000). The last scenario occurs in the absences of both chloride ion and fixed negative charge of Asp85. After dipole flipping, the protonated Schiff base in its excited state has only choice to evolve thermodynamically relaxing by releasing its proton (Kolbe et al., 2000). As a consequence, a new proton is picked up from the extracellular region through several

8 intermidiatary steps and proton translocation is detected eventually with inversed direction (Kolbe et al., 2000). Fig.7 (Kolebe et al., 2000) (A) Ion translocation in HR. Strutural features that probably undergo conformational changes during photocycle are highlighted (π bulges Ala178-Trp183 and Phe240- Phe245, blue; COOH-terminus of helix E, red; Arg108, Thr203 and PSB, sticks). (B) Photocycle of HR. The six steps necessary for vectorial catalysis are two isomerization reactions of the chromophore (I and I*), two chloride transfer steps (T), and two changes of ion accessibility at the active site. The additional passage of chloride for formation of HR520 is indicated. (C) An ion-dragging mechanism for light-driven ion pumping in archaeal rhodopsins that is controlled by ion-dipole interactions. In the absence of chloride, the two-photon mode for proton transport operates in HR and nonprotonable Asp85 mutants of BR (X is Ser or Thr). As illustrated in Fig.8, the protonated Schiff bases are connected to Arg82 and Arg108 in BR and HR respectively and to the extracellular side by and extensive three-dimentional network of hydrogen-bonds. In BR, this hydrogen-bonded network

9 is purposed as the pathway for proton transfer toward extracellular surface. Whereas in HR, it was suggested that the extracellular region is favorable for anion binding as it comprises of arginine cluster (Arg52, Arg55, Arg58, Arg60) (Kolbe et al., 2000). BR HR Asp85 Cl - Arg82 Arg108 Glu194 Glu219 Fig.8 Three-dimensional hydrogen-bonding network from protonated Schiff base (atom types colored) to extracellular side in BR and HR. The key residues are colored as residue types, i.e., nonpolar (white), polar (green), basic (blue), acidic (red), and unassigned (cyan). Putative hydrogen-bonds including the bond lengths are shown in orange. Note that HR has more putative hydrogen-bonds. In summary, the structure of HR and BR represent a wonderful evolution organized by nature. As the only replacement of the negatively charged residue (Asp85), acting as a proton receptor of BR, by chloride ion in HR leads to the alternation of ion specificities from cation to anion. The high-resolution structures of both BR and HR provide the key answers to the understanding of the similarities and differences including the interconvertable ion transports in BR and HR. Nevertheless, the ion transports are achieved through several intermediately steps, therefore, the further study of these intermediate structures would be very valuable to understand the light-driven ion transports completely.

10 References -Kolbe, M., Besir, H., Essen, L.O. and Oesterhelt, D. (2000). Structure of the lightdriven chloride pump Halorhodopsin at 1.8 Å resolution. Science, 288, Luecke, H., Schobert, B., Richter H.T., Cartailler, J.P. and Lanyi, J. K. (1999). Structure of Bacteriorhodopsin at 1.55 Å resolution. Journal of Molecular Biology, 291, Váró, G. (2000). Analogies between Halorhodopsin and Bacteriorhodopsin. Biochimica et Biophysica Acta, 1460, Sato, M., Kikukawa, T., Araiso, T., Okita, H., Shimono, K., Kamo, N., Demura, M. and Nitta, K. (2003). Ser-130 of Natrobacterium pharaonis halorhodopsin is important for chloride binding. Biophysical Chemistry, In press. -Bamberg, E., Tittor, J. and Oesterhelt, D. (1993). Light-Driven Proton or Chloride Pumping by Halorhodopsin. Proc. Natl. Acad. Sci. U.S.A., 90, Titter, J., Haupts, U., Oesterhelt, D., Becker, A. and Bamberg, E. (1997). Chloride and Proton Transport in Bacteriorhodopsin Mutant D85T: Different Modes of Ion Translocation in a Retinal Protein, J. Mol. Biol, 271, Edman, K., Nollert, P., Royant, A., Belrhali, H., Pebay-Peyroula, E., Hajdu, J., Neutze, R., and M. Landau, E.M. (1999). High-resolution X-ray structure of an early intermediate in the bacteriorhodopsin photocycle. Nature, 401,