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(HR) and Bacteriorhodopsin (BR) Belong to a Subfamily of Heptahelical Membrane Proteins, the Archaeal Rhodopsins

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(HR) and Bacteriorhodopsin (BR) Belong to a Subfamily of Heptahelical Membrane Proteins, the Archaeal Rhodopsins Chutintorn Punwong Bioph 490M 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. Chutintorn Punwong Bioph 490M 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. BR C-terminus CP HR A G B F C E D 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). A B C D E F G 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. Chutintorn Punwong Bioph 490M 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 Å2 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 Å2 (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. Chutintorn Punwong Bioph 490M 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 all- trans, 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). Chutintorn Punwong Bioph 490M 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 Cl- OD1 Trp112 PSB PSB Asp85 Asp212 Asp238 Arg82 Arg108 BR 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). Chutintorn Punwong Bioph 490M 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 Chutintorn Punwong Bioph 490M (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).
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