Crystal Structure of Escherichia coli-Expressed Haloarcula marismortui Bacteriorhodopsin I in the Trimeric Form

Bacteriorhodopsins are a large family of seven-helical transmembrane proteins that function as light-driven proton pumps. Here, we present the crystal structure of a new member of the family, Haloarcula marismortui bacteriorhodopsin I (HmBRI) D94N mutant, at the resolution of 2.5 Å. While the HmBRI retinal-binding pocket and proton donor site are similar to those of other archaeal proton pumps, its proton release region is extended and contains additional water molecules. The protein''s fold is reinforced by three novel inter-helical hydrogen bonds, two of which result from double substitutions relative to Halobacterium salinarum bacteriorhodopsin and other similar proteins. Despite the expression in Escherichia coli and consequent absence of native lipids, the protein assembles as a trimer in crystals. The unique extended loop between the helices D and E of HmBRI makes contacts with the adjacent protomer and appears to stabilize the interface. Many lipidic hydrophobic tail groups are discernible in the membrane region, and their positions are similar to those of archaeal isoprenoid lipids in the crystals of other proton pumps, isolated from native or native-like sources. All these features might explain the HmBRI properties and establish the protein as a novel model for the microbial rhodopsin proton pumping studies.     

Role of extracellular glutamic acids in the stability and energy landscape of bacteriorhodopsin

Bacteriorhodopsin (BR), a specialized nanomachine, converts light energy into a proton gradient to power Halobacterium salinarum. In this work, we analyze the mechanical stability of a BR triple mutant in which three key extracellular residues, Glu⁹, Glu¹⁹⁴, and Glu²⁰⁴, were mutated simultaneously to Gln. These three Glu residues are involved in a network of hydrogen bonds, in cation binding, and form part of the proton release pathway of BR. Changes in these features and the robust photocycle dynamics of wild-type (WT) BR are apparent when the three extracellular Glu residues are mutated to Gln. It is speculated that such functional changes of proteins go hand in hand with changes in their mechanical properties. Here, we apply single-molecule dynamic force spectroscopy to investigate how the Glu to Gln mutations change interactions, reaction pathways, and the energy barriers of the structural regions of WT BR. The altered heights and positions of individual energy barriers unravel the changes in the mechanical and the unfolding kinetic properties of the secondary structures of WT BR. These changes in the mechanical unfolding energy landscape cause the proton pump to choose unfolding pathways differently. We suggest that, in a similar manner, the changed mechanical properties of mutated BR alter the functional energy landscape favoring different reaction pathways in the light-induced proton pumping mechanism.     

A transporter converted into a sensor, a phototaxis signaling mutant of bacteriorhodopsin at 3.0 Å

Bacteriorhodopsin (BR) and sensory rhodopsin II (SRII), homologous photoactive proteins in haloarchaea, have different molecular functions. BR is a light-driven proton pump, whereas SRII is a phototaxis receptor that transmits a light-induced conformational change to its transducer HtrII. Despite these distinctly different functions, a single residue substitution, Ala215 to Thr215 in the BR retinal-binding pocket, enables its photochemical reactions to transmit signals to HtrII and mediate phototaxis. We pursued a crystal structure of the signaling BR mutant (BR_A215T) to determine the structural changes caused by the A215T mutation and to assess what new photochemistry is likely to be introduced into the BR photoactive site. We crystallized BR_A215T from bicelles and solved its structure to 3.0 Å resolution to enable an atomic-level comparison. The analysis was complemented by molecular dynamics simulation of BR mutated in silico. Three main conclusions regarding the roles of photoactive site residues in signaling emerge from the comparison of BR_A215T, BR, and SRII structures: (i) the Thr215 residue in signaling BR is positioned nearly identically with respect to the retinal chromophore as in SRII, consistent with its role in producing a steric conflict with the retinal C₁₄ group during photoisomerization, proposed earlier to be essential for SRII signaling from vibrational spectroscopy and motility measurements; (ii) Tyr174–Thr204 hydrogen bonding, critical in SRII signaling and mimicked in signaling BR, is likely auxiliary, for example, to maintain Thr204 in the proper position for the steric trigger to occur; and (iii) the primary role of Arg72 in SRII is spectral tuning and not signaling.     

The functions of tryptophan residues in membrane proteins.

Membrane proteins have a significantly higher Trp content than do soluble proteins. This is especially true for the M and L subunits of the photosynthetic reaction center from purple bacteria. The Trp residues are not uniformly distributed through the membrane but are concentrated at the periplasmic side of the complex. In addition, Trp residues are not randomly aligned. Within the protein subunits, many form hydrogen bonds with carbonyl oxygens of the main chain, thereby stabilizing the protein. On the surface of the molecule, they are correctly positioned to form hydrogen bonds with the lipid head groups while their hydrophobic rings are immersed in the lipid part of the bilayer. These observations suggest that Trp residues are involved in the translocation of protein through the membrane and that following translocation, Trp residues serve as anchors on the periplasmic side of the membrane.     

菌紫质研究的新进展

菌紫质(BR)是嗜盐菌紫膜中的唯一蛋白质,野生型的BR分子含有248个氨基酸残基,其中一个视黄醛通过希夫碱基连结在第216位赖氨酸上,它具有质子泵的功能．光照下,BR进行光循环,光循环又与质子泵过程相关联．菌紫质的结构和功能方面的研究已有很大进展,但其光循环途径和质子泵的机理还不太清楚．文章概述了近年来对菌紫质结构,光循环和质子泵机理研究的进展,尤其对争论较大的菌紫质光循环途径的四类模型作了较详细的介绍．     

Essential amino acid residues in the central transmembrane domains and loops for energy coupling of Streptomyces coelicolor A3(2) H-pyrophosphatase

The H⁺-translocating inorganic pyrophosphatase is a proton pump that hydrolyzes inorganic pyrophosphate. It consists of a single polypeptide with 14−17 transmembrane domains, and is found in a range of organisms. We focused on the second quarter region of Streptomyces coelicolor A3(2) H⁺-pyrophosphatase, which contains long conserved cytoplasmic loops. We prepared a library of 1536 mutants that were assayed for pyrophosphate hydrolysis and proton translocation. Mutant enzymes with low substrate hydrolysis and proton-pump activities were selected and their DNAs sequenced. Of these, 34 were single-residue substitution mutants. We generated 29 site-directed mutant enzymes and assayed their activity. The mutation of 10 residues in the fifth transmembrane domain resulted in low coupling efficiencies, and a mutation of Gly¹⁹⁸ showed neither hydrolysis nor pumping activity. Four residues in cytoplasmic loop e were essential for substrate hydrolysis and efficient H⁺ translocation. Pro¹⁸⁹, Asp²⁸¹, and Val³⁵¹ in the periplasmic loops were critical for enzyme function. Mutation of Ala³⁵⁷ in periplasmic loop h caused a selective reduction of proton-pump activity. These low-efficiency mutants reflect dysfunction of the energy-conversion and/or proton-translocation activities of H⁺-pyrophosphatase. Four critical residues were also found in transmembrane domain 6, three in transmembrane domain 7, and five in transmembrane domains 8 and 9. These results suggest that transmembrane domain 5 is involved in enzyme function, and that energy coupling is affected by several residues in the transmembrane domains, as well as in the cytoplasmic and periplasmic loops. H⁺-pyrophosphatase activity might involve dynamic linkage between the hydrophilic and transmembrane domains.     

Refinement of the geometry of the retinal binding pocket in dark-adapted bacteriorhodopsin by heteronuclear solid-state NMR distance measurements

The bacterial proton pump bacteriorhodopsin (BR) is a 26.5 kDa seven-transmembrane helical protein. Several structural models have been published at > or =1.55 A resolution. The initial cis-trans isomerization of the retinal moiety involves structural changes within <1 A. To understand the chromophore-protein interactions that are important for light-driven proton transport, very accurate measurements of the protein geometry are required. To reveal more structural details at the site of the retinal, we have, therefore, selectively labeled the tryptophan side chains of BR with (15)N and metabolically incorporated retinal, (13)C-labeled at position 14 or 15. Using these samples, heteronuclear distances were measured with high accuracy using SFAM REDOR magic angle spinning solid-state NMR spectroscopy in dark-adapted bacteriorhodopsin. This NMR technique is applied for the first time to a high-molecular mass protein. Two retinal conformers are distinguished by their different isotropic 14-(13)C chemical shifts. Whereas the C14 position of 13-cis-15-syn-retinal is 4.2 A from [indole-(15)N]Trp86, this distance is 3.9 A in the all-trans-15-anti conformer. This latter distance allows us to check on the details of the active center of BR in the various published models derived from X-ray and electron diffraction data. The experimental approach and the results reported in this paper enforce the notion that distances between residues of a membrane protein binding pocket and a bound ligand can be determined at subangstrom resolution.     

A comparative analysis of the spatial structure of nonspecific porins from <Emphasis Type="Italic">Yersinia ruckeri</Emphasis> using optical spectroscopy and molecular modeling

The spatial organization of outer-membrane porins is studied by optical spectroscopy and molecular modeling. It was found that the OmpF and OmpC porins from Yеrsiniа ruckeri are β-structured membrane proteins typical of the pore-forming proteins of other Gram-negative bacteria. The spatial structures of monomers and trimers of the OmpC and OmpF porins from Y. ruckeri are simulated using methods of structural bioinformatics. It was found that the structural stability of the more thermostable OmpF trimer is sustained by a greater number of hydrogen bonds and hydrophobic interactions. The main differences of the spatial structures of the test porins are observed in the structure of their outer loops. There are three tryptophan residues in the molecules of the OmpC and OmpF porins of Y. ruckeri. It is demonstrated by moleculardynamics methods that after thermal denaturation the solvent accessibility of the Trp212 residue in OmpF porin increased by two times, while the solvent accessibility of a Trp184 residue in OmpC porin was not increased. It is hypothesized that the red-shifted tryptophan fluorescence spectrum of OmpF porin during thermal denaturation is due to the behavior of the Trp212 residue.     

Tryptophan interactions in bacteriorhodopsin: a heteronuclear solid-state NMR study

The bulky and amphiphilic nature of tryptophan residues makes them particularly interesting components of proteins. In bacteriorhodopsin, four of the eight tryptophan residues are in the active site, forming parts of the retinal binding pocket. In this work, we use solid-state NMR to study the interactions of the tryptophan residues in wild-type bacteriorhodopsin, in the resting state, and in critical intermediates of the proton-motive photocycle. The range of the chemical shifts of the indole nitrogens suggests that all eight of them are hydrogen bonded. Using difference spectroscopy, we isolate several changes in these hydrogen bonds in the early and late M states. As found earlier for the peptide backbone, some perturbations found in the early M state relax in the transition to the late M state while new perturbations arise. Interestingly, Rotational Echo DOuble Resonance (REDOR) difference spectroscopy of [20-13C]retinal,[indole-15N]Trp-bR shows that indole of Trp182 is not involved in the significant hydrogen bond perturbations. We also use REDOR to measure dipolar interactions in [20-13C]retinal,[indole-15N]Trp-bR, and thereby determine the distance between the C20 of retinal and the indole nitrogen of Trp182. The internuclear distance changes only slightly from the light-adapted state (3.36 +/- 0.2 A) to the early M state (3.16 +/- 0.4 A).     

Protein-bound water as the determinant of asymmetric functional conversion between light-driven proton and chloride pumps

Bacteriorhodopsin (BR) and halorhodopsin (HR) are light-driven outward proton and inward chloride pumps, respectively. They have similar protein architecture, being composed of seven-transmembrane helices that bind an all-trans-retinal. BR can be converted into a chloride pump by a single amino acid replacement at position 85, suggesting that BR and HR share a common transport mechanism, and the ionic specificity is determined by the amino acid at that position. However, HR cannot be converted into a proton pump by the corresponding reverse mutation. Here we mutated 6 and 10 amino acids of HR into BR-like, whereas such multiple HR mutants never pump protons. Light-induced Fourier transform infrared spectroscopy revealed that hydrogen bonds of the retinal Schiff base and water are both strong for BR and both weak for HR. Multiple HR mutants exhibit strong hydrogen bonds of the Schiff base, but the hydrogen bond of water is still weak. We concluded that the cause of nonfunctional conversion of HR is the lack of strongly hydrogen-bonded water, the functional determinant of the proton pump.     

Roles of the histidine and tryptophan side chains in the M2 proton channel from influenza A virus

The M2 protein form influenza A virus forms a tetrameric ion channel, which enables proton passage across biological membranes when the N-terminal side is acidified. Among the amino acid residues in the transmembrane domain of the M2 protein, His37 and Trp41 are essential for the pH-regulated proton conductance. Current knowledge about the structures and interactions of His37 and Trp41 suggests a model for the M2 ion channel, in which the channel is closed by a network of His37 hydrogen bonds at neutral pH and is opened by a His37-Trp41 cation-pi interaction at acidic pH.     

FTIR spectroscopy of the all-trans form of Anabaena sensory rhodopsin at 77 K: hydrogen bond of a water between the Schiff base and Asp75

Anabaena sensory rhodopsin (ASR) is an archaeal-type rhodopsin found in eubacteria, and is believed to function as a photosensor interacting with a 14 kDa soluble protein. Most of the residues in the retinal binding pocket are similar in ASR except proline 206, where the corresponding amino acid in other archaeal-type rhodopsins is highly conserved aspartate that constitutes the counterion complex of the positively charged protonated Schiff base. The recently determined X-ray crystallographic structure of ASR revealed a water molecule between the Schiff base and Asp75 [Vogeley, L., Sineshchekov, O. A., Trivedi, V. D., Sasaki, J., Spudich, J. L., and Luecke, H. (2004) Science 306, 1390-1393], as well as the case for bacteriorhodopsin (BR), a typical transport rhodopsin working as a proton pump. In this study, we applied low-temperature Fourier transform infrared (FTIR) spectroscopy to the all-trans form of ASR at 77 K, and compared the local structure around the chromophore and their structural changes upon retinal photoisomerization with those of BR. The K intermediate minus ASR difference spectra were essentially similar to those for BR, indicating that photoisomerization yields formation of the distorted 13-cis form. In contrast, little amide I bands were observed for ASR. The presence of the proline-specific vibrational bands suggests that peptide backbone alterations are limited to the Pro206 moiety in the K state of ASR. The N-D stretching of the Schiff base is presumably located at 2163 (-) and 2125 (-) cm(-)(1) in ASR, suggesting that the hydrogen bonding strength of the Schiff base in ASR is similar to that in BR. A remarkable difference between ASR and BR was revealed from water bands. Although ASR possesses a bridged water molecule like BR, the O-D stretching of water molecules was observed only in the >2500 cm(-)(1) region for ASR. We interpreted that the weak hydrogen bond of the bridged water between the Schiff base and Asp75 originates from their geometry. Since ASR does not pump protons, our result supports the working hypothesis that the existence of strongly hydrogen bonded water molecules is essential for proton pumping activity in archaeal rhodopsins.     

The transmembrane 7-alpha-bundle of rhodopsin: distance geometry calculations with hydrogen bonding constraints.

A 3D model of the transmembrane 7-alpha-bundle of rhodopsin-like G-protein-coupled receptors (GPCRs) was calculated using an iterative distance geometry refinement with an evolving system of hydrogen bonds, formed by intramembrane polar side chains in various proteins of the family and collectively applied as distance constraints. The alpha-bundle structure thus obtained provides H bonding of nearly all buried polar side chains simultaneously in the 410 GPCRs considered. Forty evolutionarily conserved GPCR residues form a single continuous domain, with an aliphatic "core" surrounded by six clusters of polar and aromatic side chains. The 7-alpha-bundle of a specific GPCR can be calculated using its own set of H bonds as distance constraints and the common "average" model to restrain positions of the helices. The bovine rhodopsin model thus determined is closely packed, but has a few small polar cavities, presumably filled by water, and has a binding pocket that is complementary to 11-cis (6-s-cis, 12-s-trans, C = N anti)-retinal or to all-trans-retinal, depending on conformations of the Lys296 and Trp265 side chains. A suggested mechanism of rhodopsin photoactivation, triggered by the cis-trans isomerization of retinal, involves rotations of Glu134, Tyr223, Trp265, Lys296, and Tyr306 side chains and rearrangement of their H bonds. The model is in agreement with published electron cryomicroscopy, mutagenesis, chemical modification, cross-linking, Fourier transform infrared spectroscopy, Raman spectroscopy, electron paramagnetic resonance spectroscopy, NMR, and optical spectroscopy data. The rhodopsin model and the published structure of bacteriorhodopsin have very similar retinal-binding pockets.     

Essential amino acid residues in the central transmembrane domains and loops for energy coupling of Streptomyces coelicolor A3(2) H+-pyrophosphatase

The H+-translocating inorganic pyrophosphatase is a proton pump that hydrolyzes inorganic pyrophosphate. It consists of a single polypeptide with 14-17 transmembrane domains, and is found in a range of organisms. We focused on the second quarter region of Streptomyces coelicolor A3(2) H+-pyrophosphatase, which contains long conserved cytoplasmic loops. We prepared a library of 1536 mutants that were assayed for pyrophosphate hydrolysis and proton translocation. Mutant enzymes with low substrate hydrolysis and proton-pump activities were selected and their DNAs sequenced. Of these, 34 were single-residue substitution mutants. We generated 29 site-directed mutant enzymes and assayed their activity. The mutation of 10 residues in the fifth transmembrane domain resulted in low coupling efficiencies, and a mutation of Gly198 showed neither hydrolysis nor pumping activity. Four residues in cytoplasmic loop e were essential for substrate hydrolysis and efficient H+ translocation. Pro189, Asp281, and Val351 in the periplasmic loops were critical for enzyme function. Mutation of Ala357 in periplasmic loop h caused a selective reduction of proton-pump activity. These low-efficiency mutants reflect dysfunction of the energy-conversion and/or proton-translocation activities of H+-pyrophosphatase. Four critical residues were also found in transmembrane domain 6, three in transmembrane domain 7, and five in transmembrane domains 8 and 9. These results suggest that transmembrane domain 5 is involved in enzyme function, and that energy coupling is affected by several residues in the transmembrane domains, as well as in the cytoplasmic and periplasmic loops. H+-pyrophosphatase activity might involve dynamic linkage between the hydrophilic and transmembrane domains.     

A Blue-shifted Light-driven Proton Pump for Neural Silencing

Ion-transporting rhodopsins are widely utilized as optogenetic tools both for light-induced neural activation and silencing. The most studied representative is Bacteriorhodopsin (BR), which absorbs green/red light (∼570 nm) and functions as a proton pump. Upon photoexcitation, BR induces a hyperpolarization across the membrane, which, if incorporated into a nerve cell, results in its neural silencing. In this study, we show that several residues around the retinal chromophore, which are completely conserved among BR homologs from the archaea, are involved in the spectral tuning in a BR homolog (HwBR) and that the combination mutation causes a large spectral blue shift (λ_max = 498 nm) while preserving the robust pumping activity. Quantum mechanics/molecular mechanics calculations revealed that, compared with the wild type, the β-ionone ring of the chromophore in the mutant is rotated ∼130° because of the lack of steric hindrance between the methyl groups of the retinal and the mutated residues, resulting in the breakage of the π conjugation system on the polyene chain of the retinal. By the same mutations, similar spectral blue shifts are also observed in another BR homolog, archearhodopsin-3 (also called Arch). The color variant of archearhodopsin-3 could be successfully expressed in the neural cells of Caenorhabditis elegans, and illumination with blue light (500 nm) led to the effective locomotory paralysis of the worms. Thus, we successfully produced a blue-shifted proton pump for neural silencing.     

Structural investigation of bacteriorhodopsin and some of its photoproducts by polarized Fourier transform infrared spectroscopic methods-difference spectroscopy and photoselection

The direction of selected IR-transition moments of the retinal chromophore of bacteriorhodopsin (BR) and functional active amino acid residues are determined for light- and dark-adapted BR and for the intermediates K and L of the photocycle. Torsions around single bonds of the chromophore are found to be present in all the investigated BR states. The number of twisted single bonds and the magnitude of these torsions decreases in the order K, L, light-adapted BR, dark-adapted BR. In the last, only the C₁₄—C₁₅ single bond is twisted. The orientation of molecular planes and chemical bonds of such protein side chains, which are perturbed during the transition of light-adapted BR to the respective intermediates, are deduced and the results compared with the current three dimensional model of BR. Trp 86 and Trp 185 are found to form a rigid part of the protein, whereas Asp 96 and Asp 115 perform molecular rearrangements upon formation of the L-intermediate.     

Voltage- and pH-Dependent Changes in Vectoriality of Photocurrents Mediated by Wild-type and Mutant Proteorhodopsins upon Expression in Xenopus Oocytes

Proteorhodopsin (PR), a light-driven proton pump from marine proteobacteria, exhibits photocycle characteristics similar to bacteriorhodopsin (BR) at neutral pH, including an M-like photointermediate. However, at acidic pH, spectroscopic evidence for an M-like species was absent, and the vectoriality of proton pumping was inverted. To gain further insight into this unusual property, we examined the voltage dependence of stationary and laser flash-induced photocurrents of PR under different pH conditions upon expression in Xenopus oocytes. The current-voltage curves were linear under all conditions tested, and photocurrent reversal potentials distinctly depended on the pH gradient. PR mutants D97N and D97T exhibited transient and stationary inward currents already at neutral pH, showing that neutralization of the proton acceptor abolishes forward pumping and permits only inward proton transport. Mutation E108G, which disrupts the donor site for Schiff base (SB) reprotonation, resulted in largely reduced photocurrents, which could be strongly stimulated by azide, similar to previous observations on BR mutant D96G. When PR and BR photocurrents in response to blue or green laser flashes during or after continuous illumination were compared, direct electrical evidence for the occurrence of an M-like intermediate at neutral pH could only be obtained when reprotonation of the SB was slowed down by PR mutation E108G. For PR at acidic pH, laser flashes only produced inwardly directed photocurrents, independent from background illumination, thus precluding electrical identification of an M-like species. However, when visible absorption spectroscopy was carried out at low temperatures, occurrence of an M-like species was robustly observed at low pH. This indicates that SB deprotonation and reprotonation occur during the PR photocycle also at low pH. Our results corroborate the conclusion that in PR, the direction of proton pumping can be switched by changes in pH and membrane potential, with the protonation state of Asp-97 being the key determinant for selecting between transport modes.     

Sugar transport across lactose permease probed by steered molecular dynamics

Escherichia coli lactose permease (LacY) transports sugar across the inner membrane of the bacterium using the proton motive force to accumulate sugar in the cytosol. We have probed lactose conduction across LacY using steered molecular dynamics, permitting us to follow molecular and energetic details of lactose interaction with the lumen of LacY during its permeation. Lactose induces a widening of the narrowest parts of the channel during permeation, the widening being largest within the periplasmic half-channel. During permeation, the water-filled lumen of LacY only partially hydrates lactose, forcing it to interact with channel lining residues. Lactose forms a multitude of direct sugar-channel hydrogen bonds, predominantly with residues of the flexible N-domain, which is known to contribute a major part of LacY's affinity for lactose. In the periplasmic half-channel lactose predominantly interacts with hydrophobic channel lining residues, whereas in the cytoplasmic half-channel key protein-substrate interactions are mediated by ionic residues. A major energy barrier against transport is found within a tight segment of the periplasmic half-channel where sugar hydration is minimal and protein-sugar interaction maximal. Upon unbinding from the binding pocket, lactose undergoes a rotation to permeate either half-channel with its long axis aligned parallel to the channel axis. The results hint at the possibility of a transport mechanism, in which lactose permeates LacY through a narrow periplasmic half-channel and a wide cytoplasmic half-channel, the opening of which is controlled by changes in protonation states of key protein side groups.     

The Unique Binding Mode of Cellulosomal CBM4 from Clostridium thermocellum Cellobiohydrolase A

The crystal structure of the carbohydrate-binding module (CBM) 4 Ig fused domain from the cellulosomal cellulase cellobiohydrolase A (CbhA) of Clostridium thermocellum was solved in complex with cellobiose at 2.11 Å resolution. This is the first cellulosomal CBM4 crystal structure reported to date. It is similar to the previously solved noncellulosomal soluble oligosaccharide-binding CBM4 structures. However, this new structure possesses a significant feature—a binding site peptide loop with a tryptophan (Trp118) residing midway in the loop. Based on sequence alignment, this structural feature might be common to all cellulosomal clostridial CBM4 modules. Our results indicate that C. thermocellum CbhA CBM4 also has an extended binding pocket that can optimally bind to cellodextrins containing five or more sugar units. Molecular dynamics simulations and experimental binding studies with the Trp118Ala mutant suggest that Trp118 contributes to the binding and, possibly, the orientation of the module to soluble cellodextrins. Furthermore, the binding cleft aromatic residues Trp68 and Tyr110 play a crucial role in binding to bacterial microcrystalline cellulose (BMCC), amorphous cellulose, and soluble oligodextrins. Binding to BMCC is in disagreement with the structural features of the binding pocket, which does not support binding to the flat surface of crystalline cellulose, suggesting that CBM4 binds the amorphous part or the cellulose “whiskers” of BMCC. We propose that clostridial CBM4s have possibly evolved to bind the free-chain ends of crystalline cellulose in addition to their ability to bind soluble cellodextrins.     

Crystal structure of the eukaryotic light-driven proton-pumping rhodopsin, Acetabularia rhodopsin II, from marine alga

Acetabularia rhodopsin (AR) is a rhodopsin from the marine plant Acetabularia acetabulum. The opsin-encoding gene from A. acetabulum, ARII, was cloned and found to be novel but homologous to that reported previously. ARII is a light-driven proton pump, as demonstrated by the existence of a photo-induced current through Xenopus oocytes expressing ARII. The photochemical reaction of ARII prepared by cell-free protein synthesis was similar to that of bacteriorhodopsin (BR), except for the lack of light-dark adaptation and the different proton release and uptake sequence. The crystal structure determined at 3.2 Å resolution is the first structure of a eukaryotic member of the microbial rhodopsin family. The structure of ARII is similar to that of BR. From the cytoplasmic side to the extracellular side of the proton transfer pathway in ARII, Asp92, a Schiff base, Asp207, Asp81, Arg78, Glu199, and Ser189 are arranged in positions similar to those of the corresponding residues directly involved in proton transfer by BR. The side-chain carboxyl group of Asp92 appears to interact with the sulfhydryl group of Cys218, which is unique to ARII and corresponds to Leu223 of BR and to Asp217 of Anabaena sensory rhodopsin. The orientation of the Arg78 side chain is opposite to the corresponding Arg82 of BR. The putative absence of water molecules around Glu199 and Arg78 may disrupt the formation of the low-barrier hydrogen bond at Glu199, resulting in the “late proton release”.