Crystal Structures of Acid Blue and Alkaline Purple Forms of Bacteriorhodopsin

doi:10.1016/j.jmb.2005.06.026

J. Mol. Biol. (2005) 351, 481–495

Crystal Structures of Acid Blue and Alkaline Purple Forms of Bacteriorhodopsin Hideo Okumura1, Midori Murakami1 and Tsutomu Kouyama1,2* 1

Department of Physics Graduate School of Science Nagoya University, Nagoya 464-8602, Japan 2 RIKEN Harima Institute/ SPring-8, 1-1-1, Kouto Mikazuki, Sayo, Hyogo 679-5148, Japan

Bacteriorhodopsin, a light-driven proton pump found in the purple membrane of Halobacterium salinarum, exhibits purple at neutral pH but its color is sensitive to pH. Here, structures are reported for an acid blue form and an alkaline purple form of wild-type bacteriorhodopsin. When the P622 crystal prepared at pH 5.2 was acidified with sulfuric acid, its color turned to blue with a pKa of 3.5 and a Hill coefficient of 2. Diffraction data at pH 2–5 indicated that the purple-to-blue transition accompanies a large structural change in the proton release channel; i.e. the extracellular half of helix C moves towards helix G, narrowing the proton release channel and expelling a water molecule from a micro-cavity in the vicinity of the retinal Schiff base. In this respect, the acid-induced structural change resembles the structural change observed upon formation of the M intermediate. But, the acid blue form contains a sulfate ion in a site(s) near Arg82 that is created by re-orientations of the carboxyl groups of Glu194 and Glu204, residues comprising the proton release complex. This result suggests that proton uptake by the proton release complex evokes the anion binding, which in turn induces protonation of Asp85, a key residue regulating the absorption spectrum of the chromophore. Interestingly, a pronounced structural change in the proton release complex was also observed at high pH; i.e. re-orientation of Glu194 towards Tyr83 was found to take place at around pH 10. This alkaline transition is suggested to be accompanied by proton release from the proton release complex and responsible for rapid formation of the M intermediate at high pH. q 2005 Elsevier Ltd. All rights reserved.

*Corresponding author

Keywords: bacteriorhodopsin; proton pump; purple membrane; X-ray crystallography; purple-to-blue transition

Introduction Bacteriorhodopsin, the sole protein of the purple membrane of Halobacterium salinarum, functions as a light-driven proton pump. 1–3 It contains one molecule of retinal, which is bound via a protonated Schiff base linkage to the 3-amino group of Lys216. When the all-trans isomer of bacteriorhodopsin in the light-adapted state absorbs visible light, it undergoes a reaction cycle involving five spectroscopically distinguishable intermediates (i.e. K, L, M, N and O). A crucial event in the photocycle is isomerization of retinal around the C13]C14 double bond in the K intermediate. This initiates a series of protein conformational changes, causing proton translocation from the cytoplasmic to the

E-mail address of the corresponding author: [email protected]

extracellular side, building up the electrochemical gradient across the membrane, which is used for ATP synthesis. Over the last five years, light-induced structural changes of bacteriorhodopsin have been analyzed by using the P63 or the P622 crystals.4–14 Unfortunately, discussion about the light-induced structural changes has been controversial. Divergence in the reported structural models of photoreaction intermediates may suggest the possibility that the light-induced structural changes are affected by the lattice force (more detailed discussion is given by others.1,2,13) Crystallographic analyses of the P622 crystal of wild-type bacteriorhodopsin have suggested the following reaction scheme.6,9,13 In the K intermediate, several bonds in the retinal, including the Schiff base linkage, are largely distorted.6 This distortion has been reproduced by a simulation analysis of the primary photoreaction.15 In the K-to-L transition, a water molecule

0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.

482 interacting with the Schiff base is dragged from the proton release channel to a micro-cavity created in the cytoplasmic side of the Schiff base.13 Subsequent detachment of this water molecule from the Schiff base causes a decrease in the Schiff base pKa, inducing the proton transfer from the Schiff base to its counter ion (Asp85) that is located in the innermost part of the proton release channel (the L-to-M transition).9,13 The complete protonation of Asp85 is achieved when its pKa is increased sufficiently by re-arrangement of the hydrogenbonding network in the proton release channel.13 At neutral pH, this transition accompanies release of a proton from the proton release complex into the extracellular medium. In the M-to-N transition, the Schiff base is reprotonated from Asp96 that is located in the cytoplasmic side of the protein.15 In the N-to-O transition, a proton is taken up from the cytoplamic side and, after re-isomerization of retinal and proton transfer from Asp85 to the proton release complex, the protein returns to the initial unexcited state. Wild-type bacteriorhodopsin is able to generate a pH gradient as large as four pH units across the cell membrane.16 For better understanding of this proton pumping activity, it is indispensable to elucidate how a light-induced pH gradient causes a feedback effect on the protein structure. Although it seems practically difficult to determine the atomic structure in the presence of a pH gradient, structural data of bacteriorhodopsin at various pH values would help us to simulate the possible feedback effect of pH gradient. Such data would also be useful for elucidation of the mechanism of the color regulation of bacteriorhodopsin. It has been known that the absorption maximum of wildtype bacteriorhodopsin shifts from 570 nm to 605 nm when purple membrane is acidified to pH 2.17 The formation of the so-called blue membrane has been attributed to protonation of Asp85.18,19 The acid blue form is often considered to mimic the O intermediate having a red-shifted absorption spectrum.20 But the purple-to-blue transition or its reverse reaction proceeds in a much more complicated manner than expected from the decay process of the O intermediate. For example, the purple-to-blue transition can be induced by deionization of purple membrane and this color change is reversed upon binding of Ca2C or other divalent cations to the blue membrane.21 It has been argued by several groups that the effect of the cation binding is due to an increase of the membrane surface pH.22,23 But other groups have argued that a divalent cation binds to a specific site(s) near the membrane surface.24–29 To fully understand the mechanism of the purple-to-blue transition, we need to accumulate structural data at acidic pH. The absorption spectrum of bacteriorhodopsin scarcely changes in the alkaline region, but it has been reported that the fast rising component of the M intermediate increases significantly when the medium pH is increased to pH 10.30 This phenom-

Crystal Structures of Bacteriorhodopsin

enon has been suggested to correlate with a remarkable decrease in the rate constant of the dark adaptation at high pH.31 To explain this correlation, Balashov et al. have proposed that the pKa of Asp85 is affected by the protonation state of another residue locating near the extracellular surface.32 Their proposal has remained to be examined by structural data collected at high pH, which would provide information on pH-induced structural changes in the alkaline region. The P622 crystal, which is prepared by the membrane fusion method, is made up of stacked membranes, in each of which the trimeric bacteriorhodopsin-lipid complexes are arranged on a honeycomb lattice.33,34 This crystal has been utilized for investigation of structural changes taking place during the proton pumping cycle6,9,13 The P622 crystal is also useful for investigation of pHinduced structural changes. After searching for an appropriate procedure to prepare high-quality crystals at extreme pH values, we found that the P622 crystal prepared at pH 5.2 can be acidified or alkalized with a small deterioration of the diffraction quality. The stability of the P622 crystal seems to come from native lipids incorporated into the crystal. In the present study, diffraction data at 2.3– ˚ resolutions were collected from the P622 2.7 A crystals that had been soaked at various pH values, and structural changes associated with the purpleto-blue transition at acidic pH as well as an alkaline transition at around pH 10 were compared with those taking place during the proton pumping cycle.

Results Absorption spectra of the P622 crystal at various pH values When the P622 crystal was acidified with sulfuric acid or alkalized with sodium hydroxide in the presence of 2.1 M ammonium sulfate, its hexagonal shape remained unchanged over a wide pH range (pH 1–pH 11). In the investigated pH range, however, the color of the crystal varied with pH (Figure 1). At neutral or weakly acidic pH (pH O4), the light-adapted crystal had an absorption peak at 567–569 nm, exhibiting purple. Acidification below pH 4 caused a remarkable red-shift of the visible band and, at or below pH 3, the crystal had an absorption peak at 604 nm, exhibiting blue. It is noteworthy that this acid-induced purple-toblue transition takes place in a narrow pH region (Figure 1(b)). When the amplitude of acid-induced absorption change at 638 nm is analyzed, the experimental data are fitted well with the Henderson–Hasselbalch equation, DAZ DAmax ð1C 10nðpHKpKa Þ ÞK1 , in which the parameter n is fixed to 2. The pKa value was evaluated to be 3.5 under the solvent condition used. This result implies cooperative protonation of two ionizable residues during the purple-to-blue transition. Since

Crystal Structures of Bacteriorhodopsin

483

Figure 2. (a) X-radiation-induced absorption changes in a frozen crystal of the acid blue form. Absorption spectra were recorded before and after various periods of ˚ ) at exposure to monochromatic X-ray radiation (lZ1 A a flux rate of 2!1012 photons/mm2 per second and a beam size of 0.23 mm!0.21 mm. The numerals in this Figure indicate the accumulated exposure time expressed in seconds.

Analysis of the absorption changes between pH 9 and pH 11 suggests that the protein undergoes an alkaline transition with a pKa value of w10. This alkaline transition appears to be caused by deprotonation of a residue that makes no direct contact with the retinal chromophore.

Figure 1. (a) Absorption spectra of the P622 crystal at various pH values. Each spectrum was recorded at 300 K after a single crystal adhered to a lower glass in the crystallization kit was soaked for a few minutes in an acidic medium (O0.2 ml) containing 2.1 M ammonium sulfate and light-adapted with yellow light. The numerals represent the medium pH. (b) Acid-induced absorption change at 638 nm is plotted against the medium pH. The experimental data (filled circles) are fitted with the equation DAZ DAmax ð1C 10nðpHKpKa Þ ÞK1 in which the parameter n is fixed to 2.0 (continuous line). (c) The wavelength of the absorption maximum of the visible band is plotted against the medium pH.

a pH titration similar to that shown in Figure 2 has been observed for native purple membrane,22 it is suggested that the protein in the P622 crystal undergoes the same acid transition as observed for purple membrane. In the alkaline region, the absorption spectrum of the P622 crystal scarcely changed. But, alkalization above pH 9 caused a small blue-shift of the visible absorption band and incubation at pH O11 resulted in a gradual absorption increase at 376 nm. The latter absorption change is indicative of release of the retinal chromophore from the binding site.

X-ray-induced structural changes in the acid blue form At cryogenic temperature, the acidified crystal at pH 2 had an absorption maximum at 610 nm (slightly red-shifted as compared to the spectrum at room temperature) and its absorption spectrum was affected slightly by visible light; i.e. illumination with green light (lZ532 nm) at 100 K caused a small red shift (610 nm/614 nm) and this shift was reversed by illumination with red light (lZ 678 nm). These absorption changes can be explained by formation of a K-like red-shifted intermediate and its reverse photoreaction, respectively.35 Although the 9-cis isomer (lmaxZ455 nm) has been reported to be produced by illumination of acid blue membrane at ambient temperature,36,37 there was no indication for production of the 9-cis isomer in the P622 crystal at 100 K. In contrast to visible light, X-ray radiation caused a remarkable change in the absorption spectrum of the acid blue form. Figure 2 shows absorption spectra recorded after an acidified crystal at 100 K was exposed for various periods to X-ray radiation at a flux rate of w2!1012 photons/mm2 per second. With the increasing exposure time, the main absorption band at 610 nm decreased and, instead, the absorbance at 450 nm increased. Development

484

Crystal Structures of Bacteriorhodopsin

of this new band is described with three or more exponentials, the fastest component of which has a time constant of 80 seconds. This result indicates that the protein structure is altered by an X-ray dose of w1014 photons/mm2. A similar dose of X-ray radiation has been shown to cause significant absorption changes in the neutral purple form (lmaxZ574 nm/450, 480 and 515 nm) and its M intermediate (lmaxZ414 nm/640 nm).6,13 These absorption changes were previously attributed to isomerization and/or chemical modification of the retinal-Lys216 chain. It can be argued here that the retinal chromophore in any state of bacteriorhodopsin, including the acid blue form, is very susceptible to X-ray damage. Structure of the acid blue form ˚ resolutions were Diffraction data at 2.3–2.6 A collected from the P622 crystals that had been acidified with sulfuric acid to pH 2, 3 and 4 and then cooled to 100 K (Table 1). These diffraction data were collected at an X-ray dose of 2!1015–3! 1015 photons/mm2 per data set. This X-ray dose is higher than the safe level of X-ray dose mentioned above, though it is much lower than a critical level (1016 photons/mm2) above which de-carboxylation of Asp85 becomes significant.6 To check how much the protein structure was modified by X-ray absorption, we collected a series of complete diffraction data sets from a frozen crystal of the acid blue form. A difference electron density map that was derived from the first and the second data sets exhibited no significant peak, suggesting that

the structural change induced during the data collection is very small. In the structural analyses of the acid blue form, therefore, no special correction for X-ray damage was included. It should be kept in mind, however, that the configuration of the retinal-Lys216 chain might alter upon X-ray absorption. For a crystal soaked at pH 4, the cell dimensions were identical with those observed for the neutral purple form (Table 1). The 2FoKFc density map derived from diffraction data collected at pH 4 was explained well by the model of the neutral purple form after a rigid-body refinement (data not shown). This result indicates that at pH 4, the majority of the protein has the same conformation as that of the neutral purple form. It was observed, on the other hand, that acidification to pH 3 or pH 2 caused a noticeable shrinkage of the crystal along the c axis (cZ ˚ /109.4–109.0 A ˚ ). Since the cell dimensions 112.3 A along the a and b axes remained unchanged, the honeycomb lattice in the P622 crystal seemed to be preserved at these pH values. But, acidification to pH 1 resulted in a significant deterioration of the crystal quality, so that no reliable structural information was obtained. It is noteworthy that the crystal shrinkage takes place in a narrow pH range (between pH 4 and pH 3), which coincides exactly with the pH range where the significant color change was observed (Figure 1(b)). This coincidence strongly suggests that the purple-toblue transition is accompanied by such a structural change that causes the crystal shrinkage. It will be later shown that the crystal shrinkage is due mainly

Table 1. Data collection and final refinement statistics Date set Data collection ˚) Resolution (A ˚ ) a, b Unit cell (A c Data completion (%) No. unique reflections Multiplicity Rsymb (%) I/s Refinement ˚) Resolution limit (A Protein residues Number of lipids Number of water Rcrystc (%) Rfree (%) RMS deviation ˚) Bond length (A Bond angle (8) ˚) Average B values (A Protein Water Lipid

pH 2

pH 3

pH 4

pH 5.2

pH 9

pH 10a

54.2–2.3 102.6 109.0 99.7 15,575 6.6 7.4 6.3

51.3–2.5 102.7 109.4 98.2 11,962 6.8 8.2 7.2

51.3–2.6 102.4 112.4 99.3 9934 6.6 7.7 6.9

46.7–2.3 102.3 112.3 100.0 15,703 9.9 4.9 5.9

51.3–2.7 102.5 112.9 98.9 9912 7.5 8.7 6.9

69.0–2.6 102.6 112.9 96.0 10,826 8.6 9.9 5.5

15.0–2.3 215 5 25 23.9 28.7

15.0–2.5 215 5 25 24.3 26.5

15.0–2.6 228 5 38 23.3 27.3

15.0–2.3 228 5 41 24.8 27.1

15.0–2.7 228 5 41 24.0 27.3

15.0–2.6 228 5 42 27.6 29.1

0.008 1.16

0.007 1.01

0.008 1.17

0.008 1.16

0.008 1.18

0.007 1.16

41.6 56.7 88.5

45.3 58.2 101.6

56.6 67.0 107.1

49.0 59.1 104.6

51.1 63.8 107.4

44.0 55.4 103.4

a The refinement of the high-pH conformer was performed using the intensity data obtained by subtracting the contribution of the neutral purple P form P P data at pH 10. P from diffraction b Rsym Z hkl i jIi K hIij= hkl i Ii , where Ii is the intensity of an individual reflection and hIi is the mean intensity obtained from multiple observations of symmetry-related reflections. P P c Rcryst Z hkl ðFobs K jFcalc jÞ= hkl jFobs j (5% randomly omitted reflections were used for Rfree).

485

Crystal Structures of Bacteriorhodopsin

to a conformational change in the BC loop. The structural data observed at pH 3 and pH 2 were identical with each other, indicating that no significant structural change took place between pH 3 and pH 2. (In this study, diffraction data ˚ resolution from a crystal at pH 2 collected at 2.3 A were mainly used for the model building of the acid blue form, while diffraction data from a crystal at pH 3 were occasionally used to improve the model.) Compositional heterogeneity in the acid blue form It has been reported that a nearly equal amount of the all-trans and the 13-cis isomers are contained in acid blue membrane.38,39 In the structural analysis of the acid blue form, therefore, the compositional heterogeneity arising from a rapid thermal interconversion between the all-trans and the 13-cis isomers should be taken into account. To deal with this problem, we firstly constructed two monoconformation models in which the retinal chromophore was assumed to take either the all-trans or the 13-cis, 15-syn configuration. Their refined structures indicated that, except for the retinal-Lys216 chain and its neighbors, the protein structure was not strongly dependent on how the retinal was constrained (Figure 4). This result implies two possibilities: (1) the equilibrium between the two isomers is shifted largely towards one of the isomers in the acidified crystal; or (2) the structural difference between the two isomers is not very large. In the former case, we can argue that the trans isomer, which better explains the 2FoKFc map than the other isomer does, is the major component in the acidified crystal that had been light-adapted before rapid freezing. To investigate the second possibility, we built a double-conformation model by combining the two mono-conformation models refined above. When the two conformers in this model were alternately refined by the simulated annealing method, the structural difference between them became larger in loop regions characterized by high temperature factors. The double-conformation model is difficult to interpret uniquely, because each isomer may have multiple conformations in flexible regions. Nonetheless, as the main-chain structure in the protein interior is nearly identical for the two conformers, it is unlikely that a large structural change is induced during the interconversion between the two isomers. This result is consistent with our recent observation that only a few residues in the vicinity of the Schiff base and Arg82 undergo small structural changes during the light/dark adaptation at neutral pH (unpublished data). At the present resolution, it is difficult to determine unambiguously a possible structural difference between the two isomers contained in the acid blue form. (The refinements using many initial models provided little information as to the retinal compositional heterogeneity.) In the following sections, the essential feature of the

acid-induced structural change is described by using the mono-conformation model of the trans isomer, which is nearly identical with the averaged structure of the two conformers in the doubleconformation model. Acid-induced structural changes in the overall structure When the trimeric structure of the acid blue form is compared with that of the neutral purple form (Figures 3(a) and 4–6), the following differences are observed: (1) the extracellular half of helix C moves towards helix G; (2) helices D, E and F tilt in such a manner that their extracellular terminal ends move outward from the center of the trimeric structure; (3) the BC loop, which forms an antiparallel b sheet in the neutral purple form, becomes disordered in the acid blue form. (Gly106 in the N-terminal end of helix D, Gly155 near the C-terminal end of helix E and Pro186 in the middle of helix F act as swivel points for the tilting of helices D, E and F, respectively.) Large movements are also observed for residues in the DE and FG loops that are exposed to the extracellular membrane surface. In contrast to these movements, the backbones of helices A, B and G remain unchanged (Figure 3(b)). It should be noted that whereas the retinal Schiff base scarcely moves, a pronounced movement of the ionone ring of retinal accompanies the tilting of helices D, E and F (Figure 3(a)). In the cytoplasmic half of the protein, a few residues (i.e. Asp38 and Glu166) undergo noticeable conformational changes. Their side-chains are re-oriented towards the center of the protein at and below pH 3, where they are presumably protonated. Movements of water molecules during the purple-to-blue transition The movement of the extracellular half of helix C towards helix G results in narrowing of the innermost part of the proton release channel. For example, the distance between Asp85 Od1 and ˚ upon the purpleAsp212 Od2 decreases by 0.9 A to-blue transition. As a consequence, the microcavity near the retinal Schiff base shrinks and a water molecule (Wat602) that interacts with the retinal Schiff base in the neutral form is squeezed out from the active site (Figure 4). (Here, the water molecules in the proton pathway are numbered according to their z positions (Figure 5); Wat602 corresponds to Wat402 in the model reported by Luecke et al.40) In the acid blue form, the retinal Schiff base makes direct interactions with the carboxyl groups of Asp85 and Asp212. The reduced distance between Asp85 and Asp212 suggests that either of these residues is protonated in the acid blue form. Other water molecules in the proton release channel also undergo significant movements upon the purple-to-blue transition. A water molecule (Wat603) interacting with Asp85 moves so as to

486

Crystal Structures of Bacteriorhodopsin

(Wat606) that mediates interactions between Arg82 and the paired structure of Glu194 and Glu204 in the neutral purple form moves so as to interact with Wat605 existing near Tyr57 (Figure 5). These movements of water molecules are associated ˚ ) of the with a downward movement (by 1.5 A guanidinium ion of Arg82. With respect to a water molecule (Wat600) that is seen between the carboxyl group of Asp115 and the carbonyl of Leu92, it becomes undetectable when the intra-helix hydrogen bond between Leu92 and Phe88 is broken at low pH (Figure 6). Binding of anion to the proton release channel

Figure 3. Acid-induced structural change in the trimeric structure of bacteriorhodopsin. (a) Ribbon models of the acid blue form at pH 2 (gold) and the neutral purple form at pH 5.2 (pansy), with the retinalLys216 chain in the ball-and-stick presentation. The extracellular half of the trimer is viewed from the extracellular side and, for clarity, the loop between helices B and C is omitted. The green arrows indicate acidinduced movements of the extracellular terminal ends of helices C through F. This Figure is drawn with SwissPdbViewer.66 (b) The magnitude of acid-induced movement of Ca atom is plotted against the residue number. The two curves represent the data calculated for two conformers in a double-conformation model of the acid blue form. Residues 64–78 are disordered in the acid blue form. For this structural comparison, the model of the neutral purple form is moved along the c axis so as to minimize the overall RMS deviation between the neutral purple form and the acid blue form.

interact with the carboxyl group of Asp212. A water molecule (Wat604) that interacts with Wat603 and Asp212 at neutral pH becomes invisible in the 2FoK Fc map of the acid blue form. A water molecule (Wat605) that interacts with the phenyl OH group of Tyr57 scarcely moves, while a water molecule

Figure 5 shows acid-induced structural changes near the proton release complex, which is here considered to be composed of Glu194, Glu204 and the nearby water molecules. In the neutral purple form, the carboxyl groups of Glu194 and Glu204 are connected by a strong hydrogen-bonding interaction. The paired structure of these glutamate residues is broken in the acid blue form, in which the carboxyl group of Glu204 directs towards helix F and the carboxyl group of Glu194 moves largely towards Arg134. The latter movement, which is accompanied by a large conformational change in the FG loop, creates an open space near the guanidinium ion of Arg82. An electron density peak seen in this open space is attributed to SO2K 4 , which can stabilize the protein structure by forming a salt-bridge with Arg82. In the structural model shown in Figure 5(a), this ion is placed in such a position that it interacts with the OH group of Tyr83 and the carboxyl group of Glu194. But, as judged from the high temperature factor of the bound ion, it does not seem likely that the binding site of SO2K 4 is very specific. It is possible that this ion often migrates to another position where a water molecule (Wat607) is placed in Figure 5(a). In this is able to interact with the position too, SO2K 4 guanidinium ion of Arg82. to the proton Non-specific binding of SO2K 4 release channel may be correlated with apparent disorder of the BC loop at low pH. Indeed the electron density map of the acid blue form suggests that the N-terminal end of helix C (Pro77–Leu78) takes multiple conformations. Since this region is not far from the binding site of SO2K 4 , it is likely that the conformation of the BC loop is dependent strongly on how the bound anion interacts with the N-terminal end of helix C. It should be pointed out that the highestresolution diffraction data of the acid blue form were obtained when chloride ions, which had been contained in the mother solution, were removed during the procedure of acidification. A different procedure of acidification produced a lower-quality crystal with different cell dimensions. One plausible explanation for the variation of the cell dimensions is that the structure of the BC loop is dependent on whether sulfate ion or chloride ion binds to the proton release channel.

Crystal Structures of Bacteriorhodopsin

487

Figure 4. The 2FoKFc maps of the acid blue form at pH 2 (right) and the neutral purple form at pH 5.2 (left), contoured at 1.1s and 1.4s, respectively, and superimposed on their structural models. In the right panel, the map is fitted by monoconformation models with all-trans retinal (carbon atoms in gold) and with 13-cis, 15-syn retinal (carbon atoms in cyan). Oxygen and nitrogen atoms are drawn in red and blue, respectively. This Figure and Figures 5–8 are drawn with XtalView.64

Protein–lipid interactions at low pH In the neutral purple form, the glycolipid located in the central part of the trimeric structure takes an ordered structure by interacting with several polar residues (Thr67, Tyr69, Trp80 and Lys129). The conformation of this lipid alters greatly when a large structural change in the extracellular half of helix C is induced by acidification. Among residues in helix C, the residues (i.e. W80 and F88) extruding towards the center of the trimeic structure undergo ˚ displacements). Such large large movements (O3 A movements are allowed probably because the glycolipid has flexible phytanyl chains. Although one of its phytanyl chains interacting with helix B scarcely moves upon acidification, other parts of this lipid move very largely (Figure 7). Its head group, which is faintly visible in the density map, is suggested to have multiple conformations at low pH. But, as the head group of this lipid interacts with two monomers at least in the neutral purple form, the individual protein in the trimeric structure may not be able to behave independently of the adjacent monomers; i.e. cooperative conformational changes may take place during the purple-to-blue transition.

Besides the glycolipid mentioned above, four lipids have been identified in the neutral purple form; i.e. two phospholipids in the crevice between two monomers in the trimeric structure and two phospholipids between adjacent trimers. Among them, the lipids bound to the extracellular side of the membrane become disordered in the acid blue form. On the other hand, the lipids bound to the cytoplasmic side of the membrane undergo little conformational change upon acidification. Since most residues existing near the cytoplasmic membrane surface (e.g. K30, K40, K41, K159, K172, R175 and R225) retain their neutral conformations at low pH, it can be argued that the lipids in the cytoplasmic side have an important role in maintaining the trimeric bacteriorhodopsin–lipid complex, as has been suggested.34 Crystal structure of the alkaline purple form of bacteriorhodopsin ˚ resolutions X-ray diffraction data at 2.6–2.7 A were collected from light-adapted crystals that had been soaked at pH 9–10. The cell dimensions of these crystals were slightly larger than those

Figure 5. Acid and alkaline-induced structural changes in the proton release channel. 2FoKFc maps of the acid blue form (a) at pH 2, (b) the neutral purple form at pH 5.2 and (c) the high-pH conformer at pH 10, contoured at 1.2s and overlaid on their structural models. Carbon atoms are drawn in gold, purple or yellow; nitrogen, oxygen and sulfur atoms are in blue, red and green, respectively. The structure in (c) represents the high-pH conformer that would become the major component above pH 10.

488

Crystal Structures of Bacteriorhodopsin

Figure 6. Acid-induced structural changes in helix C. Carbon, nitrogen, oxygen and sulfur atoms in the acid blue form at pH 2 are drawn in cyan, blue, red and green, respectively. Amino acid residues and water molecules in the neutral purple form at pH 5.2 are in gray.

Figure 7. Acid-induced structural changes in glycolipids located at the central region of the trimeric structure. Carbon atoms in the lipids are drawn in gold, while carbon atoms in the protein are in purple, magenta or yellow. Nitrogen, oxygen and sulfur atoms are in blue, red and green, respectively. Upper panels, views along the membrane. Lower panels, views from the extracellular side. The 2FoKFc maps of the acid blue form at pH 2 (right) and the neutral purple forms at pH 5.2 (left) are contoured at 0.7s and 1.2s, respectively.

489

Crystal Structures of Bacteriorhodopsin

observed at pH 5.2 (Table 1). Irrespective of the crystal expansion, the structural data from a crystal at pH 9 were fitted well by the model of the neutral purple form after a rigid-body refinement. The result of the simulated-annealing refinement indicated that nearly all atoms, including water molecules, underwent no noticeable displacements between pH 5.2 and pH 9. But, small conformational changes were observed for a few residues in the BC loop (Gly72–Gln75) and the N terminal region (Thr5–Gly6). Since these residues are located in the protein–protein contact region, their small structural changes are likely to be responsible for the crystal expansion at high pH. When the crystal was alkalized to pH 10, several residues near the proton release complex were found to undergo noticeable conformational changes. Although the diffraction data from a crystal at pH 10 were fitted considerably well with the structure model at pH 9, the FoKFo difference map deduced from diffraction data at pH 9 and pH 10 clearly indicated re-orientation of the side-chain of Glu194 upon alkalization to pH 10 (Figure 8). Since the spectral data (Figure 1(c)) suggested that the alkaline transition took place at around pH 10, it seemed likely that the alkaline transition was partial at pH 10. To determine the high-pH conformation that would become the major component above pH 10, the contribution of the neutral purple form was subtracted from the diffraction data at pH 10. (In the crystal that had been soaked for 30 minutes at pH 10, about one-third of the protein was suggested to take the neutral purple conformer.) The result of the refinement (Figure 5(c)) shows that the high-pH conformer has the side-chain of Glu194 directed towards Tyr83. It also indicates that the re-orientation of Glu194 is accompanied by a significant movement of the indole ring of Trp189. As compared to the neutral purple form, the high-pH

conformer contains two additional water molecules between Glu194 and Glu204 (Wat641 and Wat642) and one between the indole NH of Trp189 and the peptide carbonyl group of Gly122 (Wat609). It should be pointed out that, besides residues near the proton release complex, no large structural change was induced upon the alkaline transition.

Discussion The previous crystallographic studies of the trans photocycle intermediates of bacteriorhodopsin have shown that the conformation of the proton release channel alters largely when the M intermediate (MRT) is generated at room temperature.10–13 When the structure of MRT generated at room temperature in the P622 crystal13 is compared with that of the acid blue form (Figure 9), many common features can be found: (1) the intra-helix hydrogen-bonding interaction between Phe88 and Leu92 is broken, leading to a pronounced movement of the extracellular half of helix C towards helix G; (2) the micro-cavity existing between the Schiff base and Asp85 is narrowed; (3) a water molecule (Wat602), which interacts with the Schiff base in the initial unexcited state of the neutral purple form, is squeezed out from the active site; (4) the guanidinium ion of Arg82 directs downward (towards the extracellular side); (5) the positions of Wat603, Wat605, Wat606 are nearly identical in these two structures. At first sight, the structural similarity between the acid blue form and MRT may be surprising, because the protonation state of the Schiff base as well as the retinal configuration is different for these two states (i.e. the Schiff base is protonated in the acid blue form, while it is deprotonated in MRT). But, this similarity provides an important insight into the structural role of

Figure 8. The FoKFo difference map associated with the alkaline transition (i.e. the density at pH 10 minus the density at pH 9), contoured at G3.6s (negative and positive densities are in gray and violet blue, respectively) and superimposed on the structural models of the high-pH conformer (gold, blue, and red) and the neutral purple form (green). Spheres represent water molecules.

490

Crystal Structures of Bacteriorhodopsin

Figure 9. Structural comparison between the acid blue form and the M intermediate (MRT) generated at room temperature.13 Carbon, nitrogen and oxygen atoms in the acid blue form at pH 2 are drawn in gold, blue and red, respectively. All atoms in MRT are in gray.

Asp85. It has been reported that Asp85 is protonated in both the blue form and the M intermediate, while it is deprotonated in the light-adapted state of the neutral purple form.17–19 Structural comparison of these three states indicates that the distance between the two aspartate residues in the active site (i.e. Asp85 in helix C and Asp212 in helix G) is reduced when Asp85 is protonated. This structural correlation suggests that the electric repulsion between Asp85 and Asp212, both of which are deprotonated in the initial unexcited state at neutral pH, is an important factor determining the mutual positions of helices C and G and, therefore, the volume of the micro-cavity at the active site and the distribution of water molecules. As the blue form has no water molecule in the vicinity of the Schiff base, its excitation may not be followed by directional movements of water molecules, which have been suggested to be crucial for the proton pumping activity.9 Since the protonation state of Asp85 remains unchanged through the N and O intermediates,41,42 it seems plausible that the conformation of the proton release channel found in MRT does not essentially alter in the late stage of the photocycle. If the structure of the active site is not different for the acid blue and the O intermediate, it would be expected that in the O intermediate too, the protonated Schiff base is hydrogen bonded to the carboxyl group of Asp85 or Asp212. Glaeser and his colleagues have recently reported the structures of the D85S mutant of bacteirorhodopsin, which is able to accommodate an anion (chloride, bromide or nitrate) in the vicinity of the Schiff base.20,43,44 In the presence of halide ions, this mutant exhibits purple. It has been shown that a halide ion binds to the active site and interacts directly with the protonated Schiff base.43 On the

other hand, the anion-free form of this mutant exhibits blue and, therefore, it is thought to correspond to the acid blue form of wild-type bacteriorhodopsin. They observed a large change in the tilt angle of helix E and less significant tiltings of helices A through D upon the purple-to-blue transition.43 Significant changes in the tilt angles of helices D and E are also observed in the acid transition of wild-type bacteriorhodopsin (Table 2). When the blue species of the D85S mutant and the acid blue form of wild-type bacteriorhodopsin are compared in detail, however, no strong correlation is found between these structures. Firstly, the blue species of the D85S mutant contains a water molecule in the vicinity of the Schiff base, while the Schiff base interacts directly with Asp85 in the Table 2. Relative tilts and RMS deviations between transmembrane helices of the neutral-purple form and the acidic-blue form Helix A B C-Cytoa C-Extraa D E F G

Tilt angle (deg.)

Displacement ˚) (A

˚) RMSD (A

1.42 1.56 0.57 2.39 3.49 2.98 2.37 0.75

0.28 0.06 0.07 0.45 0.16 0.12 0.06 0.04

0.57 0.48 0.26 1.32 1.02 1.08 0.78 0.28

Acid-induced changes in the tilt angles and displacements of the individual helices were calculated with Helix-Angles (Campbell, R. L. (2000). http://biophysics.med.jhmi.edu/rlc/work/scripts/ helix_angles). RMS deviations were calculated for Ca with SwissPDB viewer.66 a C-Cyto and C-Extra represent the cytoplasmic and the extracellular half, respectively, of helix C.

Crystal Structures of Bacteriorhodopsin

acid blue form of wild-type bacteriorhodopsin. This difference is explained by the volume change of the micro-cavity existing near the Schiff base, which is enlarged by deletion of oxygen and carbon atoms from the carboxyl group of Asp85. A more significant difference is seen in the structure of the proton release complex, which is here supposed to be comprised of Glu194, Glu204 and the nearby water molecules. In the anion-free form of the D85S mutant, the guanidinium ion of Arg82 contacts the pair of Glu194 and Glu204. Under their crystallization condition (i.e. at pH 5.6), the paired structure of Glu194 and Glu204 seems to possess a negative charge so as to attract the guanidinium ion of Arg82. This is not the case in the acid blue form of wild-type bacteriorhodopsin, in which the guanidinium ion of Arg82 interacts with SO2K 4 that is bound to a site(s) created by re-orientations of the carboxyl groups of Glu194 and Glu204. (It seems likely that the same binding of SO2K 4 would be reproduced if the D85S mutant is acidified by sulfuric acid.) These glutamate residues are probably protonated in the acid blue form but, owing to the bound anion, the total net charge in the protein interior appears to be negative; i.e. two positive charges (the protonated Schiff base and Arg82) versus three negative charges (Asp212 and SO2K 4 ). Since Glu194 and Glu204 are reported to be deprotonated in the O intermediate,41,42 the charge distribution is not essentially different for the acid blue form and the O intermediate. From this similarity, it may be argued that the acid blue form mimics the O intermediate. However, it is unlikely that binding of an anion to the proton release channel takes place during the proton pumping cycle. As a matter of fact, the rate of the purple-to-blue transition or its reverse transition is much lower (tw 10K2–10 seconds) than the transition rate from the O intermediate to the initial unexcited state (twa few milliseconds).45 The slow rate of the purple-to-blue transition (or its reverse reaction) may be due to a large energy barrier for detachment (or binding) of an anion from (to) the proton release channel. Indeed the BC loop was found to be disordered in the acid blue form. It is possible that a large fluctuation of the BC loop is necessary for entrance of SO2K 4 into the proton release channel. To explain the multiple kinetics of the purple-to-blue transition or its reverse reaction, however, we may also have to take account of re-arrangement of lipid molecules during the purple-to-blue transition (Figure 6). Since the intra-trimer interactions are mediated by lipid molecules, it is likely that a conformational change of one subunit affects the structures of the other subunits in the same trimeric structure. The result of pH titration (Figure 1) suggests that the purple-to-blue transition accompanies cooperative protonation of two residues. One of them can be assigned to Asp85 or Asp212, because their mutual distance is reduced significantly in the acid blue form. This assignment is not contradictory to earlier studies suggesting that protonation of Asp85 is the

491 major cause of the acid-induced color change.17–19 But, the present result does not exclude the possibility that one proton is shared by Asp85 and Asp212 in the acid blue form. With respect to the second residue that is protonated simultaneously with Asp85 (or Asp212), a likely candidate is the proton release complex. It should be noted that in the neutral purple form, the carboxyl group of Glu194 makes a direct contact with the carboxyl group of Glu204. This conformation has been observed in any 3D crystals of wild-type bacteriorhodopsin.40,46–48 The paired structure of Glu194 and Glu204 can be stabilized by sharing one proton (i.e. by a low-barrier hydrogen-bonding interaction49) or by migration of hydronium ion within the proton release complex.50 It has recently been reported that the proton of the proton release complex is delocalized over a water cluster around Glu194 and Glu204.51 Although the stabilization mechanism of the paired structure of glutamate residues has not yet been established, we can argue that proton uptake by the proton release complex at low pH (i.e. protonation of both the glutamate residues) destabilizes the paired structure of Glu194 and Glu204, leading to opening of the proton release channel and an increased accessibility of proton to Asp85. This argument is supported by the recent observation that replacement of four extracelluar glutamate residues (including Glu194 and Glu204) by neutral residues results in an increased exposure of the extracellular region to the solvent, which leads to high accessibility of hydroxylamine to the retinal Schiff base.52 It is also consistent with the previous observation that the cooperative nature observed for the purple-to-blue transition disappears when either Glu194 or Glu204 is replaced by a neutral residue.53 It is worthwhile to note that this cooperativity disappears also when Arg82 is replaced with lysine or histidine.54 Since the side-chain of this residue is shown to undergo a pronounced structural change during the purpleto-blue transition (Figure 5), it is very likely that Arg82 plays an important role in mediating a strong coupling between the proton release complex and Asp85, as has been suggested by the structural analysis of the M intermediate.10–13 The diffraction data from alkalized crystals show that the proton release complex undergoes a different type of structural change at around pH 10. It was shown here that the transition from the neutral form into the high-pH conformer is accompanied by re-orientation of the carboxyl group of Glu194 towards Tyr83 and by incorporation of two water molecules between Glu194 and Glu204. This result is consistent with the previous report that the proton release complex loses a proton with a pKa of 9.7.31 Similar re-orientation of the side-chain of Glu194 towards Tyr83 has been shown to take place upon formation of MRT.13 But, unlike the structural change initiated by the photoisomerization of retinal, the alkali-induced structural change accompanies a limited movement of the peptide backbone of Arg82. Due to this

492 restriction, the side-chain of Arg82 cannot move enough to make a salt-bridge with Glu204. It appears that the driving force causing re-orientation of Glu194 is different for the alkali-induced transition and the formation of the M intermediate. In the latter case, a large structural distortion in the FG loop, which is caused as a result of sliding of helix G, has been suggested to induce re-orientation of Glu194.13 In the alkali-transition, on the other hand, a likely driving force is an electric repulsion between Glu194 and Glu204, which becomes significant when both of them are deprotonated. It has been reported that the fast rising component of M becomes significant when the pH increases above pH 10.30 This phenomenon can be explained readily because the energy barrier for re-arrangement of the hydrogen-bonding network in the proton release channel becomes smaller when the paired structure of Glu194 and Glu204 is already broken in the initial unexcited state. The purple-to-blue transition can be induced by deionization of purple membrane and its reverse color change occurs upon addition of Ca2C or other cations to the deionized membrane.21 The present result excludes the previous argument that the color change is due to binding of a divalent cation to the active site, but it does not necessarily rule out the possibility that there is a specific binding site near the membrane surface. By analyzing the interaction between Mn2C and a nitoxyl radical probe bound to several mutants, Eliash et al. have shown that the highest-affinity binding site of Mn ion is located at ˚ from the spin label to residue 74C in the BC 9.8 A loop.26 It is possible that binding of a divalent cation to a site located near the BC loop contributes to enforce the antiparallel b-sheet seen in the BC loop, which is disordered in the acid blue form. It has been reported, on the other hand, that the purple-toblue transition caused by deionization is missing in lipid-depleted purple membrane.55 Their observation seems to suggest that the lipid–protein interactions affect the equilibrium between the purple form and the acid blue form. It is shown here that the glycolipid located in the central part of the trimeric structure undergoes a large structural change during the purple-to-blue transition. Since the head group of the glycolipid is located near the BC loop, it is possible that this lipid is involved in forming the cation binding site discussed above. The efficiency of light energy conversion by bacteriorhodopsin is proportional to an electrochemical potential of protons that the protein generates across the cell membrane. To design a proton pump with a high efficiency of energy conversion, one needs to understand how the active conformation of the protein is maintained in the presence of a pH gradient and membrane potential. From the observation that the neutral purple form is stable between pH 4 and pH 9, it may be argued that wild-type bacteriorhodopsin is capable of maintaining the active conformation in the presence of a pH gradient as large as five pH units. The

Crystal Structures of Bacteriorhodopsin

stability of the neutral form seems to come partly from the strong hydrogen-bonding interaction between the two glutamate residues (i.e. Glu194 and Glu204) existing near the extracellular membrane surface. It is noteworthy that the paired structure of these glutamate residues is preserved in archaerhodopsin, a light-driven proton pump isolated from Halorubrum sp. aus-1.56 It is conceivable that this structure has been acquired to stabilize the neural purple form and therefore to maximize the efficiency of energy conversion. It is noteworthy, on the other hand, that the paired structure of the glutamate residues is missing in proteorhodopsin, a retinal protein recently discovered in eubacteria.57 Proteorhodopsin is able to pump protons at high pH but it undergoes a significant color change at around neutral pH,58 implying that this protein may not be able to generate a large pH gradient. It is interesting to ask whether this new protein has the same physiological function as archaeal proton pumps.

Materials and Methods Protein purification and crystallization Purple membrane was isolated from Halobacterium salinarum JW3 and purified as described.59 Crystals were grown by the membrane fusion method as described,33 with slight modifications. Briefly, purple membrane sheets (5 mg/ml) were vesicularized at 32 8C in the presence of 2.5 mg/ml of octylthioglucoside, 1.0 M ammonium sulfate, 0.16 M NaCl, 12% (w/v) trehalose, 0.04% (w/v) NaN3 and 0.08 M sodium citrate (pH 5.2).60 The resultant vesicular assemblies of bacteriorhodopsin were concentrated at 10 8C by the sitting-drop, vapordiffusion method using a reservoir solution containing 1.9–2.0 M ammonium sulfate. Incubation for three to six months yielded hexagonal crystals exhibiting birefringence. For investigation of pH-induced structural changes, the mother solution was firstly replaced with a solution containing 2.1 M ammonium sulfate and a saturated concentration of octylthioglucoside and then the crystals were acidified with sulfuric acid or alkalized with sodium hydroxide in the presence of 2.1 M ammonium sulfate. Measurements of absorption spectra and absorption kinetics Absorption spectra of the acidified or alkalized crystals were measured using a micro-spectrophotometer, in which monochromatic light from a double monochromator (Shimadzu UV350A) was focused on a single crystal by a combination of quartz lenses and two diaphragms in a home-made microscope and the measuring light passing through the crystal was focused again on a pinhole attached before a photomultiplier tube.61 Absorption spectra were corrected using the following equation: AZKðlogðIsample K ID ÞK logðIblank K ID ÞÞ, where Isample and Iblank are photo-currents measured in the presence and the absence of a crystal, respectively, and ID is the dark current that was measured when the measuring light was blocked at the entrance of the microscope. For absorption measurements at various pH values,

493

Crystal Structures of Bacteriorhodopsin

crystals adhered to a lower glass of the crystallization kit were soaked in an acidic or alkaline medium containing 2.1 M ammonium sulfate. This lower glass was mounted in the micro-sepctrophotometer and its position was adjusted so that the measuring light passed through the center of a single crystal. Each absorption spectrum of the light-adapted crystal was recorded a few minutes after the soaking solution was exchanged by a new solution whose pH had been adjusted with sulfuric acid or sodium hydroxide. For absorption and diffraction measurements at cryogenic temperature, a single crystal that had been soaked in an acidic or alkaline medium containing 2.1 M ammonium sulfate and 30% (w/v) trehalose was picked up with a cryo-loop and flash-cooled with liquid propane (at its melting temperature). Absorption spectra were measured after a frozen crystal was mounted on a goniometer head attached to the micro-spectrophotometer, by which the crystal orientation was adjusted so that its c-axis became parallel to the measuring light beam. The temperature of the crystal was controlled in a flow of cold nitrogen gas from a cryostreamer (Oxford CC-12). Data collection and scaling X-ray diffraction measurements were performed at the beamlines SPring8-BL38B1 and -BL44B2, where a frozen crystal kept at 100 K was exposed to a monochromatic ˚ with an X-ray flux rate X-ray beam at wavelength of 1.0 A of 1!1012–2!1012 photons/mm2 per second. Diffraction data were collected using a CCD detector (Rigaku Jupiter or MarResearch marccd165), with an oscillation range of 18 and an X-ray flux of 4!1013 photons/mm2 per image. Indexing and integration of diffraction spots were carried out with Mosflm 6.1.62 The scaling of data was done using SCALA in the CCP4 program suites.63 Structural refinement of the acid blue form and the alkaline purple form Crystal structures at pH 4, 5.2 and 9 were determined on the assumption that only the trans isomer is contained in the crystal. In structural analyses of the acid blue form (i.e. crystal structures at pH 2–3), on the other hand, the compositional heterogeneity (i.e. co-existence of the trans and the 13-cis isomers) was taken into account. Firstly, trial models in which the retinal takes either the all-trans or the 13-cis, 15-anti configuration were built on the basis of the 2FoKFc map64 and refined using CNS.65 Secondly, the two trial models were combined to build a doubleconformation model in which the two isomers are equally contained. Alternate simulated-annealing refinements of the double-conformation model resulted in an Rcryst of 23.9% and an Rfree of 28.7% (Table 1). But, the structural difference between the two conformers was small, suggesting that the result of the double-conformation refinement may not be so meaningful. In this study, therefore, the trans conformer refined by the singleconformation refinement was used for structural comparison with the neutral purple form. The structure of the major component at pH 10 was built on the basis of the FoKFo difference map between diffraction data at pH 10 and those at pH 9. Its refinement was performed on the assumption that the crystal at pH 10 contained a 1:2 mixture of the neutral purple form and the high-pH conformer.

Protein Data Bank accession codes Crystallographic coordinates of acid blue and alkaline purple forms of bacteriorhodopsin have been deposited in the Protein Data Bank (accession codes 1X0I and 1X0K, respectively).

Acknowledgements This work was supported by grants-in-aid from the Ministry of Education, Science, and Culture of Japan and partly by the National Project on Protein Structural and Functional Analyses.

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Crystal Structures of Bacteriorhodopsin

29.

30.

31.

32.

33.

34.

35. 36.

37.

38.

39.

40. 41.

42.

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Edited by R. Huber (Received 24 March 2005; received in revised form 5 June 2005; accepted 8 June 2005) Available online 28 June 2005