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* Graduate Group in Biophysics, University of California, Berkeley, California 94720; Life Sciences Division, Donner Laboratory, Lawrence Berkeley National Laboratory, Berkeley, California 94720; Department of Molecular and Cell Biology, Stanley/Donner ASU, University of California, Berkeley, California 94720-3206; and Department of Bioengineering, University of California, Berkeley, California 94720
Correspondence: Address reprint requests to Robert M. Glaeser, E-mail: rmglaeser{at}lbl.gov.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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, 1995; Soulimane et al., 2000; Tsukihara et al., 1996; Xia et al., 1997; Zhang et al., 1998), these include the sarcoplasmic Ca2+ ATPase (Toyoshima et al., 2000), halorhodopsin (hR) (Kolbe et al., 2000), and bacteriorhodopsin (bR) as well as some of its mutants (Belrhali et al., 1999; Luecke et al., 2000, 1999a,b; Rouhani et al., 2001). Although informative, these studies each lack key information necessary to fully understand the respective mechanisms of these pumps. For example, progress made on the structure of Ca2+ ATPase still shows the protein only in both its resting, substrate-free (Toyoshima and Nomura, 2002) and substrate-bound (Toyoshima et al., 2000) states. Even for bacteriorhodopsin, for which considerable information is available regarding conformational changes that occur during the pumping cycle (Facciotti et al., 2001; Lanyi and Schobert, 2002; Luecke et al., 2000, 1999a; Rouhani et al., 2001; Royant et al., 2000; Sass et al., 2000; Schobert et al., 2002), the question whether this protein is a proton pump or a hydroxyl-ion pump (Oesterhelt and Stoeckenius, 1973; Perkins, 1992) remains under discussion (Betancourt and Glaeser, 2000; Herzfeld and Lansing, 2002; Luecke, 2000).
Like wild-type bR, bR(D85S) contains an all-trans retinal chromophore bound via a protonated Schiff-base linkage to K216 on helix G. In wild-type bR, the ion pumping cycle begins with photoisomerization of the retinal to the 13-cis configuration. The cycle then progresses thermodynamically downhill through a series of spectroscopically identifiable intermediate states that ultimately return the protein to its resting state and the retinal to its initial all-trans configuration. As is wild-type bR, bR(D85S) is also a light-driven ion pump; however, the single amino acid substitution permits the protein to pump anions from the extracellular side of the membrane into the cell and unlike wild-type bR its catalytic cycle does not involve Schiff-base deprotonation (Brown et al., 1996; Sasaki et al., 1995).
As a means to gain more insight about the ion pumping process, we previously crystallized a halide-pumping mutant of bacteriorhodopsin, bR(D85S), in its halide-free state (Rouhani et al., 2001). Here, we describe the crystal structure of bR(D85S) with a bromide ion bound in its ground-state binding site. In conjunction with the previously determined structure of the halide-free state (Rouhani et al., 2001), this study reveals structural changes that occur during the initial substrate-binding step of halide-ion transport, and suggests some design principles that may apply more generally to other ion pumps.
Specifically, these results, in conjunction with the previously solved structures of intermediate states in the wild-type photocycle, suggest that bR(D85S) facilitates active transport via a four-step process. First, the substrate must diffuse into the binding site past a dynamic gate consisting of the side chain of R82. Second, the halide ion makes an ion pair with the protonated Schiff base in the binding site, and in so doing induces a conformational change that latches the dynamic gate in a closed conformation. Third, the energy of the absorbed photon forces a small displacement of the protonated Schiff base, through isomerization of the retinal chromophore. The resulting charge separation raises the potential energy of the substrate to a level higher than it would have on the opposite side of the membrane and thus leads to ion transport. Finally, the protein relaxes back to its ground state.
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MATERIALS AND METHODS |
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and solubilized in 1.2% octylglucoside overnight. The solubilized membrane was then concentrated to 15 mg/ml. A quantity of 10 µl protein was then used to hydrate 10 mg dry monoolein in a 0.2 ml polymerase chain reaction (PCR) tube for each crystallization setup. This mixture was then centrifuged at 10,000 x g at 22°C for 3 h. After centrifugation, 100 µl of the mother liquor consisting of 100 mM sodium acetate, pH 4.6, 200 mM KCl, and 10% PEG 4000 was layered on top of the hydrated gel. Crystals grew to a final size of 5 µm x 30 µm x 120 µm in the period of 3 weeks. Aliquots of the gel were then transferred to a lipase solution (Nollert and Landau, 1998) consisting of 100 mM sodium acetate, pH 4.6, 200 mM KCl, and 50 mg/ml lipase, and allowed to incubate overnight. Individual crystals were then transferred to a solution containing no halide, 100 mM sodium acetate, pH 4.6, and 10% PEG 4000 to remove the bound chloride, after which they were transferred sequentially to cryoprotectant solutions containing 100 mM sodium acetate, pH 4.6, 1.25 M KBr, and 12%, 16%, 20%, and 25% PEG 4000, respectively.
Data collection and model building
Crystals were flash frozen in liquid nitrogen and irradiated on beamline 8.3.1 of the advanced light source (ALS) with a 30-µm collimated x-ray beam. Five crystals were used to collect the halide data set. The halide-bound crystals belong to the C2221 space group with average unit cell dimensions in angstroms (Å) of a = 52.50, b = 121.33, and c = 73.87, whereas the previously described halide-free crystals have a unit cell of a = 51.80, b = 121.30, and c = 85.70. A Quantum 210 CCD detector was used to collect images. Data reduction was performed by the Elves scripts (Holton, 2002). Diffraction data were integrated with MOSFLM (Leslie, 1992) and subsequently scaled with SCALA (Collaborative Computation Project, 1994). Molecular replacement using 1KGB (Facciotti et al., 2001) as the starting search model, without the retinal, water, and lipid molecules, was performed by the program CNS Version 1.1 (Brunger et al., 1998). Refinement with CNS and model building using the program O (Jones et al., 1991) together with annealed simulated omit, |Fo| - |Fc|, and 2|Fo| - |Fc| maps reduced the values of R and Rfree to their final values (Table 1). The final model was analyzed with PROCHECK (Laskowski et al., 1993) and MOLPROBITY (Richardson, 2002) and exhibits good stereochemistry.
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RESULTS |
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; Sasaki et al., 1995; Tittor et al., 1997), that residue D212 becomes protonated upon halide binding. This protonation, although necessary for binding of a halide ion, is energetically costly at pH values above the pKa of D212, which is thought to be 6.9 in bR(D85T) when measured in saturating sodium chloride (Tittor et al., 1997).
The halide binding site of bR(D85S) differs from the halorhodopsin-binding site, where D238, the homolog to residue D212 in bacteriorhodopsin, remains negatively charged when a halide ion is in the binding site. This is because the halorhodopsin aspartate does not interact directly with the halide ion, but serves instead as a counterion to the Schiff base and indirectly, through an ordered water molecule, to R108 (Kolbe et al., 2000). Another difference is that in bR(D85S) only one water molecule interacts with the bound bromide ion, whereas three water molecules had been reported in the binding site of halorhodopsin (Kolbe et al., 2000). These differences in polar interactions within the binding site, and the need to protonate D212, are likely to account for the lower halide binding affinity, over a narrower pH range, of the D85S mutant relative to halorhodopsin.
Structural rearrangements caused by halide binding
A comparison of the halide-bound and halide-free (Rouhani et al., 2001) forms of bR(D85S) reveals that, after bromide binding, the structure on the cytoplasmic side remains as it was in the halide-free form. On the extracellular side, however, bromide binding induces a variety of side-chain movements (Fig. 2 A). One of the most notable movements involves the side chain of R82, which is best modeled with two distinct and nearly equally occupied conformations (Fig. 3). In what we presume is the functional conformation, CB-CD-CG swing downward in concert with a small shift in backbone position, whereas the guanidinium group itself rotates to a more upward-facing orientation, reminiscent of the conformation adopted in the ground state of wild-type bacteriorhodopsin. In the second rotamer conformation, the side chain of R82 adopts a downward facing structure reminiscent of that adopted in the halide-free form. Residues W138 and W189 also change rotamer conformations between the halide-free and halide-bound states by flipping about 
1 and about both 1 and 2, respectively, resulting in a local reorganization of the interhelical hydrogen bonds. In the bromide-bound structure, the OH of Y83 (helix C) accepts a hydrogen bond from the NE1 of W189 (helix F), whereas in the halide-free state Y83 forms a hydrogen bond with the backbone carbonyl of I119 (helix D). The reorganization of Y83 and the formation of its hydrogen bond with W189 is facilitated by rotations about both 1 and 2 for W189, during which the residue breaks a 
-bond with the indole ring of W138. These changes in hydrogen bonding allow W138 to break a hydrogen bond with the carbonyl oxygen of G122 (helix D) and rotate about 1 to form a new hydrogen bond with the carbonyl oxygen of P186 (helix F). The net result is that substrate binding eliminates two hydrogen bonds that helix D makes with its neighboring helices, while establishing one new cross-helix-bundle hydrogen bond between helices C and F. These rotamer switches involving R82, W138, and W189 in the bromide-bound form of bR(D85S) regenerate the same bonding arrangement that occurs in the functionally equivalent ground state of wild-type bR.
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DISCUSSION |
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), an energy-driven cycle causes the protein to alternate between inward and outward facing conformations. In its simplest form, one might imagine that these open states provide direct access to the binding site via a static, solvent-accessible tunnel. However, in neither the bromide-free nor the bromide-bound structures is there a true, solvent-accessible tunnel that leads all the way to the binding site (Fig. 4, A and B). Instead, the side chains of R82 and its immediate neighbors cap the short tunnel that does exist. Our analysis suggests, therefore, that refinements need to be made in how the Jardetzky model is used to explain the ability of bR(D85S) to pump anions. We suggest, first of all, that entry of the substrate anion must rely on a fluctuating gate that opens transiently, providing access of substrate to its binding pocket by a mechanism similar to that which permits binding of molecular oxygen to hemoglobin and myoglobin (Karplus and McCammon, 1986).
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). As was shown by Honig and Hubbel (1984), the cost of burying an ion pair in a region of low-dielectric constant is no greater than that of burying a single ion. Thus, the energetic cost associated with transporting an anionic substrate out of the high-dielectric, aqueous medium may have already been prepaid by burying R82 during the protein-folding step.
The use of the buried formal charge of R82 to prepay the Born energy of the anionic substrate during transit to the binding site contrasts with the mechanism used by ion channels to overcome the Born energy barrier for ion transport. The structures of the potassium and chloride channels (Doyle et al., 1998; Dutzler et al., 2002) have revealed that polar peptide-backbone residues of the "pore-loop" sequence are used, rather than buried counterions, to create an environment for a dehydrated ion that is almost equivalent, energetically, to that of its fully hydrated state (Berneche and Roux, 2001).
A key difference between these two mechanisms for lowering the Born energy barrier is that the pore design lacks any way to prevent backflow of the transported ion, and thus it is compatible only with passive (downhill) diffusion of substrate ions. On the other hand, a design that uses rotamer switching of a charged side-chain residue, R82 in the case of bR(D85S), as a flexible gate would allow substrate-induced conformational changes to latch the gate in a single, frozen conformation and thereby prevent backflow during subsequent steps in the pumping cycle. In halorhodopsin, the homologous residue, R108, is also known to block extracellular access to the binding site and would, therefore, also need to move to allow anion access to the binding site (Kolbe et al., 2000). Thus, R108 in halorhodopsin could also serve as a dynamic ion-pairing gate. In addition, the recently published structure of the Ca2+ ATPase in the substrate-free state (Toyoshima and Nomura, 2002) also reveals an access tunnel that is capped by a potential counter ion, in this case the carboxyl group of E309. Upon Ca2+ binding, E309 appears to switch rotamer conformations (Toyoshima et al., 2000) in a way that again suggests this residue plays a role that is functionally equivalent to what we propose for R82 in bR(D85S).
Although we suggest that the bromide binding induced helix repacking could functionally close the extracellular side of the protein, the repacking that is observed does not return all of the helices to the positions occupied in the resting state of wild-type bR. We suspect that the observed repacking thus represents an incomplete closure of the extracellular side in which dual occupancy of rotamer conformations can still exist for the gate, R82. As a result substantial backflow of halide ions could still occur in this mutant. The poor pumping efficiency of bR(D85S), accompanied by a measured component of backflow of chloride ion (Kalaidzidis and Kaulen, 1997), may thus be explained by the differences in helix packing that remain between bR(D85S) and wild-type bR in their respective resting states. The inability of bR(D85S) to completely close the extracellular side is not surprising since a single protein engineering step, (mutating D85 to S) can not be expected to optimize all aspects of halide-ion transport to the extent that evolution has done for halorhodopsin.
The structures of bR(D85S) and hR both reveal that charge-charge interaction is a key component of ion binding in these pumps. The same mechanism of ion binding is also seen in the Ca2+ ATPase, a cation pump, where there is a direct charge-charge interaction between substrate and a cluster of aspartate and glutamate carboxyl groups (Toyoshima et al., 2000). We suggest that direct charge-charge interactions in the binding site could serve an important mechanistic function for ion pumps, beyond merely trapping the substrate in the binding site. The use of externally supplied energy to switch either the position or the orientation of a side-chain counter ion (or in our case the protonated Schiff base of the retinal group), by even a small amount, can raise the potential energy of the substrate ion and even draw it on to the next site.
As a concrete example, we can model what the consequence might be after isomerization of the retinal group in the bR(D85S) mutant by assuming the structural changes to be similar to what occurs in the M intermediate of bacteriorhodopsin. This assumption will only be valid if the reaction coordinate of the halide-pumping mutant, when expressed in terms of a progression of structural changes in the protein, remains similar to that of wild-type bacteriorhodopsin. This is similar to the assumption made when interpreting the structural features of trapped photointermediates of proton-pumping mutants within the context of the wild-type photocycle (Luecke et al., 2000, 1999a).
It is generally accepted that the photon energy is initially stored in the retinal chromophore in the form of torsion of the normally planar polyene chain (Braiman and Mathies, 1982; Lanyi and Schobert, 2002; Schobert et al., 2002; Smith et al., 1985), and that the Schiff-base nitrogen atom is gradually displaced from its original position as this torsion is relaxed (Facciotti et al., 2001; Luecke et al., 1999a). We have therefore used the atomic model of a late M intermediate (Luecke et al., 1999a) to estimate the amount of charge separation that might occur between the protonated Schiff base and the bromide anion in the D85S mutant. Alignment of the late M structure to our atomic model of the bromide-bound resting state of bR(D85S) was carried out in two ways. In the first, the RMSD between the retinal ring of the two models was minimized, whereas in the second, the RMSD between selected CA atoms of the two models was minimized.
According to the first method of alignment, the distance between the halide anion (were it to remain fixed) and the midpoint of the N–H bond of the Schiff-base cation would increase to 
1.4 times their separation in the ground state, while in the second method of alignment the distance increases by a factor of 1.6 (Fig. 5). If we take even the smaller amount of displacement, the Born energy cost for this small amount of charge separation would actually be 40% of the favorable Coulomb interaction that is involved in forming the initial ion pair. The bromide ion could avoid the high cost of this charge separation by moving to a position that is closer to the displaced Schiff-base cation, of course, ultimately moving to the cytoplasmic side of the retinal group. Even then, however, the anion would remain at a higher potential energy than in the resting state, for it would no longer be solvated in this new position by the S85 or D212 polar groups.
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). iii), The substrate must then traverse the remaining portion of the protein. In the case of bR(D85S), it is reasonable to suppose that a repacking of helix F and helix G occurs on the cytoplasmic side of the protein, producing an N-like intermediate similar to that observed with the F219L mutant (Vonck, 2000) or the constitutively N-like structure of the D96G/F171C/F219L triple mutant (Subramaniam and Henderson, 2000). The helix packing observed in the N-like structures is believed to increase the access of water molecules to the cytoplasmic side of the Schiff base, and thus may open up a path for the diffusion of the halide ion out to the cytoplasmic side of the membrane. The suggestion that the extracellular side must open to create an exit for the halide ion has also been proposed as a requirement for halorhodopsin's pumping cycle (Kolbe et al., 2000). iv), After the substrate has left, the protein will be free to return to its ground-state conformation.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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This work was supported in part from a National Institutes of Health grant GM51487.
Submitted on December 4, 2002; accepted for publication March 17, 2003.
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H. Jang, P. S. Crozier, M. J. Stevens, and T. B. Woolf How Environment Supports a State: Molecular Dynamics Simulations of Two States in Bacteriorhodopsin Suggest Lipid and Water Compensation Biophys. J., July 1, 2004; 87(1): 129 - 145. [Abstract] [Full Text] [PDF]
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Z. Balint, M. Lakatos, C. Ganea, J. K. Lanyi, and G. Varo The Nitrate Transporting Photochemical Reaction Cycle of the Pharaonis Halorhodopsin Biophys. J., March 1, 2004; 86(3): 1655 - 1663. [Abstract] [Full Text] [PDF]
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. Distances to the key residues with which the ion interacts are drawn with green dashed lines. The atoms and bonds shown in gold belong to the retinal. The atom at the binding site was initially modeled by a water molecule. However, after refinement, the B value for the fully occupied molecule was reduced to an unphysical value of 0.25. A bromine atom was subsequently used to model the density feature and refined to a B value of 29.5 and an occupancy of 74%. Another bromide ion (shown in 
