The 1.9 A X-ray Structure of a Closed Unliganded Form of the Periplasmic Glucose/Galactose Receptor from Salmonella typhimurium*

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1 THE JOURNAL OF BIO~ICAL CHEMISTRY by The American Society for Biochemistry and Molecular Biology, Inc. Vol. 269, No. 12, Issue of March 25, pp , 1994 Printed in U.S.A. The 1.9 A X-ray Structure of a Closed Unliganded Form of the Periplasmic Glucose/Galactose Receptor from Salmonella typhimurium* Maria M. FloccoS and Sherry L. Mowbray$ (Received for publication, September 14, 1993, and in revised form, January 5, 1994) From the Department of Molecular Biology, Swedish University of Agricultural Sciences, Uppsala Biomedical Center,.. Box 590, S Uppsala, Sweden The three-dimensional structure of a ligand-free closed formof the glucose/galactose binding protein from Salmonella!yphimurium has been determined at a resolution of 1.9 A. The crystallographic R-factor for the refined structure is 17.9%. The model contains all the atoms of the SO9 residues of the protein sequence, a calcium ion, and 174 water molecules. The root mean squart (r.m.s.) deviations for the whole molecule are: A for bond lengths and 2.44 for bond angles, indicating a good stereochemistry for the model. This struc- ture shows that the protein is able to close in the absence of ligand, adopting a conformation similar to the liganded form but slightly more open. Water molecules satisfy the hydrogen bonding ability of the hydrophilic side chains of the binding site in a manner which is reminiscent of the sugars hydrogen-bonding patterns. Since packing forces are weak, the crystallization event is unlikely to trigger a change from an open to a closed conformation. Instead, the latter must be one of the species in equilibrium in solution which is selected by packing in the crystal lattice. * This work was supported in part by Swedish Natural Science Research Council grants K-KU (to S. L. M.) and K-KU (to M. M. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked aduertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The atomic coordinates and crystallographic data have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, ivy $ Supported in part by the Swedish University of Agricultural Sciences. 8 TO whom correspondence and reprint requests should be addressed. The abbreviations used are: GBP, the glucose/galactose-binding protein; r.m.s., root-mean-square; R-factor = H 11 Fobs I - I FCalc I( / I Fobs I. a resolution of 1.7 A (Zou et al., 1993), and the structure of the complex with glucose is known to a resolution of 2.4 A (Mowbray et al., 1990). The structure of a very similar GBP from Fherichia coli bound to glucose is known to a resolution of 1.9 A (Vyas et al., 1988). The protein has two similar Culp domains linked by a hinge made up of three polypeptide strands. The ligand is almost completely buried in a binding site located in the cleft between the two domains. There is evidence from a number of sources, including fluorescence emission (Boos et al., 1972; McGowan et al., 19741, fluorescence energy transfer (Zukin et al., 1977, 19791, small angle scattering (Newcomer et al., 19811, and NMR spectroscopy (Luck and Falke, 1991a, 1991b) that conformational changes occur upon ligand binding to the periplasmic proteins. The x-ray structures of the unliganded forms of four related binding proteins, L-leucine- and leucine/isoleucine/valine-binding proteins (Sack et al., 1989a, 1989b1, maltose-binding pro- tein (Sharff et al., 1992), and lysine/arginine/ornithine-binding protein (Oh et al., 1993) reveal a more open conformation for these species. The latter two proteins have also been studied in closed liganded forms (Spurlino et al., 1991; Oh et al., 1993). However, the details of the mechanism of activation of GBP Soluble proteins found in the periplasm of Gram-negative remain unclear and the three-dimensional structure of the bacteria serve as the primary receptors for transport and in open form of GBP is unknown. some cases for chemotaxis in response to compounds such as In this article we report a high resolution crystal structure of sugars, ions, and small peptides. The periplasmic receptors an unliganded closed conformation of the glucose/galactosebecome activated by binding the ligand directly and can then be binding protein from S. typhimurium. recognized by the appropriate membrane receptor for chemotaxis (Manson, 1992; Stewart and Dahlquist, 1987) or interact EXPERIMENTALPROCEDURES with the membrane proteins responsible for the transport of Protein purification was carried out as outlined in Mowbray et al. the ligand across the inner membrane. (1990). The endogenous sugar bound was removed by a modification of The glucose/galactose-binding protein, GBP, is the primary the technique described in Miller et al. (1980); the concentration of receptor for the mgl transport system (Anraku, 1968a); com- it guanidine HCl was increased to 3 M for the denaturation step and petes with the ribose-binding protein for the same membrane calcium was added to the renaturation buffer to give a final composition chemotaxis receptor (Strange and Koshland, 1976). GBP has a of 1 mm CaCl,, 10 mm Tris-HC1 at ph 7.4. Initial screening of the crystallization conditions for unliganded GBP was carried out following molecular mass of approximately 30 kda and binds glucose and galactose tightly, with Kd values of approximately M (Anraku, 1968b). The x-ray structure of GBP from Salmonella typhimurium bound to galactose has been solved and refined to 8931 the Sparse Matrix Sampling method (Jancarik and Kim, 1991) at room temperature. Hanging drops for the vapor diffusion technique were made by mixing equal amounts of the protein and the different crystallization solutions assayed. To achieve a crystal size suitable for x-ray structural studies, the small crystals obtained in the screening experiments were used as seeds for the macroscopic seeding technique performed at 4 C essentially as explained in Mowbray and Petsko (1983). Diffraction data were collected at room temperature using a R axis IIc imaging plate detector mounted on a Rigaku rotating anode x-ray generator (CuK, radiation, 50 kv, 180 ma, graphite monochromator). The software of the R axis IIc system (Sato et al., 1992) was used for control of the data collection and processing of the oscillation frames to obtain a set of reflections. Crystallographic refinement was carried out using the program X- PLOR (Briinger et al., 1987). The parameters used for the Powell minimization and temperature-factor optimization routines of X-PLOR as well as the refinement protocol were the same as described in Zou et al. (1993). Manual refitting of the model to the electron density maps

2 8932 Unliganded Closed TABLE I Statistics for the x-ray data set The crystals have the symmetry of the space group C2 with unit cell dimensions a = A, b = A, c = A, p = 123.9". The R-merge value reported by the averaging program of the R axis IIc data processing software was 4.59%. The cell dimensions for tbe galactosebound GBP crystals are a = A, b = A, c = A, p = 123.2". Resolution No. of unique range reflections Completeness in shell Completeness cumulative Average IldI) A % 9% during refinement was done with the interactive computer graphics program 0 (Jones et al., 1990; Jones and Kjeldgaard, 1992). The real space fit feature of the program 0 (Jones et al., 1991) was used to assess the quality of fit of the model to the electron density. The least-squares option of the program 0 was used to align the unliganded GBP structure with that of the galactose-bound GBP. Further comparison of these two forms of the protein was accomplished by calculating least squares planes through the core p-sheets of domains 1 and 2 and evaluating the angles which relate them as described in Zou et al. (1993). Glucose J Galactose Receptor all the side chain atoms. There are no peaks in the IFobsI - I Fcalc I electron density map contoured at 3.5 u anywhere in the structure. Two small peaks in the binding cleft are observed when the contour level of the I Fobs I - I FcalE I map is lowered to 3.3 u. They cannot be accounted for by introducing more solvent molecules and are probably due to the fact that the water RESULTS network in the sugar-free binding site could be modeled in Protein Purification and Crystallization-Both the un- several alternative ways. The real space fit analysis gives an treated and the guanidine-treated purified protein gave a average correlation coefficient for all atoms of 0.894, indicating single band on a Coomassie Blue-stained SDS-gel electrophore- a good fit of the model to the electron density. sis. The native GBP gave a doublet on an isoelectric focusing gel The average temperature factor for pfotein atoms is 20.5 A" with bands at estimated PI values of 5.50 and 5.55 while the (18.8 A2 for main-chain atoms and 22.5 A2 for side chain atoms) guanidine-treated protein gave only a single band at an esti- and for solvent atoms is 35.5 A'. Residues 1 and 2 have the mated PI = Either protein gave a single band at PI 5.50 highestemperature-factor values in agreement with the when sugar was included in the isoelectric focusing gel. These results indicate that there is a change in the overall surface charge upon binding of sugar to GBP, and that the untreated protein is a mixture of sugar-bound and sugar-free species. Small single crystals were obtained from a GBP solution at poorer definition of the electron density observed in the maps and the low values for the respective correlation coefficients calculated in the real space fit analysis. The refined model exhibits little deviation from the ideal values for bond lengths and angles as well as Chedral angles. approximately 8 mg/ml protein (in the renaturation buffer) The bond lengt! r.m.s. deviations are A for the main with a crystallization solution consisting of 30% polyethylene glycol 4000, 0.1 M "is-hc1 buffer at ph 8.64, and 0.2 M sodium acetate. These crystals were used as seeds for macroscopic seeding with an initial concentration of polyethylene glycol in chain and A for the whole molecule, with no bond lengths deviating by more than 0.06 A. The bond angle r.m.s. deviations are 2.40" for the main chain and 2.44" for the whole molecule. Overall Description of Three-dimensional Structure and the reservoir of 18% (buffered with 0.1 M Tris-HC1, ph 8.64). Comparison with Liganded GBP Structure-The GBP molecule Stepwise increases of 3% gave a final concentration of polyeth- is organized in two similar domains. Both the NH2-terminal ylene glycol of 30%. The crystal used in data collection grew to and the COOH-terminal domains are composed of a core of 0.21 x 0.10 x 1.2 mm in about 4 weeks. Data Collection, Phasing, and Structure Refinement-A total parallel p-sheet flanked by two layers of a-helices (Fig. MI. Each domain is made up by two non-contiguous segments of the of 59,612 observations with I > dl) yielding 21,036 unique polypeptide chain. They are thus connected by three short segreflections within the resolution limits A were re- ments of amino acid chain referred to as the hinge. A cleft corded from a single crystal. Some statistics for the data set are between the two domains has been previously identified as the given in Table I. The crystals of unliganded GBP were nearly isomorphous with the previously studied galactose-bound GBP (see legend to Table I). Hence, all the non-hydrogen atoms of the protein (sugar was omitted), a calcium ion, and 153 water molecules of the latter were used as the starting model in the program X-PLOR. The initial R-factor was 42.8% for 20,142 reflections with Fobs > dfobs) in the resolution range A. At the first round of refinement, Powell minimization to convergence and optimization of B-factors lowered the R-factor to 18.4%. Subsequently, cycles of refinement were alternated with manual re- fitting of the model to the 2 IFobs I - I Fcalc I and I - I Fcalc I electron density maps (contoured at 1 and 3 u, respectively). After the first round of refinement both electron density maps confirmed that the sugar was absent. The shape of the electron density shown in Fig. 1 is clearly different from those observed in earlier studies with liganded proteins (Mowbray et al., 1990; Mowbray and Cole, 1992). Five water molecules were added to account for the observed electron density at the binding site. The nature of the binding site and the shape of the electron density were not consistent with other components of the crystallization medium. A total of six rounds of refinement involved modeling of the water network at the binding site, changes in the position of several other water molecules and minor modifications in the conformation of some of the side chains. Analysis of the Current Model-The structure of the unliganded GB: has been refined to an R-factor of 17.9% at a resolution of 1.9 A. The present model contains all the atoms of the 309 residues of the protein sequence, a calcium ion, and 174 water molecules. The 2 IFobsI - IFcalcI electron density map contoured at 1 u shows continuous electron density for all backbone atoms except for the amino terminus which has density only for the C atom. There is clear electron density for almost binding site for the sugars (Vyas et al., 1988; ZOU et al., 1993). A calcium binding loop (similar to the EF-hand motif (Vyas et al., 1988)) is present in the COOH-terminal domain. The three-dimensional structures of the galactose-bound and the glucose-bound forms of GBP from S. typhimurium are nearly identical. Alignment of their 309 C-a atoms gives an r.m.s. deviation of 0.15 A. The conformation of the unliganded GBP described here is very similar to the conformations of both liganded forms of the protein (see Fig. 2B). The backbones of the unliganded and of the galactose-bound GBPs can be superimposed with an r.m.s. deviation of A for the 309 C-a. The superposition of the NH2-terminal domains (residues and ) of the two species gives an r.m.s. deviation of A for 147 C-a atoms and that for the COOH-terminal domains (residues and ) has an r.m.s. deviation of A for 162 C-a atoms. Improved alignment of the

3 Closed Unliganded Glucose Galactose Receptor 8933 FIG. 1. Stereo diagram of the I Fobe I - I Fed= I electron density map (contoured at 3 u) at the binding cleft after the first round of refinement of the initial model (see "Data Collection, Phasing, and Structure Refinement'' under "Results"). All the residues involved in the hydrogen-bond network are shown. NHz-terminal domains using a cutoff of 0.3 A (as expdained in Mowbray (1992)) gives an r.m.s. deviation of A for 142 C-a atoms (residues 1,82, and do not meet th? cutoff criterion). For the COOH-terminal domains the 0.3 A cutoff tion (see Fig. 2B) clearly show a different relative rotation of yields an r.m.s. deviation of A for 164 C-a atoms (residues the domains for the unliganded protein. However, it is difficult and do not meet the cutoff criterion but the to associate specific bond rotations with it, largely because of adjacent residues and of the NHz-terminal the small changes involved. A comparison of the torsion angles domain do). Residues 257 and 293 are located at the hinge and the fragment between residues 150 and 153 is located at one of the lips of the binding cleft. Residues 82 and 308 are located in regions of crystal contact. The greater similarity in the conformations of the COOH-terminal domains with respect to the NH2-terminal domains was also observed in the comparison of the liganded GBPs from S. typhimurium and from E. coli (Zou et al., 1993). Both the numbers above and the conformations of the individual side chains indicate that the guanidine-treated GBP is in its native conformation. The lower r.m.s. deviations observed when either the NHzalong the entire C-a backbones (calculated with the program DSSP; Kabsch and Sander (1983)) of the unliganded and the galactose-bound proteins gives an average angular difference of 1.6", which is well below the uncertainty in the values calculated for these angles (-6") due to coordinate error. Although the galactose-gbp complex and the sugar-free GBP have been crystallized in the same space group, their cell constants are slightly different (see legend to Table I), and a few differences in the crystal contacts are observed. There are $2 interactions (distance A for Van der Waals and A for hydrogenbond interactions) for the whole protein in each case. The unliganded GBP makes 22 contacts in domain 1, 18 of which are terminal or COOH-terminal domains are aligned separately indicate that the angle of relative rotation of the two domains is different in the two forms of the protein. A method of measuring the relative orientation of the domains which exploits the limited flexibility and near planarity of the core P-sheets in the periplasmic binding proteins has been described in Zou et al. (1993) (see Fig. 2A ). By using this method it was found that the angle between the lines perpendicular to the least-squares planes defined through the core p-sheets of domains 1 and 2 (z and z' axes in Fig. 2 A ) is 83.5" and the angle between the straight lines along the respective central p-strands ( y and y' axes in Fig. 2 A ) is 199.8'. The first quantity reflects the fact that the plane of the core p-sheet of domain 1 is nearly perpendicular to that of domain 2; the two planes are approximately related by a rotation about the long axis of the molecule. This quantity is almost identical to the one found for the galactose- radius 1.5 A) of residues 152 and 153 increase by 11 and 10 A', respectively. These results as well as inspection at the graphics worksta- the same as in the galactose-bound form of the protein. All of the 20 contacts in domain 2 are also found in the liganded species. In the light of the results of the C-a backbone alignments reported above, the few differences observed in the crystal contacts of the two proteins do not seem to give rise to important local conformational changes, except perhaps in the neighborhood of residue 308. There is a hydrogen bond between Gln-308 and Thr-159 of a symmetry-related molecule in the liganded GBP which is lost in the sugar-free protein. In con- trast, the crystal contacts around Gly-82 are nearly identical in the two structures, although this residue could not be matched within the 0.3 A cutoff for alignment of the NHz-terminal domains. In the sugar-bound forms of GBP (crystallized at ph 7.0) the distance betweep the OD-2 atom ofasp-121 and the OE-2 atom of Glu-165 (2.5 A) was found to be shorter than expected for the bound GBP (83.6"). The value of the second angle indicates that pair of negatively charged side chains (Zou et al., 1993). In the the strands of each domain point in approximately opposite unliganded form of GBP (crystallized at ph 8.6) the side chain directions, both toward the center and approximately along the long axis of the molecule. The number calculated for the closed unliganded GBP is 1.8" larger than the one for the galactose- GBP complex (198.0"), indicating that the sugar-free protein adopts a slightly more open conformation. The residues of domain 1 and domain 2 which are located at ofasp-121 was not so well defined in the electron density map; two possible conformations could be deduced. For the most populated conformation, which is similar to the one found in the liganded GBP, the distance between the OD-2 atom of Asp- 121 and the OE-2 atom of Glu-165 is 2.7 A. These observations seem to support the suggestion (Zou et al., 1993) that the pk of the lips of the binding cleft are thus slightly further apart in the one of these acidic groups is considerably perturbed, and a unliganded protein than in the ligand-bound form. For ex- proton would be retained at the ph of crystallization (7.0) of the ample, the distance be!ween the C-a atoms of residues 70 and 151 increases by 1.33 A, that of residuy 69 and 150 by 1.20 A galactose-gbp complex; at the higher ph of crystallization (8.6) of the unliganded species the carboxylate group begins to be and that of residues 71 and 152 by 0.94 A, all of these values are deprotonated, and the Asp-121 side chain (but not that of Gluwell beyond the limits of coordinate error. At the same time the 165) becomes more disordered. solvent accessible areas (Lee and Richards (1971); probe of Binding Site-A cleft between the two structural domains

4 8934 Closed Unliganded Glucose I Galactose Receptor a

5 A B rl1 Asn-256 Closed Unliganded Glucose IGalactose Receptor 8935 Water molecule S5 has also been found in all the liganded forms of GBP studied so far. In the present structure it makes hydrogen bonds with the side chain amide group ofasn-211 and with another water molecule (Fig. 3A). In the galactose-bound protein, water S5 binds to the hydroxyl group at position 3 of galactose and to the side chain of Asp-14 (Fig. 3B). The corresponding water molecules in the glucose-bound GBPs from E. coli (Vyas et al., 1988) and from S. typhimurium (Mowbray et al., 1990) make hydrogen bonds with the hydroxyl groups at positions 3 and 4 of glucose. The water molecules named S169, S170, and 5171 are well defined (they can be modeled at $111 occupancy with temperature factors between 23 and 26 A2). The interactions of water molecule S169 with Arg-158 and with Asp-236, those of water 5170 with Asn-211, with Asp-236 and with the conserved water molecule S5, and the interactions of water S171 with Asn-91 and with His-152 all mimic the interactions of the hydroxyl groups at positions 2,3, and 6, respectively, of galactose and of glucose in the liganded proteins. Another water molecule, named S174, accepts a hydrogen bond from Asn-256 as the hydroxyl group of C-1 of galactose and glucose do in the ligande! proteins. It has, however, a higher temperature factor (44 A2) than the other solvent molecules at the binding site indicating more mobility. Two water molecules are modeled as having two possible positions, each occupied half of the timeo(occupancy = 0.5, temperature factors between 12 and 20 A2). These are labeled S172A and S173A for one of the alternative models and S172B and S173B for the other. Water S172A makes a hydrogen bond with Asp-14 in a similar fashion to the hydroxyl group of C-4 of galactose, whereas at the alternative position S172B this bond is lost. Water S173B makes hydrogen bonds with Asn-91 (like the ring oxygen atoms of galactose and glucose) and with asp154 (like the hydroxyl group of C-1 of galactose and glucose) while at the alternative location S173A it makes hydrogen bonds only with other water molecules. Overall, 11 hydrogen bond interactions are established between protein side chains and water molecules when model A is considered; 9 of them involve side chains of the COOH-terminal domain and 2 involve side chains of the NH2-terminal domain. The corresponding numbers when model B is used are 12, 10, and 2. In the sugar-bound GBP, 12 hydrogen bonds are formed between sugar and side chains and 1 between sugar and water. The Fngths of these bonds are close to ideal and are generally 0.1 A longer than the ones measured in the galactose-gbp complex for each type of atom pair ( ,O... 0, N*... 0, N... 0). FIG. 3. A, schematic representation of the hydrogen bonding pattern in the binding cleft of unliganded GBP. Broken-line arrows link hydrogen-bond partners. The lengths of the bonds are indicated in A on the arrows. Water molecules that are well defined are shown as solid line boxes. Less ordered water molecules which have been modeled with half occupancy are shown as broken line boxes. B, same representation for galactose-bound GBP. The carbon atoms of the galactose molecule are numbered. constitutes the sugar binding site of GBP. In the present unliganded protein structure water molecules occupy the binding site (Fig. 2A 1. While a unique water network cannot be defined, two primary modes of water binding account well for the density observed. A web of hydrogen bonding between these water molecules and side chains of residues located in the loops between the COOH termini of the P-strands and the NH2 termini of is reminiscent of the hydrogen-bond patterns observed in the crystal structures of the galactose-gbp complex (Fig. 3, A and B) and of the glucose-gbp complex (Mowbray et al., 1990). DISCUSSION It has been generally accepted that the periplasmic binding proteins are open in the absence of ligand and closed after ligand binding. The three-dimensional structure of a ligand- free GBP from S. typhimuriurn shows that this protein is in fact able to close in the absence of ligand, adopting a conformation that is only slightly more open than that of the liganded forms. These results support the view that the conformational change which is associated with sugar binding is a shifting of equilibria rather than a necessarily "triggered" event. A network of water molecules satisfy the hydrogen bonding capacity of the side chains of the binding site of the closed unliganded form in a fashion which mimics the sugars. The total number of hydrogen bonds involved is essentially identical to the number made by galactose or glucose in the liganded protein. Differences in hydrogen bonding will probably not explain the apparent predominance of the open form in the absence of ligand. It is presumably the interaction between apolar surfaces of the sugar and non-polar groups of the protein which

6 8936 Closed Unliganded Glucose I Galactose Receptor favors the closed form in the presence of ligand. tration and the fact that the binding of the two proteins may be The fact that the closed unliganded form is found in the weaker than is found in the transport case. crystal proves that at least small concentrations must be pre- Acknowledgments- We thank Prof. Winfried Boos for providing the sent in solution which can be trapped in the crystal lattice. The results prior to publication. One of us (M.M.F.) is grateful to Malin equilibrium between open and closed unliganded forms of the Junerup and Brian Shilton for valuable hints at the stage of protein same protein has been invoked to explain the one open, one purification. closed crystal structure of human apolactoferrin (Anderson et al., 19901, an iron binding protein composed of two lobes each REFERENCES Anderson, B. F., Baker, H. M., Norris, G. E., Rumball, S. V & Baker, E. N. (1990) with a fold similar to the periplasmic binding proteins. A closed Nature 344, Anraku, Y. (1968a) J. Bwl. Chem. 243, unliganded form of the arabinose binding protein has been Anraku, Y. (1968b) J. Biol. Chem. 243, mentioned by Quiocho (19911, but no structural information or Binnie, R. A,, Zhang, H., Mowbray, S. L. & Hennodson, M. A. (1992) Protein Science 1, further description of the protein is given. Boos, W., Gordon, A. S., Hall, R. E. & Price, H. It has also been believed that only the closed liganded form D. (1972) J. Biol. Chem. 247, of the binding proteins can be recognized by the membrane Briinger, A. T., Kuriyan, J. & Karplus, M. (1987) Science 235, Jancarik, J. & Kim, S.-H. (1991) J. Appl. Crystallogr. 24, receptor for transport. Recently, however, Bohl et al. have Jones, T. A. & Kjeldgaard, M. 0. (1992) 0-The Manual, Version 5.8.1, Uppsala, simulated the behavior of the maltose-binding protein-depend- Sweden Jones, T. A,, Bergdoll, M. & Kjeldgaard, M. (1990) in Crystallographic and Modent transport system using a set of rate equations for the reding Methods in Molecular Design (Bugg, C. E. & Ealick, S. E., eds) pp actions that are likely to occur during the transport event. The 195, Springer-Verlag. New York simulations agree with the available experimental results only Jones, T.A., Zou, J.-Y., Cowan, S. W. & Kjeldgaard, M. (1991)Acta Crystallogr. A47, 11cL119 when interactions of both the sugar-loaded and the sugar-free Kabsch, W., & Sander, C. (1983) Biopolymers 22, binding proteins with the membrane components are taken Lee, B. & Richards, F. M. (1971) J. Mol. Biol. 55, Luck, L. A. & Falke, J. J. (1991a) Biochemistry 30, into account. This kinetic study does not indicate which con- Luck, L. A. & Falke, J. J. (1991b) Biochemistry 30, formation of the unliganded protein would be implicated. It is Manson, M. D. (1992) Adu. Microbiol. Physiol. 33, tempting to speculate that a closed unliganded species is suit- McGowan, E. B., Silhavy, T. J. & Boos, W. (1974) Biochemistry 13, Miller, D. M., Olson, J. S. & Quiocho, F. A. (1980) J. Biol. Chem. 255, able for this kind of unproductive encounter with the translo- Mowbray, S. L. (1992) J. Mol. Biol. 227, cation complex, since the overall conformations of the closed Mowbray, S. L. & Cole, L. B. (1992) J. Mol. Biol. 225, Mowbray, S. L. & Petsko, G. A. (1983) J. Biol. Chem. 258, unliganded and the sugar-bound species are very similar. The Mowbray, S. L., Smith, R. D. & Cole, L. B. (1990) Receptor 1, amounts of the membrane proteins are too small, and binding Newcomer, M. E., Lewis, B. A. & Quiocho, F. A. (1981) J. Bid. Chem. 256, to. the periplasmic receptor is too weak, to shift the equilibrium Oh, B.-H., Pandit, J., Kang, C.-H., Nikaido, K., Gokcen, S., Ames, G. E-L. & Kim, toward the closed unliganded form to the same extent that can be achieved with crystal packing. Studies of GBP and the related ribose-binding protein show that these proteins interact in a similar fashion with their common membrane receptor in chemotaxis despite slightly different relationships between the two domains in each structure (Binnie et al., 1992; Mowbray, 1992; Yaghmai and Hazelbauer, 1992). This property suggests that distinct conformations of the membrane receptor will give rise to signaling in each case. It is possible, therefore, that the closed unliganded form of GBP can bind to the membrane receptor as well, but does not have a significant effect on chemotaxis both because of its low concen- 2 E. Bohl, H. A. Shuman, and W. Boos, personal communication. S.-H (1993). J. Biol. Chem. 268, Quiocho, F. A. (1991) Curr. Opin. Struct. Biol. 1, Sack, J. S., Saper, M. A. & Quiocho, F. A. (1989a) J. Mol. Biol. 206, Sack, J. S., Trakhanov, S. D., Tsigannik, I. H. & Quiocho, E A. (1989b) J. Mol. Bid. 206, Sato, M., Yamamoto, M., Imada, K., Katsube, Y., Tanaka, N. & Higashi, T. (1992) J. Appl. Crystallogr. 26, Sharf, A. J., Rodseth, L. E., Spurlino, J. C. & Quiocho, F. A. (1992) Biochemistry 31, Spurlino, J. C., Lu, G.-Y. & Quiocho, F. A. (1991) J. Biol. Chem. 266, Stewart, R. C. & Dahlquist, F. W. (1987) Chem. Rev. 87, Strange, P. G. & Koshland, D. E., Jr. (1976) Prw. Natl. Acad. Sci. U. S. A. 73, Vyas, N. K., Vyas, M. N. & Quiocho, E A. (1988) Science 242, Yaghmai, R. & Hazelbauer, G. L. (1992) EMBO J. 12, Zou, J.-Y., Flocco, M. M. & Mowbray, S. L. (1993) J. Mol. Biol., in press Zukin. R. S.. Hartie. P.R. & Koshland. D. E., Jr. (1977) Proc. Natl. Acad. Sci. U. k A. 74, Zukin, R. S., Hartig, P. R. & Koshland, D. E., Jr. (1979)Biochemistry 16,

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