Structural Basis for Multiple Ligand Specificity of the Periplasmic

THE JOLWUAL OF Bromic& CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Voi. 269, No. 42, Issue of October 21, pp. 26323-26330,1994 Printed in U.S.A

Structural Basis for Multiple Ligand Specificity of the Periplasmic Lysine-, Arginine-, Ornithine-binding Protein*

(Received for publication, June 14, 1994)

Byung-Ha Oh$§, Giovanna Ferro-Luzzi Amesll, and Sung-Hou KimSII From the Department of Chemistry and Structural Biology Division ofLawrence Berkeley Laboratory and the ~ ~ p a r t ~ e n ~ of ~ o l e c ~ l u r and Cell 3iology, U n ~ v ~ r s i t y of C a ~ ~ ~ o r n ~ a , Berkeley, ~ a ~ ~ f o ~ ~ 94720

The substrate-binding site of a protein with multiple specificity should satisfy geometric and energetic complementarity for several different substrates. The structural basis of the multiple ligand specificity of the periplasmic lysine-, arginine-, ornithine-binding protein (LAO) was investigated by determining and analyzing the structures of the protein unliganded and liganded with each of the three high-affinity ligands (L-lysine, L- arginine, and L-ornithine) and with one low-affinity ligand (L-histidine). The geometric complementarity is achieved primarily by virtue of the large size of the 1i- gand-binding site which can accommodate the maximum common volume of the four ligands plus three water molecules. The opti~zation of energetic complement^^ is achieved by the relocation of protein-bound water molecules and by the movement of the Asp-11 side chain. The structure of the LAO-histidine complex indicates that the 30-fold reduced affinity of the protein for histidine is primarily due to unavailability of one ionic interaction of the histidine side chain with the protein which is present in the other three complexes.

Many proteins have specificity for more than one substrate. For example, subtilisin, a proteolytic enzyme, is quite undis- criminating about the nature of the side chains adjacent to the susceptible peptide bond (Markland and Smith, 1971). Other enzymes are very selective, such as thrombin, a proteol~ic enzyme pa~icipating in blood clotting, which cleaves only arginine-glycine bonds of fibrinogen fBlomb&ck et al., 1967; Pow- ers et al., 1989; Stubbs et al., 1992). Numerous examples of multiple substrate specificity can be found in the literature including nitrogenase (Hardy, 19791, lipoamidase (Oizumi and Hayakawa, 1990), Sermhr protein kinases (Proud et al., 1991), tyrosine protein kinases (Donella-Deana et al., 19901, tyrosyl- protein sulfotransferase (Niehrs et al., 1990), and isoprenylated protein endoprotease (Ma et al., 1992). The substrate-binding sites of such proteins must be able to accommodate each of several substrates geometrically and energetically.

Bacterial periplasmic binding proteins are essential compo- nents that serve as initial receptors for active transport sys- tems (Ames, 1986; Furlong, 19871, and chemotaxis (Koshland, 1988) in some cases. A salient feature of the binding proteins is

DK43747 and DK12121 (to G. F.-L. A.) and AI30725 (to S.-H. K.), the * This work was supported by National Institutes of Health Grants

United States Department of Energy, and the KECK Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduer-

this fact. tisement" in accordance with 18 U.S.C. Section 1734 solely to indicate

g Present address: Dept. of Life Science, Pohang University of Science and Technology, Pohang, Korea.

11 To whom correspondence should be addressed. nl.: 510-486-4333; Fax: 510-486-5272.

a remarkably large "opening and closing" domain movement due to a hinge motion at the peptide segments connecting the two lobes of the proteins. The closing motion encloses a substrate completely in between the two lobes (Sharff et al., 1992; Oh et al., 1993). The liganded closed conformation interacts with the membrane-bound transport complex or chemotaxis receptor. Several binding proteins are known to bind more than one ligand with virtually the same finity. The glucose/galactose-binding protein (GBPI1 binds the D-glucose and D-galactose epimers with dissociation constants (Kd) of 0.2 and 0.4 m ~ , respectively (Miller et al., 1983). The arabinose-binding protein ( B P ) binds a- or P-L-arabinose, the best ligands, with a Kd of 0.1 nm and the D-galactose epimer with a Kd of 0.2 mM (Miller et al., 1983). The leucin~isoleu~n~valine-binding protein (LW-BP) binds the 3 hydrophobic amino acids with Kd values of 0.1 to 1 n m (Amanuma et aE., 1976). LAO has an affinity for L-lysine, L-arginine, and L-ornithine (Kd values of 15,14, and 30 nM, respectively: Nikaido and Ames (1993)) about 10 times higher than any of the sugar-binding proteins for their ligands; L-histidine also binds to LAO, but less tightly than the other 3 amino acids, with a Kd of 500 n~ (Nikaido and Ames, 1993). The histidine-binding protein, HisJ, which is closely related to LAO (Higgins and Ames, 1981), has a reversed hierarchy of substrate affinities, and binds histidine better than arginine, lysine, and ornithine (Ames and Lever, 1972).

Both LAO and HisJ interact with the membrane-bound complex of the histidine permease tQ.M.P complex) resulting in the s u b E ~ u e n t t r a n s ~ ~ a t ~ o n of the bound ligand. Transport through such permeases involves signaling through the membrane-bound complex and ATP hydrolysis concomitant with transport (Petronilli and Ames, 1991; Davidson et al., 1992). The interaction and signaling between the binding proteins and the membrane-bound complex must be sensitive to the specific conformation of the binding protein. In order to under- stand the molecular mechanism of transport it is of interest to investigate whether binding proteins with multiple specificity assume conformations with each of the ligands that can be discriminated by the membrane-bound complex. LAO is an excellent system for studying the basis of multiple

ligand specificity and affinity because it binds 4 amino acids which differ in shape and number of atoms, but all bind LAO with fairly high affinity. In this paper, we present the analysis of five crystal structures of LAO from S u l m o n e l ~ a t ~ p ~ i m u r i u m as determined and refined at resolution higher than 2.06 A: unliganded LAO and the liganded forms of LAO complexed with each of lysine, arginine, ornithine, and histidine. The results are discussed in relation to those obtained with other binding proteins with multiple substrate specificity.

ABP, arabinose-binding protein; LIV-BP, leucine/isoleucine/valine-bind- The abbreviations used are: GBP, glucose/galactose-binding protein;

ing protein; LS-BP, leucine-specific binding protein; r.m.s., root mean square.

26323

26324 ~ ~ ~ t i p ~ e Ligand ~ ~ e c i ~ c i t y of a Lysine-, Arginine-, Urni~~. ine-~inding Protein

TABLE I Crystallographic refinement statistics of unliganded and liganded LAOs

LAO LA0:lysine LA0:arpinine LA0:ornithine LA0:histidine - -

Maximum resolution (A)

Completeness of data (F > IC, %) R factor (4,O A, F > lo, %) Number of water molecules Average temperature factor (A2) R.m.s. bond deviation from ideal (A) R.m.s. angle deviation from ideal (") R.m.s. improper angle

Rmerge On IhkI (%)

(excluding hydrogens and water oxygens)

deviation from ideal ("1

1.9 6.26

92.5 19.7 90 33.6

0.014 2.76 1.32

1.8 4.05

93.0 16.5

22.1 214

0.013 2.54 1.14

2.06 5.14

82.6 16.1

20.0 106

0.014 2.67 1.29

2.06 6.09

89.2 15.8

24.2 143

0.014 2.68 1.19

2.06 6.44

88.3 16.2

25.4 134

0.013 2.70 1.22

E ~ E ~ I M E ~ ~ P R O C E D ~ E S Protein Preparation and Crystal~uu~ion-The protein purification,

crystallization, and crystal cell parameters of unliganded LAO and lysine-liganded LAO were reported earlier (Nikaido and Ames, 1993; Oh et al., 1993). The crystals of lysine-liganded LAO were shown to contain a small fraction (about 20%) of LAO-arginine molecules. Since the ligand is mostly lysine, this liganded LAO will be referred as LAO- lysine throughout the text.

Unliganded LAO was prepared as described (Nikaido and Ames, 1993). To prepare LAO liganded with arginine, ornithine, or histidine, each of these substrates was added to the aqueous solution of unliganded LAO in a slight excess above equimolar concentration. ~nliganded LAO and the three liganded LAOs were crystallized under the same conditions as for the crystallization of LAO-lysine, In brief, drops of 7 pl of protein solution (15 mg/ml) were mixed in an equal volume with the precipitant containing 0.1 M sodium acetate, 0.05 M sodium cacodylate (pH 6.51, and 25% polyethylene glycol (PEG 4000), and then were equili- brated against 20 ml of the precipitant through vapor diffusion. All four liganded LAOs crystallized in the same space group P2,2,2, with similar cell parameters. Despite the similarities the crystal packing of the LAO-o~ithine and LAO-~stidine complexes (which are isomorphous with each other with cell parameters of a = 37.49 a, b = 58.19 A, c = 117.65 A) are completely different from that of the isomorphous LAO- lysine and LAO-arginineocomplexes (with cell parameters ofa = 37.79 A, b = 59.63 A, c = 115.93 A).

Crystal Structure Determination and Refinement-All diffraction data were collected at room temperature using CuKa radiation on a Rigaku R-AXIS-IIc imaging plate system operated at 50 kV and 100 mA. A detailed account of the structure determination of unliganded LAO and of the LAO-lysine complex was reported previously (Oh et aZ., 1993). Subsequently the structures were further refined by the iterative process of including water molecules into isolated electron densities of the F, - F, maps, and followed by atomic positional refinement using conjugate gradient minimization methods as implemented in the X- PLOR program package (Briinger, 1991).

The structure determination of LAO-arginine was straightforward since the crystals of LAO-lysine and LAO-arginine were isomorphous. The LAO-lysine structure with lysine deleted was refined by the conjugate gradient minimization method against the diffraction data obtained for LAO-arginine, and bound arginine was subsequently put in place according to the clear electron density in Wa - F, map.

For the structure determination of LAO-histidine and LAO-ornithine complexes, the molecular replacement method was employed, because their crystal packing was completely different from that of the LAO- lysine or LAO-arginine complex. The structure of LAO-histidine was determined using the LAO-lysine structure with the lysine deleted as a probe molecule in the molecular replacement; subsequently, the structure of LAO-ornithine was solved by refining the LAO-histidine structure without the bound ligand against the diffraction data obtained for the ~0-orn i th ine . In both cases, the respective ligand was put in place manually on a graphics c o ~ p u ~ r according to the clear electron density of the 2 F e - F, maps. In the case of the LAO-ornithine complex, the 2F0 - F, map calculated for the initial coordinates determined by the molecular replacement clearly indicated that the backbone and side chain conformation of Asp-11 is different from that of the other four structures. All molecular replacements and structure refinements were car- ried out using X-PLOR (Brtinger, 1991). The final crystallographic statistics of the structures are summarized in Table I.

RESULTS

As reported previously (Oh et ad., 1993), LAO is composed of two lobes, one bigger (lobe I, residues 1-88 and 195-238) than the other (lobe 11, residues 93-189, connected by two short peptide segments. In the liganded protein conformation, the ligand is held at the interface between the two lobes (Fig. la). The detailed ligand-binding mode for lysine is shown in Fig. lb . Ionic interactions play a primary role in neutralizing the charged group of the ligand in LAO, in contrast with the situ- ation with all other binding proteins, in which the charged group of the ligand is stabilized exclusively by uncharged polar and van der Waal's interactions (Quiocho, 1990). LAO also employs hydrophilic and hydrophobic interactions for tight binding of the ligand as shown in Fig. l b and schematically in Fig. IC.

The ligand-binding pocket can be considered in three parts. Region I makes many hydrogen bond and electrostatic interactions with the common amino and carboxyl termini of the ligands; region I1 consists of two aromatic and two aliphatic side chains which make exclusively hydrophobic interactions with the aliphatic or aromatic portion of the ligands; and region 111, like region I, makes many hydrogen bond and electrostatic interactions with the charged and/or polar portion of the ligands.

In comparing the structures of the liganded LAOs, the C" atoms of residues 5-235 are superimposed by a least squares fit. The r.m.s deviation between any pair of the liganded structures is less than 0.18 A. Thus the overall conformation is essentially the same for all four liganded forms. Recognizable differences are located only in and around the binding pocket, as described below. The superposition of C' atoms of the four liganded LAO structures shows that the ammonium and carboxyl groups, common to all four ligands, are positioned essentially identically, and the planes of the aliphatic chains of Lys, Arg, and Om superpose well with the plane of the His side chain (Fig. 2, a-d). In comparing the structures of unliganded LAO with liganded LAOS, the C" atoms of lobe I (residues 5-88 and 195-235) are superimposed. T h e r.m.s deviation between unliganded LAO and any of the liganded structures is less than 0.28 A. The superposition also shows that, within experimental error, all protein residues involved in ligand binding (Tyr-14, Phe-52, Asp-30, Ser-69, Ser-70, Ser-72, Arg-77, Leu-117, Thr- 121, and Asp-161), except for Asp-11, do not change their positions in the four liganded LAOs (partly shown in Figs. 2, a d ) ; variations in the protein-~gand interactions occur only at the side chain portion of the ligands, as discussed below.

Binding Site of the LAO-Lysine Complex-As mentioned earlier, this structure contains 20% arginine and 80% lysine in the ligand-binding site. We assume that the positions of the lysine ligand and other atoms at the ligand-binding site in this structure are the same as those of the pure LAO-lysine complex except for 80% occupancy of the ligand. Superposition of the

M ~ l ~ i p l e Ligand Specificity of a Lysine-, Arginine-, Ornithine-binding Protein 26325

a

FKC. 1. Ligand-binding site of LAO. a, stereo view of the LAO structure, unliganded (thin lines) and liganded with iysine (thick line8). The lobes I1 (bottom domaim) of the two structures are s u p e ~ m ~ s ~ . The long straight l ine passing through the two connecting strands between lobe I (top domains) and lobe I1 represents the axis about which lobe I makes a 52" rotation with respect to lobe I1 upon ligand binding. b, stereo view of the atomic interactions between lysine and LAO obtained from the 1.8-A resolution structure of the LAO-lysine complex. The ligand is shown as ball and stick, protein atoms as lines, and water molecules as circles. The two water molecules which appear to be intrinsic to the protein are labeled 401 and 402. c, schematic drawing of the ligand-binding pocket of LAO divided into three regions; for clarity, Leu-117, Ser-69, and Ser-70 are not shown (see text for explanation).

structures of the lobes 1 of unliganded LAO and of the LAO- lysine complex reveals that two water molecules, 401 and 402, are present at nearly the same places in the protein regardless of the presence or absence of the ligand (Figs. l b and 2uf. The temperature factors of the oxygen atoms of these water molecules in unliganded LAO are 37.3 A' and 32.8 A2, respectively, which is comparable to the average temperature factor of the protein atoms of LAO, which is 33.5 A'. The relatively tight binding of the two water molecules in the unliganded conformation appears to be the result ofthe tridentate hydrogen bonds connecting them t o Asp-11 0, Ser-69 Oy, Ser-70 Or and Asp-30 Os2, as well as between themselves (Figs. l b and Za). Upon lysine binding, the side chains of Tyr-14, Asp-11, and Ser-72 move toward the ligand (Fig. 3b in Oh et uZ. (1993)), but the rest of the residues do not significantly change their positions, indicating that lobe I has a binding site which can readily fit lysine both geometrically and energetically. Upon ligand binding, water

molecules 401 and 402 undergo a shift of 0.10 and 0.37 A from their positions in unliganded LAO, respectively. As in unliganded LAO, water molecule 401 maintains the same three hydrogen bonds with the Asp-11 0, Ser-69 Oy, and water molecule 402 (Figs. l b and 2a). Water molecule 402 also maintains its interactions with Asp-30 (2.82 A from Asp-30 0") and Ser-70 (2.75 A from Ser-70 Or). In addition, water molecule 401 gains a hydrogen bond with the N' of bound lysine.

Binding Site of the LAO-Arginine Complex-In the LAO- arginine complex, two additional side chain atoms of arginine, as compared to lysine, need to be accommodated in the binding site. This is achieved primarily by the simple displacement of water mofecule 401 into the bulk solvent (Fig. 2%). As a conge- quence, the arginine Nq2 replaces three hydrogen bonds in which water molecule 401 was engaged and gains one favorable ionic interaction with Asp-11. Water molecule 402 moves 0.65 A closer toward the arginine N'@ upon ligand binding and main-

26326 Multiple Ligand Specificity of a Lysine-, Arginine-, Ornithine-binding Protein

1

1

b

A 6

1

3 FIG. 2. Stereo views of superimposed structures. a, superimposed structures of the unliganded LAO (yellow) and of the LAO-lysine complex

(red). The 2F,, - F, electron density map calculated for the final structure of the unliganded LAO is shown. The contour level is 1.20. Water molecule 401 changes from a trigonal coordination in the unliganded LAO to tetragonal coordination (shown in dotted lines in Fig. lb) in the LAO-lysine complex. For clarity, Tyr-14, Asp-30, Ser-72, Arg-77, Thr-121, and Asp-161 interacting with each of the four ligands in the same scheme as shown in Fig. l b are not shown here and in the following figures. b, superimposed structures of the LAO-lysine (red) and LAO-arginine complexes (yellow). The Wo - F, map was calculated for the final structure of LAO-arginine complex. The contour level is 1.20. The interaction between the Ser-69 0 and the IT of arginine is shown as a dotted line. c, superimposed structures of the LAO-lysine (red) and LAO-ornithine (yellow) complexes. The W, - F, map was calculated for the final structure of LAO-ornithine complex. The contour level is 1.20. The empty space resulting from the lack of one carbon in ornithine as compared to lysine is occupied by the Asp-11 side chain rotated significantly toward the N' of ornithine. Interactions with the IT are shown as dotted lines. The Asp-11 side chain and the peptide bond betweenhp-11 and Thr-12 were fitted according to the electron density of the W,, - F, map calculated for the initial coordinates determined by molecular replacement. d , superimposed structures of the LAO-lysine (red) and LAO-histidine (yellow) complexes. Hydrogen bonds associated with the imidazole ring are shown as dotted lines. The Wo - F, map was calculated for the final structure of LAO-histidine complex. The contour level is 1.20.

tains the same interactions with Asp-30 (3.17 8, from Asp-30 OM) and Ser-70 (3.10 8, from the Ser-70 07) as in unliganded LAO. Fig. 2b shows that the bulky guanidino group of arginine causes minor changes in the conformation of the Asp-11 side chain. As shown schematically in Fig. 3, a and b, the side chain of Asp-11 is rotated slightly and pushed away 0.45 A (distance between the Asp-11 Cy positions in the LAO-lysine and LAO- arginine structures) toward the bulk solvent, as compared with the LAO-lysine complex. This Asp-11 reorientation is appar- ently necessary in order to maintain a proper distance for ionic interaction between its OB2 and the NV1 atom of arginine. Even though this could be considered a substantial movement, it does not result in repositioning of any of the other atoms, since the Asp-11 0'' faces toward and is pushed into the bulk solvent

(Fig. 4). It is also noted that the Ser-69 0, not used in LAO- lysine, makes a hydrogen bond with the Ne of bound arginine.

Binding Site of the LAO-Ornithine Complex-omithine, the smallest of the 4 amino acids, occupies the least space at the binding site. Surprisingly, the empty space thus generated is filled up by the Asp-11 side chain rather than by an additional water molecule. As shown in Figs. 2c and 3c, the Asp-11 side chain rotates and forms a salt bridge with the ornithine N'. The change in the side chain conformation would be impossible without the concomitant rotation of the peptide bond between Asp-11 and Thr-12 because of a steric crash between the carbonyl oxygen and the carboxyl group of Asp-11. The large 122 ' rotation of the peptide bond takes place as a consequence of the changes in backbone torsion angle in the two residues: the

Multiple Ligand Specificity of a Lysine-, Arginine-, Ornithine-binding Protein 26327

C

I ,

T

t€s

I

!"P

d

-. \

i

FIG. 2.-Continued.

PWsi angles of Asp-11 (-94.5 "1112.7 ") change to -88.3 "I -15.1 ", and the PWsi angles of Thr-12 (-85.0 "/32.2 ") change to 58.3 "/9.8 ", all of which are values for energetically favorable main chain conformation. The positive charge of the bound ornithine is neutralized by Asp-11 06', and further stabilized by the Ser-69 0 and water 401 (Figs. 2c and 3c). Water molecules 401 and 402 show a movement of 0.52 A (toward the N' of bound ornithine) and 0.37 A, respectively, as compared with their position in unliganded LAO. As in LAO-lysine, water molecule 401 gains a hydrogen bond with the N' of bound ornithine. Water molecule 401 is involved in the same two hydrogen bonds with the Ser-69 O y and water molecule 402 as in unliganded LAO (Fig. 3c). As a consequence of the peptide bond rotation, water molecule 401 loses a hydrogen bond with the Asp-11 0, but gains a new hydrogen bond with the Asp-11 O*'. Water molecule 402 also maintains its hydrogen-bonds with Asp-30 (2.89 A from Asp-30 Os') and Ser-70 (2.81 A from Ser-70 Ov).

Binding Site of the LAO-Histidine Complex-Histidine is different from the other 3 amino acids in that its side chain is uncharged and it contains a ring structure. The different

shape, however, can also be accommodated without any global or local conformational changes, since the amino acids involved in ligand-binding in the LAO-histidine complex structure are nearly identically positioned as those in the LAO-lysine or LAO-arginine complex (Fig. 2d). Water molecule 403 makes a hydrogen bond to the Nsl of histidine as shown in Figs. 2d and 3d. This water molecule does not interact with any of the other three ligands, but interacts with the adjacent water molecule and mediates the interaction between the Tyr-14 Oq and Leu- 117 N atoms (Fig. l b ) in the liganded LAOS. It should be noted that water molecule 403, in contrast to water molecules 401 and 402, is present only in the closed conformation of LAO since the Tyr-14 01 (present in lobe I) and Leu-117 N (present in lobe 11) are far apart from each other in the unliganded open coy- formation of LAO. Water molecules 401 and 402 move 0.60 A (toward N' of bound histidine) and 0.33 A, respectively, as compared to unliganded LAO; water molecule 401 is involved in three (or possibly four) hydrogen bonds (Fig. 3d), and water molecule 402 maintains its interactions with Asp-30 (2.84 A beheen Asp-30 Os2 and 402) and Ser-70 (2.98 A between Ser-70 0 7 and 402).

26328 Multiple Ligand Specificity of a Lysine-, Arginine-, Ornithine-binding Protein

FIG. 3. Schematic drawings of the variations in protein-ligand interactions. a, LAO-lysine; 6, LAO-arginine; e, LAO-ornithine; and d, LAO-histi~ne. For histidine, the unprotonated form of the imidazole ring is assumed. Dotted lines indicate hydrogen or ionic bonds, and their distances in A are shown near the lines. Asp-11 is drawn differently in each figure to emphasize its conformational change in the different structures. "he distance between the Asp110 and water molecule 401 in LAO-~s t id i~e indicates that the two atoms may be too far apart to form a hydrogen bond.

FIG. 4. Solvent accessibility of Aap-11 in liganded LAO struchres. Amino acid residues in arginine and in L A O ~ ~ i t h ~ n e are shown as thick and thin lines, respectively. The solvent acces- sible surface of Asp-11 in LAO-arginine was calculated using a 1.4-A radiue fox a probe water molecule, and i s represented by a dotted surface. All atoms within 4 a from any atom of Asp-11 or Thr-18 are shown. Although Thr-12 in LAO-ornithine also changes conformation of its side chain slightly due to the peptide bond rotation between Asp-11 and Thr-12, it does not require repositioning of nearby protein atoms. The carbonyl oxygen atom of Asp-11 in the LAO-ornithine complex becomes exposed to the bulk solvent as a result of the peptide bond rotation between Asp-ll and Thr-18, whereas its side chain becomes buried. Although not shown, the f Y 1 of Asp-11 in LAO-lysine and ~ 0 - ~ s t i d i n e is similarly q e d to the bulk solvent as in LAO-arginine.

Multiple Ligand Specificity of a Lysine-, Arginine-, Ornithine-binding Protein 26329

may form a hydrogen bond with the “OH4 group when it is either in the equatorial position (D-glucose) or in the axial position (D-galactose), thus explaining the negligible difference in the affinity of GBP for these two epimers. In the crystallographic studies on ABP complexed with each of L-arabinose, D-galactose, and D-fucose, it was concluded that the two protein- bound water molecules at the ligand binding site directly modulate the specificity of ABP by mediating interactions of ligands with the protein.

As an example of multiple specificity for uncharged amino acids, model building studies of LN-BP in the “open” conformation with L-leucine bound to the NH,-terminal domain indicated that a depression in the LIV-BP binding site could accommodate the side chain of either L-isoleucine or L-valine, as well as L-leucine (Sack et al., 1989). On the basis of this analysis of binding site specificity an attempt was made to interconvert LIV-BP and LS-BP; the lack of success indicated that as yet undefined residues from the COOH-terminal domain of the two proteins must also contribute to determining their specificity (Adams et al., 1991). The structure of the “closed” form of LIV-BP may be necessary to obtain precise and complete infor- mation on the full extent of the ligand-protein interactions. The studies presented in this paper on multiple specificity for charged amino acids complement those for uncharged sugars and amino acids summarized above.

General Characteristics of the Ligand-binding Pocket-The ligand-binding pocket is large enough to accommodate the maximum common volume of all four ligands plus three water molecules. A similar observation has been made in the crystal structures of the Fab fragment of an antiprogesterone antibody complexed with each of five different steroids. This study showed that the antigen-binding surface is large and that each steroid can be oriented in one of the two pockets of the binding site, thus explaining the multiple specificity for this group of steroids (Arevalo et al., 1993). As for the ligand-binding pocket of LAO, it can be divided into three regions: region I interacts with the terminal carboxyl and amino groups common to the 4 amino acids; region 111 interacts with the positive charge of the side chains common to all three high affinity ligands; and region I1 accommodates those portions of the side chains that are hydrophobic and different in the four ligands.

Role ofProtein-bound Water Molecules-Water molecules 401 (or 402 in LAO-arginine) and 403 interact with both the ligand and the protein, and thus appear to contribute directly to the ligand specificity and affinity of proteins by mediating interactions between a protein and its ligands, as Quiocho et al. (1989) concluded from the crystallographic study of ABP. Binding of different ligands to ABP causes positional shifts of the two water molecules involved in hydrogen bonds between the protein and the ligands, as was also observed in the liganded LAO structures. In the ABP-L-arabinose and ABP-D-fUCOSe structures the two water molecules are found at nearly the same positions. However, in the ABP-D-galactose structure, one of the two water molecules is dislodged into the bulk solvent by the -CH,OH group of D-galactose, and the other water molecule shifts its position and retains two hydrogen bonds and forms a new hydrogen bond with the protein.

However, in LAO all five of the hydrogen bonds between the protein atoms and water molecules 401 and 402 in the liganded LAOS (Figs. l b and 3) are not newly created by ligand binding (except for the one between water molecule 401 and the Asp-11 Osl in LAO-ornithine), but are present even in unliganded LAO. In the presence of a ligand, they simply gain one more hydrogen bond with the ligand (Figs. 2 and 3). Thus, the major role of these water molecules in LAO is to contribute to neutralizing the charges and partial charges of the protein atoms.

Water molecule 403 also gains one hydrogen bond with the bound histidine, but it is also involved in two other hydrogen bonds that are present in all the liganded LAOs (Fig. lb). The major role of water molecule 403, therefore, is to neutralize the partial charges of protein atoms that are brought together by the closing motion of the protein, rather than specifically mediating the interactions between the bound ligand and the protein. From these observations, it is more reasonable to conclude that the destiny and position of the protein-bound water molecules, at least in LAO, are primarily determined by the shape and electrostatic properties of the protein surface in the pocket, rather than directly influencing the interactions between the protein and ligand. On the other hand, it should be also noted that the mobility of the bound water molecules allows the same hydrogen bonds with the protein to be maintained, despite the difference in ligand structures; a new hydrogen bond is also established with a ligand or with the protein in the case of LAO-ornithine.

Electrostatic Interaction for High Ligand Afinity-The affinity of LAO for histidine (ICd of 500 nM) is about 30- and 17-fold lower than that for lysine or arginine (Kd of about 15 nM), and ornithine ( ICd of 30 nM), respectively. One striking difference in the binding mode of histidine as compared to that of the other three ligands is the absence of an ionic interaction with the Asp-11 side chain. The length of the bound histidine is the same as that of ornithine; thus the bound histidine leaves a void volume at the binding site. Unlike the case of ornithine binding, in which the rotation of the Asp-11 side chain toward the void volume provides an ionic interaction with ornithine, such a rotation does not occur upon binding of histidine. The proto- nation state of the imidazole ring of histidine does not appear to be the reason for the lack of the ionic interaction. Aplausible explanation is that the imidazole nitrogen of histidine cannot be drawn to a proper distance and angle to the carboxylic oxygen of Asp-11 for a favorable interaction. The distance between the histidine N” and the Asp-11 Os’ in the LAO-histidine is 3.84 A, while the distance between the ornithine N’ and Asp-11 0” in the LAO-ornithine complex is 2.97 A. We suggest that the absence of an ionic interaction between the histidine ligand and the protein may be the primary reason why the LAOs affinity for histidine is far below that for the other three substrates, all of which make an ionic interaction with Asp-11.

It was previously noted that the W spectrum of LAO changes upon liganding and varies with the nature of the ligand (Nikaido and Ames, 1993). These observations can be explained in terms of the data presented here, and indeed sup- port them. Tyr-14, which is presumably responsible for the changes in UV absorption, is stacked upon the aliphatic car- bons of liganded arginine and lysine, resulting in very similar spectra. On the other hand, when histidine is the ligand, Tyr-14 is stacked upon its imidazole ring, resulting in a very different spectrum. The even more different spectrum observed in the presence of ornithine is likely to be due to exposure of Tyr-14 to the more polar environment of the ammonium group of ornithine and of the carboxyl group of Asp-11.

Structural Basis for Mutiple Ligand Specificity-The unliganded and the four liganded structures of LAO indicate that the structural basis for LAO’S multiple substrate specificity has three aspects. 1) The binding site of LAO is large enough to accommodate the maximum common volume of the 4 amino acids plus water molecules 401, 402, and 403. Because of the large size of the binding site, lysine, ornithine, or histidine readily fit into the site, and in the case of arginine, the fit is accompanied by the simple displacement of one water molecule. 2 ) All the residues in the ligand-binding pocket (except one residue in the LAO-ornithine complex) undergo only small con-

formational changes to achieve geometric fit and favorable interactions with the iigands. Asp-11, which undergoes a large con€ormational change upon o ~ i t ~ i n e binding, is on the surface of the protein, so that its side chain can be pushed into, or the carbonyl carbon can be rotated toward, the bulk solvent as shown in Figs. 3 and 4, thus causing no steric hindrance while satisfying alternative hydrogen bonding with the bulk solvent. 3) Protein bound water molecules can be displaced by a ligand, as observed in the LAO-arginine structure, to provide room for a better geomet~c fit, or their position can be shifted, as observed in all the rest of the Iiganded structures, to maintain andor to establish energetically favorable interactions between the ligands and the protein. The higher affinities of LAO for the 4 amino acids, as compared to sugar-binding proteins such as GBP and ABP or uncharged amino acid-binding protein such as LIV-BP, are probably due to multiple ionic interactions between the ligands and LAO. The coordinates of LAO-arginine, LAO-ornithine, and LAO-histidine will be deposited in the Brookhaven Protein Data Bank. The coordinates of unliganded LAO and LAO-lysine have been deposited,

A c k ~ o ~ ~ e ~ g ~ n ~ - W ~ thank Carolyn Chi for help with the protein purification.

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Structural Basis for Multiple Ligand Specificity of the Periplasmic