topology of an hiloride-binding protein*

THE JOURNAL OF BIOLCGICAL CHBSOSTRY Vol. 269, No. 4, Issue of January 28, pp. 2806-2813, 1994 Printed in U S A .

Topology of an hiloride-binding Protein* (Received for publication, April 15, 1993, and in revised form, September 10, 1993)

Chaomei LinSOll, Thomas Kieber-EmmonsSII**, Annabelle P. VillalobosSOSS, Mary H. Foster*, Curtis Wahlgrenll, and Thomas R KleymanSQm From the Departments of medicine and lWhysiology, the University of Pennsylvania, the IlWistar Institute, and the $Veterans Administration Medical Center, Philadelphia, Pennsylvania 19104

Amiloride and structurally related compounds inhibit many transport proteins, enzymes, and drug or hormone receptors, although the topology of amiloride binding sites on these proteins has not been defined. We have previously raised and characterized a monoclonal anti- amiloride antibody (mAb BA7.1) which is similar to epithelial Na+ channels in its specificity of binding of amiloride and amiloride analogs, suggesting that their amiloride binding sites may be similar in topology. mAb BA7.1 was used as a model system to analyze the three- dimensional conformation of an amiloride binding site. The photoactive amiloride analog I'-methoxy-S'-nitrobenzamil specifically labeled the heavy chain of mAb BA7.1, suggesting that the heavy chain participates in amiloride binding. The nucleotide sequences of the variable regions of the heavy and light chains of mAb BA7.1 were determined and amino acid sequences deduced to analyze the structure of the amiloride binding site. A comparative modeling approach was used to construct a model of the amiloride binding domain of mAb BA7.1, and a docking procedure was used to place amiloride within this domain. The model indicated that planar aromatic amino acid residues form a pocket into which amiloride, a planar molecule, inserts. Constraints on amiloride binding predicted by this model correlated with the measured specificity of binding of amiloride analogs with mAb BA7.1. These results provide a potential guide for the identification of motifs or amino acid contact residues present within other amiloride-sensi- tivs proteins.

High resistance Na+ transporting epithelia express Na+ channels that are functionally restricted to their apical plasma membrane. Na+ crosses the apical membrane by passive diffu- sion through this channel and is actively extruded from the cell by a Na+/K+-ATPase present on the basolateral plasma membrane (Sariban-Sohraby and Benos, 1986). The diuretic amiloride is an inhibitor of epithelial Na+ channels. This drug and

*This work was supported in part by grants from the American Cancer Society (to T. K.-E.), the Cystic Fibrosis Foundation (to T. R. K.), the Southeastern Pennsylvania Chapter of the American Heart Asso- ciation (to T. R. K.), and was performed during the tenure of an Estab- lished Investigatorship Award from the American Heart Association (to T. R. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

to the GenBankTMIEMBL Data Bank with accession numberfs) L24802 The nucleotide sequeme(s) reported in this paper has been submitted

(heavy chain) and L24803 flight chain). ll Recipient of a postdoctoral fellowship award from the Southeastern

Pennsylvania Chapter of the American Heart Association. ** To whom correspondence should be addressed: Wistar Institute,

3601 Spruce St., Philadelphia, PA 19104. $$ Present address: RW Johnson Pharmaceutical Research Institute,

Protein Development Center, Raritan, NJ 08869-0602.

structurally related compounds were initially developed as K+- sparing diuretics, which is result of Na+ channel inhibition (Kleyman and Cragoe, 1988b). Subsequent studies have shown that amiloride and specific amiloride analogs inhibit or interact with a variety of proteins, including many Na+-selective transport proteins, enzymes, and drug or hormone receptors (Kley- man and Cragoe, 1988a). It is not known whether amiloride binding sites on these proteins share common topologic features. Amiloride could serve as a probe to begin to dissect topologic relationships among these proteins.

Amiloride is pyrazinoylguanidine with amino groups on the 3- and 5-positions and a chloro group on the 6-position of the pyrazine ring (Fig. 1). Analogs with appropriate modifications have increased affinity and specificity for certain transport systems. For example, the introduction of hydrophobic substituents on the terminal nitrogen of the guanidinium moiety enhances activity against a class of epithelial Na+ channels (H-type), whereas the addition of hydrophobic groups on the 5-amino moiety enhances activity against the NHEl isoform of the Na+/H+ exchanger (Oh and Benos, 1992; Haggerty et al., 1988; Simchowitz et al . , 1992). Higher concentrations of amiloride inhibit other Na+-selective transporters, such as L-type epithelial Na+ channels, Na+/H+ exchangers (apical membrane isoforms), Na+/Ca2+ exchangers, Na+-coupled cotransporters, and Na+/K+-ATPase (Haggerty et al . , 1988; Kleyman and Cra- goe, 1988a; Oh and Benos, 1992; Simchowitz et al., 1992).

Examination of the interaction of amiloride with the epithelial Na+ channel indicates that amiloride and Na+ share a common binding or recognition site at or near the pore of the channel (Cuthbert and Shum, 1974; Cuthbert, 1981; Li and Lindemann, 1984), although conflicting studies have been reported (Benos et al . , 1980; for review see Garty and Benos, 1988). Kinetic studies of the interaction of amiloride with several Na+-selective transport proteins, including Na+/H+ exchangers (Kinsella and Aronson, 1981), Na+/Ca2+ exchangers (Kaczorowski et al., 19851, and Na+-hexose cotransporters (Cook et al., 1987) suggest that amiloride binds to a Na+ recognition or binding site on these transport proteins. These observations led to speculation that amiloride binding sites on Na+-selective transport proteins may share common structural features (Garritsen et al., 1991). Differences in the sensitivity of transport proteins to amiloride analogs might arise through the interaction of the hydrophobic substituents with hydrophobic or hydrophilic domains at, or in proximity to, the amiloride binding site. Alternatively, modifications of amiloride by the introduction of substituents might alter or mask a feature required for receptor binding leading to changes in the apparent affinity of amiloride analogs for specific transport proteins.

If amiloride binding sites on Na+-selective transport proteins share common topology, it is not evident fmm an analysis of sequence alignments of amiloride-sensitive transport proteins as extensive homology has not been observed, except within members of the same family ( i e . Na+M+ exchanger isoforms NHE1, NHEB, and NHE4) (Orlowski et al., 1992). However,

2805

2806 Topology of an Amiloride Binding Site

FIG. 1. Structure of amiloride. Numbers indicate positions of sub-

used for the model of amiloride were assigned automatically within the stituents on the pyrazine ring. The potential atom types and charges

program Insight 11. The potential atom types for the pyrazine ring are np for the ring nitrogens, cp for the ring carbons, n2 for the amino nitrogens, hn for the amino hydrogens, and cl for the halo group. The guanidinium group was assigned potential atom types for arginine, n2 for the amino nitrogens, hn for the respective hydrogens, n l for the double-bonded nitrogen, cr for the guanidinium carbon, c' for the carbonyl carbon, and 0' for the carbonyl oxygen. Charges were assigned accordingly by Insight 11. For the pyrazine ring, np is -0.220, n2 is -0.665, hn is 0.280, cp at the 6th position (cp6) is 0.212, cp5 and cp3 are 0.215, cp2 is 0.110, and cl is -0.102. The overall charge for the pyrazine ring is neutral. For the guanidinium group, the carbonyl carbon is 0.38 with the carbonyl oxygen -0.38, the nitrogens are -0.500, the amino hydrogens are 0.36, with the hydrogen from the double-bonded nitrogen 0.280. The guanidinium carbon is 0.45. The overall charge on amiloride as assigned by Insight I1 is 0.95. The omega bond in the guanidinium group derived from the x-ray structure is 171 degrees (Ball, 1991),

force constant (K value) of 1,000 kcal. The planarity was further re- which was retained as planar between 170 and 180" using a torsional

tained by the addition of distance constraints of 2.12-2.23 A between the carbonyl oxygen and the closest amino hydrogen and between the hydrogen off the double-bonded nitrogen and the closest amino hydrogen, as observed in the crystal Structure of amiloride and in arginine guanidinium groups in general. The force constant was also 1,000 kcal.

these proteins could fold in a manner that results in amiloride contact residues being similar in three dimensions which are otherwise cryptic in linear sequence alignments. Attempts to identify specific amino acid residues on amiloride-sensitive transport proteins that participate in amiloride binding have relied primarily on chemical reagents that modify specific amino acid residues and alter ion transport in an amiloride- protectable manner (Ganaphthy et al., 1987; Grill0 and Aron- son, 1986; Park and Fanestil, 1980, 1983; Park et al., 1983b). Although this approach has led to identification of putative amino acid residues that might contact amiloride, it does not provide for a topology of the amiloride binding site.

Anti-amiloride antibodies could serve as a model system to identify structural features required for amiloride binding and provide a framework for predicting putative structural charac- teristics of amiloride-sensitive transport proteins that participate in drug binding. The hypothesis that an antigen binding site on an anti-ligand antibody may mimic a receptor (or binding site) for that ligand has received experimental support from numerous studies (for recent reviews, see Nisonoff, 1991; Taub and Greene, 1992; Zanetti et al., 1991). Studies of the structure of antibody hypervariable regions indicate that it is possible to predict with a high degree of accuracy the configuration of antibody hypervariable loops from primary sequence data (Martin et al., 1991). Antigen binding to specific antibodies can be analyzed at a molecular level through model building, and this information can aid in the development of a conceptual framework of a receptor binding site (Zanetti et al., 1991; Vaux and Fuller, 1991). Examination of relationships between antibodies and ligand receptors has relied primarily on analysis of primary sequence data (Taub and Greene, 1992; Williams et al., 1988; Pride et al., 1992). In addition, analysis of the spatial orientation of contact residues on antibodies has been used successfully to identify topologic relationships between antibodies and ligand receptors (Williams et al., 1991b).

We previously raised a monoclonal antibody (mAb)' BA7.1

The abbreviations used are: mAb, monoclonal antibody; H chain,

against an amiloride analog that was coupled to albumin through its guanidinium group with a hydrocarbon spacer. This anti-amiloride antibody mimicked the H-type epithelial Na+ channel in its specificity of binding to amiloride and amiloride analogs (Kleyman et al., 1988a, 1989b), suggesting that the amiloride binding site of this antibody is similar in structure to the amiloride binding site on the Na' channel. Furthermore, polyclonal anti-amiloride antibodies raised against the amiloride-albumin conjugate have similar, though not identical, specificity of binding to amiloride analogs (Kleyman et al., 1989b) and were used in the generation of anti-idiotypic antibodies that recognized the epithelial Na' channel (Kleyman et al., 19911, providing further evidence of the structural similarities between the drug binding site on these antibodies and on the Na+ channel. We have now developed a model of the antigen binding site of the anti-amiloride mAb BA7.1 based on the primary sequences of the light (L) and heavy (H) chain variable (V) regions. Our model of the antigen binding site correlates with the specificity of binding of amiloride analogs to mAb BA7.1 and indicates that planar amino acids play a prominent role in stabilizing the interaction of amiloride with the antibody. The residue types are similar to those suggested to affect amiloride binding to amiloride-sensitive proteins, indicating that mAb BA7.1 mimics some of the salient features of the amiloride binding site on amiloride-sensitive proteins.

EXPERIMENTAL, PROCEDURES MaterialeDNA polymerase, Moloney mouse leukemia virus reverse

transcriptase, T4 ligase, and DNA Sequenase kit were obtained from U. S. Biochemical Corp. Amiloride analogs were a giR from Dr. E. J. Cragoe, Jr., Nacogdoches, "X. All other chemicals were reagent grade.

Antibody Preparation-Hybridoma cells secreting mAb BA7.1 (Kley- man et al., 1989b) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. The peritoneal cavities of BALB/c male mice were primed with 0.5 ml of 2,6,10,14-tetramethylpentadecane at least 1 week prior to inoculation with 2-5 x lo6 BA7.1 hybridoma cells. Ascites was subsequently collected, cellular debris removed by centrifugation (12,000 x g for 5 min), and stored at -80 "C with 0.05% (w/v) NaN,. mAb BA7.1 was purified by protein A affinity chromatography as described previously (Harlow and Lane, 1988).

Enzyme-linked Zmmunosorbent Assay-Microtiter plates (96 wells) were coated with 1 pg/well of an amiloride-rabbit serum albumin conjugate (amiloride coupled to rabbit serum albumin through its guanidinium group (Kleyman et al., 1986)) in 50 pl of a 0.1 M NazCOs/ NaHCOs buffer, pH 9.5, and incubated at 4 "C overnight. Wells were rinsed and incubated with 100 pl of phosphate-buffered saline, pH 7.4, containing 0.1% (v/v) Tween 20 and 5% (w/v) nonfat dry milk for 1 h at 37 "C to prevent nonspecific protein binding. Wells were then incubated with mAb BA7.1 (1:104 dilution from ascites fluid), with or without amiloride analogs, in 50 pl of phosphate-buffered saline with 0.1% Tween 20 at 37 "C for 2 h. Wells were rinsed extensively and incubated with 50 pl of a 1:103 dilution of an anti-mouse IgG raised in goat and conjugated to horseradish peroxidase (Boehringer Mannheim) for 30 min at room temperature. Wells were rinsed, and peroxidase activity was detected with the addition of 50 pl of 0.07% (w/v) o-phenylenedi- amine in 0.1 M sodium citrate, pH 4, containing 0.1% (v/v) H202. The reaction was stopped by adding 50 pl of 0.05% NaNs in 0.1 M sodium citrate, pH 4. Absorbance at 490 nm was measured using a Microplate Reader, Bio-Rad model 3550, with Microplate software.

Photoaffinity Labeling-Affinity-purified mAb BA7.1 was dissolved in phosphate-buffered saline at a concentration of 100 pg/ml. The photoactive amiloride analog 2'-methoxy-5'-nitrobenzamil (NMBA) was used to label mAb BA7.1 as described previously (Kleyman et al., 1989a). Briefly, NMBA was added to the solution in the presence or absence of the amiloride analogs benzamil and dimethylamiloride and incubated on ice in the dark for greater than 30 min. The mixture was then irradiated for 20 min with light from a mercury arc lamp (Zeiss HBO, 100 watts) filtered through a 313-nm narrow band pass filter

heavy chain; L chain, light chain; V region; NMBA, 2"methoxy-5'- nitrobenzamil; PAGE, polyacrylamide gel electrophoresis; CDR, complementarity-determining region; EEDQ, N-ethoxycarbonyl-2- ethoxy-1,Z-dihydroquinoline; MPA, 54N-methyl-N-propy1)amiloride.

lbpology of an Amiloride Binding Site 2807

(Oriel) with constant stirring at 4 "C under a stream of Nz. After irra- diation, 20 pg of cytochrome c and 500 pl of acetone were added to the reaction mixture and allowed to incubate for 30 min at 4 "C to precipi- tate the antibody. Following a 10-min, 12,000 x g centrifugation, the protein pellet was dissolved in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer containing 3% (w/v) SDS, 15% (w/v) sucrose, and 92.5 m~ Tris.HC1, pH 6.9. Samples were reduced with 10 m~

dithiothreitol for 15 min followed by an incubation with 20 m iodo- acetamide at mom temperature. Following 7.5% SDS-PAGE, proteins were transferred to nitrocellulose, and NMBA was detected with a polyclonal anti-amiloride antibody as described previously (Kleyman et al., 1989a).

Oligonucleotide Synthesis-The m o w Cy primer was the 15-mer oligodeoxynucleotide 5'-GGCCAGTGGATAGAC-3'. The mouse Cy probe was 5 ' - T G G G G ( C G ) T 3 ' . The mouse CK primer was 5 ' - C T G C T C A C T 3 ' , and the CK probe was 5'-AGATG- GATACAGTTGGT-3'. Oligonucleotides were prepared using the phos- phoramidite method with a DNA synthesizer (model 391, Applied Bio- systems) and were purified with an oligonucleotide purification cartridge. Probes were 5'-end radiolabeled with T4 polynucleotide ki- nase and [y-32P]ATP as described previously (Sambrook et al., 1989) and purified over NENSORB-20 cartridges (DuPont-NEN). V, and V, Sequence Analysis-lbtal RNA was extracted from mAb

BA7.1 hybridoma cells and isolated by the guanidinidCsC1 method as described previously (Chirgwin et al., 1979). Double-stranded cDNA was synthesized using RNA as a template and C, or Cy oligonucleotide primers, Moloney m o w leukemia virus reverse transcriptase, and Escherichia coli DNA polymerase I (Gubler and Hoffman, 1983; Levy et al., 1987; Guillaume et al., 1990). Double-stranded cDNA was blunt- ended with T4 DNA polymerase and ligated into SmaI-digested and dephosphorylated M13. E. coli DH5-aF'IQ were transformed with re- combinant phagemids and clones screened with CK (L chain) and Cy (H chain) oligonucleotide probes (Sambrook et al., 1989). Positive plaques were purified and sequenced by incorporation of dideoxynucleotides using the Sequenase version 2.0 DNA sequencing kit. Nucleotide sequences were confirmed by sequencing a second independent clone.

M&ling of mAb EA7.1-Following procedures we have used in previous studies (Williams et al., 1988, 1989, 1991a, 1991b; lbmiyama et al., 1992), the conformations of the CDRs of mAb BA7.1 H chain and L chain were compared with known immunoglobulin crystal structures (Bernstein et al., 1977) to develop possible models for the localized structural folds of CDRl and CDR2. For CDR3, several antibodies of known crystal structure were least squares superimposed to define invariant residue positions (lbmiyama et al., 1992). These positions define the amino-terminal beginning and the carboxyl-terminal end that are shared among the putative CDR3s of varying lengths. The systematic superpositioning of the CDR3 over short sequences defined a consensus region in which the structure was conserved among the antibody templates. This consensus region defined the framework onto which a model for the CDR3 loop of mAb BA7.1 was built.

The crystallographic database was searched to identify loops of the same size as the CDR3 loop of mAb BA7.1. The spatially conserved Cartesian positions at the amino- and carboxyl-terminal regions of CDR3 were held fixed in this search procedure. A Cartesian distance matrix was constructed for combinations of the residues on the amino- and carboxyl-terminal regions of CDR3 and compared with a precalcu- lated Cartesian distance matrix data base of high resolution protein structures (Jones and Thirup, 1986). The 20 best matches were examined using the program Insight I1 (version 2.1, Biosym Technologies), and an appropriate choice was made based upon similarities in chirali- ties of side chains a t the ends of the CDR3 loop and sequence similarities within the CDR3 loop.

The CDRa and framework regions of the templates were mutated to those of mAb BA7.1 using Insight 11. The side chain angles of the substituted residues were set according to angles identified in a data base of side chains. Each CDR and framework region was individually changed, followed by 1,000 cycles of energy minimization to eliminate close contacts among atoms. As in our previous studies, the program Discover (version 2.8, Biosym Technologies) was used for conformational calculations with supplied consistent valence force field parameters. A distance dielectric model with a dielectric of 4 was employed in waling the electrostatic interactions. This dielectric is arbitrary but represents a compromise between a purely in U ~ C U O calculation (devoid of solvent in the binding site) and a solvent effect. After model building, the structure was energy optimized with the above scheme to convergence. As a control on the adequacy of using a dielectric of 4, we also used a dielectric of 10. These different dielectric values only affected the electrostatic energy contribution to the total energy and not the struc-

ture of the model. Docking of Amiloride to mAb BA7.1-In the placement of amiloride

into the combining site of BA7.1, the molecular modeling approach assumed that the antigen-binding site is relatively rigid and that the intramolecular energy change upon hapten binding is small compared with the interaction energy between the hapten and the antigen binding site, so that the given binding mode for each molecule is its optimal binding mode. This need not be the case, and we have not addressed the issue of whether induced fitting or major conformational adjustments of mAb BA7.1 are required for amiloride binding (Cheetham et al., 1991; Voss et al., 1992; Rini et al., 1992).

The procedure of fitting amiloride to mAb BA7.1 is tantamount to identlfylng common surface features with subsequent docking of the complementary surfaces (Des et al., 1986). The binding surface on mAb BA7.1 was defined as sites accessible to a probe sphere of radius greater than 1.5 A but not accessible to a probe sphere of radius greater than 4.0 A (Sudarsanam et al., 1992). The probe spheres were rolled on the binding surface of mAb BA7.1 to identify the possible positions that can be occupied by the atoms of amiloride, reducing a continuum of loci to a set of discrete points. These site points are localized at atom positions accessible to the probe spheres on each residue on the surface of mAb BA7.1 and were assigned a pseudoatom type, based on ability to simulate plausible hydrogen bonding or dispersion and on electrostatic interactions between any ligand and mAb BA7.1. The pseudoatom types were generically assigned and were dependent on the associated atom on BA7.1. For example, i fa nitrogen on BA7.1 is associated with a site point, the pseudoatom type can be an oxygen to simulate a potential hydrogen bond or electrostatic interaction, or it can be a carbon atom implying a dispersion or polarization interaction. The assignment of a pseudoatom type is not a prerequisite for placement of the ligand. It is used as a tool to help identify relationships between a ligand (i.e. amiloride) and potential site points that occupy a binding site.

The site points were used as a guide in the placement of amiloride in the antibody combining site. A docking pattern was provided by matching the various combinations of the distance matrix (a distance M e r - ence map) between site points and amiloride atom positions which can be discarded or accepted depending on steric conflicts between amiloride placement within the antibody combining site. The structure of protonated amiloride used in the docking studies was based on a crystal structure of an unprotonated form of amiloride (Ball, 1991) and on a similar protonated structure determined by NMR and theoretical calculations (Smith et al., 1979) (see Fig. l legend). The pK. values of amiloride and benzamil are 8.7 and 8.1, respectively (Kleyman and Cragoe, 1988a). It is the protonated form of amiloride which interacts with the Na' channel (Kleyman and Cragoe, 1988a), and it is assumed that the protonated species binds to mAb BA7.1.

Amiloride was least squares fitted to the site points that had the requisite distance match. A scoring function was used to determine the close contacts between amiloride and mAb BA7.1 (Des et al., 1986). Sites associated with the CDR loops forming the classical antibody combining site were examined. The docking procedure was strictly based upon geometric alignment of ligand atoms with combining site points. Following previous studies on docking approaches (Des et al., 1986, 1988), a score was generated for each unique alignment of the ligand in which four ligand center pairs and four antibody combining site point center pairs were within a specified distance limit of 1.5 A. Scoring was based on distances of ligand atoms to combining site atoms. Distances that were too close ( ~ 2 . 3 A) were given negative scores. Dis- tances that were too far b5.0 A) were given zero scores. Distances that were not too close but less than a maximal optimum distance (3.5 A) were given a score of 1.0. All other distances were rated by an exponen- tial function that compared the observed distance with the maximal optimum distance (3.5 A) and assigned a value between 0 and 1 (Des et al., 1986). The cumulative dock score for a given orientation was a sum of the individual scores for each atom.

A docking run with mAb BA7.1 and amiloride generated 23 orientations with cumulative docking scores from 31 to 68. Intermolecular interaction calculations for each of the orientations indicated that the docked orientation with a score of 35, under the constraints imposed, was the best match. Complexes that exhibited a reasonable score by the scoring function were subjected to energy minimization. A molecular dynamics calculation over 50 ps using the program Discover was performed. The dynamics run was not intended to be a detailed study but rather to alleviate further any close contacts within the antibody and between amiloride and the antibody. The calculation was initialized and equilibrated for 20 ps at 600 "C at constant pressure and cooled to 300 "C over 30 ps. A time step of 1 fs was used. The resulting structure was energy minimized using conjugate gradients to convergence. In the


Competitive inhibition of mAb BA7.1 binding to RSA-amiloride TABLE I

by amiloride derivatives (compounds I and 3) and triamterene (cornpound 2)

mAb BA7.1 binding to RSA-amiloride was determined by ELISA as described under “Experimental Procedures,” using a 1: lo4 dilution of the mAb BA7.1 and varying concentrations of the compounds illustrated in the table. Bound antibody was detected by incubating wells with peroxidase-conjugated anti-mouse IgG, followed by o-phenylene- diamine as a substrate and measurement of absorbance at 492 nm.

104M 0

molecular dynamics simulation a torsional restraint of 5 kcal x mol-’ x rad-2 was employed around the omega bond, with a distance-dependent dielectric. In the minimization and dynamics run no constraints were placed on the BA7.1 antibody or binding site, but amiloride was re- strained in a planar configuration in these calculations. Charges and nonbonded parameters for amiloride were assigned from atom types from the consistent valence force field. The acylguanidinium group emulated both charge and nonbonded parameters of a protonated arginine side chain.

RESULTS mAb BA7.1 was raised against amiloride coupled to bovine

serum albumin through a hydrocarbon spacer arm on the terminal nitrogen of its guanidinium moiety. Previous studies demonstrated that this antibody recognizes a specific epitope on amiloride, the 3,5-diaminopyrazinyl moiety (Kleyman et al., 1989b). An amiloride analog with a benzyl group on the 5 9 - sition of the pyrazine ring hindered access to this epitope and was not recognized by the antibody. An amiloride analog with the addition of a benzyl substituent on a terminal nitrogen of the guanidinium group (benzamil) has an affinity for the antibody which is similar to the parent compound. The substitution of an iodo for a chloro group in the 6-position of the pyrazine ring (iodoamiloride) abrogated binding to mAb BA7.1. We examined whether additional modifications of the structure of the guanidiniumcarbonyl group affected binding to the antibody. Binding of mAb BA7.1 to amiloride-rabbit serum albumin was measured in the presence of amiloride analogs. One amiloride analog had two substituents on a terminal nitrogen of the guanidinium group (compound 3, “able I). A second compound had a nitrogen placed between the carbonyl and guanidinium groups (compound 1, Table I). Both are poor inhibitors of epithelial Na+ channels (Kleyman et al., 1988a). Neither compound was efficiently recognized by mAb BA7.1 (Table I). The compound triamterene is a heterocyclic weak base and lacks a guanidiniumcarbonyl group. Triamterene is a poor inhibitor of epithelial Na+ channel, relative to amiloride (Cuthbert and Shum, 19741, and did not bind mAb BA7.1 (compound 2, Table I).

Photoaffinity Labeling-Photoactive analogs of amiloride have been used to identify the amiloride-binding subunit of the epithelial Na+ channel (Kleyman et al., 1989a; Benos et al., 1987). One of these analogs, NMBA, undergoes photoincorporation into protein by the mechanism of aromatic nucleophilic photosubstitution (Cornelisse and Havinga, 1975). NMBA was used to affinity label mAb BA7.1 purified from ascites. The

t

0 1 10 35 100 1000

[NMBA] nM F’Ic. 2. Dose response of photoincorporation of NMBA. mAb

BA7.1 was photolabeled for 20 min with increasing concentrations of the photoactive amiloride analog NMBA, a derivative of benzamil. Pho- tolabeled proteins were analyzed by SDS-PAGE and detected by immunoblot with a polyclonal anti-amiloride antibody. Migration of the immunoglobulin H chain is indicated by the arrow. Only the H chain of mAb BA7.1 was labeled.

antibody was photolyzed in the presence of increasing concentrations of NMBA and then subjected to SDS-PAGE. A polyclonal anti-amiloride antibody that recognizes NMBA-labeled proteins was used to identify affinity-labeled antibody by immunoblot (Kleyman et al., 1989a). NMBA labeled the H chain of mAb BA7.1 (Fig. 2), suggesting that the H chain participates in amiloride binding. The specificity of photoincorporation was demonstrated by photolabeling with NMBA in the presence or absence of varying concentrations of the amiloride analog benzamil, which was added to inhibit specific photoincorporation of NMBA into mAb BA7.1, as well as dimethylamiloride, which should not bind mAb BA7.1 (Kleyman et al., 1989b). Benzamil inhibited photoincorporation of NMBA into mAb BA7.1 H chain in a dose-dependent manner, whereas dimethylamiloride did not block photoincorporation (Fig. 3).

Cloning and Sequencing of the L and H Chain cDNA of mAb BA7.1-mAb BA7.1 H and L chain V region cDNAs were synthesized and introduced into M13. Individual subclones were isolated on the basis of hybridization to mouse Cy and CK probes. Sequence analysis of these clones revealed that the H chain is encoded by a VH gene of the 5558 gene family, a DFL16.1 D gene segment, and a JH4 gene. The L chain is encoded by a member of the Vu19 subgroup and a Ju2 gene. The H chain and L chain sequences are illustrated in Fig. 4. Amino acid residue position numbering in the text is preceded by H or L to denote heavy or light chain, respectively.

Model of m.Ab BA7.1 Antigen Binding Domain-Sequence comparisons with known immunoglobulin crystal structures indicate that the structure of the anti-lysozyme antibody HY- HEL-5 deposited in the Brookhaven Protein data base (Bern- stein et al., 1977) provides a canonical structure (Chothia et al., 1992) for the CDRl and CDR2 of the H chain of mAb BA7.1 (Fig. 5a ). An important feature in defining this canonical structure is the proline residue a t position H52a, the glycine a t residue position H55, and the alanine residue at position H71 (valine can substitute for alanine). In our search procedure to model the CDFB of BA7.1, the loop of the CDW of mAb BA7.1 was found to be most like that of the anti-phosphoryl antibody McPC603. The CDFB loop of McPC603 was spliced into the HY-HEL-5 template using the program Insight 11.

The L chain was built in the same manner. The lengths of the CDRs of the mAb BA7.1 L chain most closely match that of the antibody F19 (Fig. 5b). However, previous studies of the structural similarities between antibody templates identified a dis- cordance between sequence alignment and structural alignment (Kieber-Emmons and Wiener, 1992). A comparison of structures at the beginning and ends of the beta strands span- ning framework 1 of the L chains of antibodies F19 and FVB (Fig. 5b) indicated that they are not structurally equivalent. Superpositioning the Cartesian coordinates of these residues onto each other indicated that the ‘IT tract of F19 (amino acid

Topology of an Amiloride Binding Site 2809

a. Benzamil t

b. Dimethylamiloride t-

0 50 5005000

[Competing Drug] nM FIG. 3. Specificity of photoaffinity labeling of mAb BA7.1 H

chain. mAb BA7.1 was photolabeled for 20 min with 20 n~ NMBA in the presence of varying concentrations of benzamil (panel a ) or dimethylamiloride (panel b) . Photolabeled proteins were analyzed by SDS- PAGE and immunoblotting as described under “Experimental Proce-

arrow. Benzamil inhibited labeling of the H chain of mAb BA7.1. Di- dures.” Migration of the immunoglobulin H chain is indicated by the

methylamiloride did not inhibit photoincorporation of NMBA.

residues L7 and L8) bulges out with respect to the SP tract found at the same sequence position in FVB (Kieber-Emmons and Wiener, 1992). The structure for the FVB template was adopted for this region of mAb BA7.1 L chain (through framework 11, whereas the remainder of the structure was adopted from F19. Docking of Amiloride to mAb BA7.1-A model of amiloride

bound to mAb BA7.1 is illustrated in Fig. 6. This model indicates that the chlorine atom on amiloride might be stabilized by an electrostatic interaction with the histidine residue at position HlOOh of the H chain. Analysis of the van der Waals contacts for this histidine and the chlorine atom indicate that substitution of atoms with larger radii might cause steric conflicts in binding to the antibody, in agreement with our previous observation that an amiloride analog with an iodo substitution at this position (ie. iodoamiloride) did not bind mAb BA7.1 (Kleyman et al., 198913). The antibody combining pocket is sur- rounded by tyrosine residues located at positions L32 and L94 on the L chain, position HlOOi on the H chain, and a phenylalanine residue at position H95 on the H chain. The 5-amino group of amiloride is stabilized by hydrogen bonding andor electrostatic interactions with the backbone carbonyl of glycine L89 and the hydroxyl group of serine L34. The pyrazine ring has stacking interactions with tyrosine L96, and the guanidinium moiety has stacking interactions with tyrosine L94. The carbonyl moiety hydrogen bonds with the backbone amide of tyrosine L94. Theoretical structure analysis (Smith et al., 19791, confirmed by x-ray analysis (Ball, 1991) of amiloride indicates that the 3-amino group of amiloride is involved in hydrogen bonding with the carbonyl moiety. Although the 3-a- mino group is not involved in hydrogen bonding to mAb BA7.1, this group appears to be stabilized by stacking interactions with tyrosine L96 and perhaps by polarization effects. The model indicates that the addition of substituents at the 5-a- mino group will abrogate binding, in agreement with previous experimental observations. In addition, the model indicates that amiloride analogs with substituents at the 3-amino group will not bind.

The model suggests that analogs with single substitutions on an amino-terminal group of the guanidinium moiety will still bind mAb BA7.1. This result also agrees with experiments

examining the binding of amiloride analogs to this antibody (Kleyman et al., 1989b). The model also indicates that two substitutions on an amino-terminal group on the guanidinium moiety might abrogate binding because of steric conflicts between substituents and tyrosine HlOOi, in agreement with experiments reported in Table I (compound 3). The addition of a nitrogen atom between the carbonyl and guanidinium moiety of amiloride may result in abrogation of binding by loss of stacking interactions with tyrosine L94 or by an increase in steric hindrance if the N-N bond is unable to rotate (see Table I, compound 1).

Photoaffinity labeling of the H chain with NMBA might occur at the histidines at positions HlOOh, H50, or H35 or tyrosine at position HlOOi (Cornelisse and Havinga, 1975). These are the potential residue types on the H chain which either surround or are in the vicinity of the antigen combining site that could incorporate into activated NMBA via aromatic nucleophilic substitution. Reactivity with histidine HlOOh would require that the photoreactive moiety, which is coupled to amiloride through the guanidinium group, be inserted into the pocket first. Such an orientation for amiloride is not preferred under the conditions of the modeling of mAb BA7.1 and is in contrast to the halide positioned toward this residue in the model presented in Fig. 6. However, we have not accounted for conformational changes or induced fitting in the binding of amiloride to mAb BA7.1. Positions H35 and H50 are not in proximity to the putative amiloride binding site. Models placing amiloride in contact position with these two histidines indicate undue steric hindrance, suggesting that NMBA is not bound to either of these residues. The most likely residue for NMBA incorporation is tyrosine HlOOi which is adjacent to histidine HlOOh that stabilizes the halide. However, in our model tyrosine HlOOi does not stabilize amiloride directly by stacking interactions or by hydrogen bonding interactions. Our model, however, suggests that NMBA incorporation on this residue could block access to the binding site.

DISCUSSION Amiloride binding site(s) on transport proteins interact with

specific amiloride derivatives, suggesting that these proteins may interact with specific regions of amiloride. Our previous studies indicate that anti-amiloride antibodies also recognize specific regions on the parent compound (Kleyman et al., 1989b). To correlate these recognition properties we have examined the sequence and structure of an anti-amiloride antibody, mAb BA7.1, that recognizes amiloride analogs in a manner similar to that of epithelial Na’ channels. A tertiary structure model of the mAb BA7.1 was developed to examine the topology of an amiloride binding site. Analysis of the binding of amiloride to the model of mAb BA7.1 indicated that planar aromatic amino residues, including tyrosine, phenylalanine, and histidine, appear to form a pocket into which amiloride, a largely planar molecule, inserts. Our model of the antigen binding site correlated with the binding specificity of mAb BA7.1 with amiloride analogs. The histidine in the H chain CDR3 at position HlOOh can participate in an electrostatic interaction with the chlorine atom on amiloride. This histidine derives from either a nonconservative substitution mutation of germ line sequences or a nontemplate-encoded nucleotide insertion (N nucleotide) during the recombination process in the H chain CDR3 (Fig. 7). Analysis of the van der Waals contact for this histidine and the chlorine atom indicated that substitution of atoms with larger radii might cause steric conflicts in binding to the antibody and correlated with the lack of binding of iodoamiloride to mAb BA7.1. The model also suggested that substitution on the amino group in position 5 on the pyrazine ring will also abrogate binding of amiloride, which has been


a 10

t t t t 20 e t GAG GTC CAG CTG CAA CAG T C T GOA CCT GAG CTG GTG AAG CCT GGG GCT TCA GTG AAG ATA Clu V a l G l n Leu G l n G l n Sor G l y Pro G l u Lou V a l Lyr Pro G l y A l a Ser Val Lyr 118

40

TCC TGC AAG GCT TCT GOT TAC TCA TTC ACT GGC TAC TAC ATC CAC TGG GTG AAG CAA AGC

3 0 t t t t t t

Jer Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Tyr f le P l s Trp V a l Lyr G l n 5.1

C D R I

5 0 52 52. 53 t s9

t t t

CAT GTA AAG AGC C T T GAG TGG A T T GGA CAT ATT AGT CCT TAC M T GGT GCT ACT ACC TAC Hls Val Lyr Ser LOU GlU Trp I 1 0 G l y H i s 218 SOr Pro Tyr Arn G l y Ala Thr Thr Tyr

t e

I COB2

6 9 79

M C CAG AAT TTC M G GAC ACG C C C AGC T T G A C T GTA G A T M G T C C F C C ACC TCA GCC TAC Asn G l n Asn Pho Lyr Asg T R r Ala Ser Leu Thr Val Asp Lys Ser 5.1 Thr Ser A l a Tyr

96

ATG GAG C T C CAC AGC CTG ACA T C T GAG GAC T C T GCA GTC TAT TAC TGT GCA AGA T T T M T net G l u Leu Ills Ser Leu Thr Ser G l u Asp S e r Ala Val Tyr T y r Cyr Ala Ar9 Phe Asn -

e t t t t t

8 2 8 2 1 82b 02c 8 3 8 6 t 4 t t e

99 100h1001100jlOOkl01 103 t t t 11 3 . t

TAC TAC GGT CAC TAT ACT ATG GAC TAC TOG G G T CAA GGA ACC TCA GTC ACC GTC TCC TCA T y r T y r G l y P i s T y r Thr Ekt Asp Tyr Trp Gly Gln G l y Thr Ser Val Thhr Val Ser ser

b 1 0 20 e t t t t e

M C A T T G T A ATG ACC C M T C T C C C M A TCC ATG TCC A T 0 T C A G T A GOA GAG AGO GTC ACC Arn 11 0 Val #tot T h r Gln Ser pro Lyr See Net Sor not sor V a l G l y G l u Arg Val Thr

t t 3 0 t t t

40 t

TTG ACC TOC AAG GCC ACT GAG AAT GTG GTT ACT TAT GTT TCC TGQ T A T C M CAG AAA'CCA Leu T R r Cyr Lys A1 a Ser G1 u A S 0 Val V a l Thr Tyr V a l Sor Trp T y r Gln GJ n Lys Pro

CDRI

t 5 0 t t e

60 t

7 0 80 t t t e t t

CGC TTC ACA GGC AGT GGA TCT GCA ACA GAT TTC ACT CTG ACE ATC AGC ACT GTG.CAG GCT Ar9 pho T h r ~ l y Sor Gly Sor Ala Thr Asp Pho Thr Lou The 118 Sor Sor Val G l n Ala

t t 90 t t t

100 t

GAA GAC CTT GCA GAT TAT CAC TCT GGA CAG GGT TAC AGC TAT CCG TM: M:G T T C GGQ G l U A l p LOU Ala Alp Tyr P i s CyS GJy G l n G l y T y r So* Tyr Pro rye Thr Ohm Gly Gly

C D I I t t

GGG ACC AAG CTG GAA ATA AAA Gly Thr L y s Leu 01 u I l o L y s

FIG. 4. Sequence of H and L chains of mAb BA'7.1. The nucleotide and deduced amino acid sequence of the variable region of mAb BA7.1 H chain are illustrated in p a n e 2 a. The nucleotide and deduced amino acid sequence of the variable region of mAb BA7.1 L chain are illustrated in panel b. CDRs are underlined. Codon numbering is according to convention (Kabat et al., 1987).

observed experimentally (Kleyman et al., 1989b). The presence nogen and seems to have emphasized the steric nature of amilo- of one substituent on a terminal nitrogen of the guanidinium ride binding. This asped is implicit in our modeling approach. moiety will not interfere with amiloride binding to mAb BA7.1, The concept of amiloride forming an initial complex with the although two substituents on a terminal nitrogen atom will Na+ channel mediated by the charged guanidinium moiety (Li abrogate binding, in agreement with data presented in Table I. et al., 1987) is not represented in the model of BA7.1. Exami-

mAb BA7.1 was generated with an amiloride analog coupled nation of charged residues in the H or L chain which line the to carrier protein through the guanidinium group as an immu- combining site indicates that BA7.1 is devoid of negative

Topology of an Amiloride Binding Site

a HY-HEL-s ~ u ~ ~ o o s ~ n ~ ~ n ~ c ~ n ~ ~ ~ ~ s c ~ n s ~ v ~ ~ s 1n7. I E U O L O O ~ O ~ E L ~ K ~ ~ ~ S ~ K I ~ ~ K ~ ~ ~ V ~ F T

FRl

s 2 r S I

2811

HY-HEL-s D v u I L u u K o R c e n e L E u I e E I ~ ~ i - 8 s - < s T n v 017. I ~ v v ~ n u u ~ o s n u ~ s ~ ~ u ~ e n ~ s ~ ~ n ~ a ~ ~ ~

C D R l c 112 C D R 2

HV-HEL-S S 0 U V V C L H 6 M V D F u o u e o e n c c c s o 3 c n ~ n ~ v e s ~ u ~ ~ ~ u n e n e 117. t S ~ U V Y C ~ R F ~ Y Y ~ O I

C D R 3

b r e

IN. I n ~ u n ~ ~ s c ~ ~ n s n s u e ~ ~ ~ ~ ~ ~ c ~ a s ~ n u u FUI ~ ~ u ~ ~ ~ ~ c n ~ n s n s ~ o ~ ~ u ~ n ~ c s n s s s u s F19 D I O ~ T O T T ~ ~ L ~ ~ ~ L ~ D R U T I ~ ~ R R ~ ~ D I ~

FI 1 COR1

FUI - V ~ H U V O O K S ~ T S ~ K R U I V D T S K L ~ ~ ~ U ~ ~ FI9 ~ Y L ~ U V O O K S ~ T S ~ K R U I V D T ~ K L ~ S ~ U ~ ~ 1n7. t ~ S U V O O K ~ L O S ~ K L l l V ~ ~ 6 U ~ D

Fl2 C D R 2

FUI R F ~ O S ~ S ~ T ~ Y ~ L T I ~ ~ ~ E ~ E D ~ ~ T V V ~ ~ ~ Ft9 R F ~ ~ ~ ~ S ~ T D Y ~ L T I S ~ L E ~ E D I ~ T V F C O ~ 017. t R F T ~ ~ ~ S ~ T D C ~ L T I ~ S U O ~ E D L R D V ~ C ~

F I 3

FUI U S S M P V T F 6 6 6 F19 0 S T T P R T F B 6 6

C D 1 3 1117. t G U F 6 6 6

structure of the anti-lysozyme antibody HY-HEL-5 provides a canonical structure for the CDR2 of BA7.1. CDRs of mAb BA7.1 are underlined. FIG. 5. Sequence comparisons of mAb BA7.1 H chain and L chain with immunoglobulins of known crystal structure. Panel a, the

Framework (FR) regions indicated in the figure are not underlined. Panel b, the L chain F19 provides a canonical structure for mAb BA7.1 L chain.

BA7.1 and in the L chain FVB. The FVB template was adopted for the framework (FR) 1 region of the model of mAb BA7.1 L chain. CDRs of mAb However, the TT tract of F19 (residues 7 and 8) adopts a configuration that differs from the SP tract found at the same sequence position in mAb

BA7.1 are underlined.

charges in this region (data not shown). The immunization strategy precludes the guanidinium moiety from initially in- serting into the binding site first. Our model suggests that the 5th and 6th positions of the pyrazine ring of amiloride (Fig. 1) initially insert into the antibody pocket. The placement of the chloro group of amiloride in this model emphasizes the possible steric restriction of iodoamiloride. Previous studies have suggested that steric factors influence the binding of the 6-iodoamiloride to the Na+ channel (Venanzi et al., 1992).

Amino acid-specific reagents have been used to identify putative residues that participate in the binding of amiloride andor Na+ to the epithelial Na+ channel, the Na+/H+ exchanger, and the Na+/glucose cotransporter. Both EEDQ and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole inhibit epithelial Na+ channels in toad urinary bladder in an amiloride protectable manner, suggesting that acidic amino acid, histidine, and tyrosine residues are in proximity to the amiloride binding site on the channel (Park and Fanestil, 1980, 1983; Park et al., 1983). Tyrosine residues are located at or near the Na+ binding site of the Na+/glucose cotransporter, as chemical modification of tyrosine residue(s1 inhibits this transporter in an Na+-protectable manner (Peerce and Wright, 1985). Similarly, studies using either diethylpyrocarbonate or EEDQ suggest that histidine and acidic amino acid residues participate in amiloride binding to the Na+/H+ exchanger in placental or renal

brush-border membranes (Ganaphthy et al., 1987; Grillo and Aronson, 1986). Site-directed mutagenesis of specific histidine residues of the Na+/H+ exchanger (NHE1 isoform) alters amiloride sensitivity, providing additional evidence of the role of histidine in amiloride binding (Wang et al., 1991). These previous studies in which alterations of specific amino acid residues result in modifications of ion transport in an amiloride protectable manner must be interpreted with caution. The altered residues might not contact amiloride but might be required for the amiloride (or Na') binding site to assume proper topology. Nevertheless, our studies suggest that mAb BA7.1 mimics some of the salient features of the amiloride (or Na+) binding site on amiloride-sensitive transport proteins. The observation that histidine and tyrosine residues play a prominent role in stabilizing amiloride binding to mAb BA7.1 indicates that a subset of these residues on amiloride-sensitive proteins participates in amiloride binding and that chemical modification of these residues could interfere with amiloride binding.

Pouyssegur and co-workers devised an alternative approach to attempt to characterize the amiloride binding site of the NHEl isoform of the Na+/H+ exchanger (Counillon et al., 1992b). They selected fibroblasts that express a Na+/H+ exchanger that is insensitive to an amiloride analog by subjecting fibroblasts to an acid load and allowing cells to recover in bi- carbonate-free medium in the presence of increasing concen-

2812

I

Topology of an Amiloride Binding Site

b

Rc. 6. Docking of d o r i d e to mAb BA7.1. Panel a , a stereoscopic view of a Cartesian trace of mAb BA7.1 is presented with amiloride and interacting residues depicted in solid sphere rendering. The H chain of mAb BA7.1 is colored orange, and the L chain is colored blue. Amiloride is colored yellow, with the chlorine halide atom colored green. Histidine lOOh of the H chain is colored red. Serine 34 of the L chain is colored light purple, with the glycine at position 89 of the L chain colored dark purple. The tyrosines at positions 94 and 96 on the L chain are colored green. Pane b, the Cartesian trace of the backbone of the antibody BA7.1 with amiloride in ita putative binding site.

D t L l 6 TT TAT TAC TAC GOT A TY r

As n BA7 T-- --- --- --- C JBI BA7.1 C-- --- -- -I Tyr Ala

Wis T h r

TAC TAT GCT ATQ G A C TAC

FIG. 7. Comparison of mAb BA7.1 H chain CDRS with the reported germ line sequences. Noncon!ervative substitution muta- tions andor N nucleotide insertion during the recombination process in the H chain CDR3 were observed.

trations of the amiloride analog MPA. One clone isolated following this selection procedure expressed a Na+/H+ exchanger with an apparent 30-fold decrease in affinity for MPA. Sensi- tivity of the mutant Na+/H+ exchanger to amiloride was not altered to the same extent. This mutant had a single point mutation changing a leucine in position 167 to a phenylalanine (Counillon et al., 1992a). Although this approach led to the identification of an amino acid residue that appears to participate in binding of the amiloride analog MPA, the amino acid residue identified in these studies is not necessarily in contact with MPA. As mentioned above, mutation or modification of an amino acid residue outside the contact site could indirectly

alter the topology of the amiloride binding site. It is natural to ask what similarities exist among proteins

that bind the same ligand. Anti-amiloride antibodies are not necessarily expected to exhibit primary sequence homologies with amiloride-sensitive transport proteins, as these transport proteins do not share extensive sequence homology among themselves. Indeed, this is what we have observed in searching protein sequence data bases (data not shown). However, the most salient features of ligand binding are the residues that contact the ligand (Williams et al., 1991a, 1991b). Among the various proteins that bind amiloride, it is conceivable that these contact residues are conserved, although the structural realization of a binding site could be achieved in multiple ways. For example, sequence alignment between the C D B of the H chain of mAb BA7.1 with the protein Apx, a subunit of the epithelial sodium channel which has been postulated to contain the amiloride binding site (Staub et al., 1992), indicates a sequence similarity between the sequence tract NYYGH at positions H96-HlOOh of mAb BA7.1 with the tract NYF’GR at positions 195-199 of Apx. Analysis of crystallographically known structures shows that GR and GH residues can be located in turn regions, displaying similar conformational properties

Topology of an Amiloride Binding Site 2813 (data not shown).

A subset of Na+-coupled solute transporters, including those that transport glucose, proline, and glutamate, have a common motif that involves the residue tract GR. This motif, b AXXXXZXXXGR (where X is any residue, and the elongated dash is tantamount to 37-40 residues in length), has been postulated to participate in Na+ transport (Deguchi et al., 1990). An equivalent motif has been observed in a Na+/PO, cotransporter which contains a PR tract at the analogous GR position (Werner et al., 1991). Since Na+ symport is common among these four transport proteins, these conserved amino acids could be essential for Na+ recognition or binding, although there is no direct evidence to support this hypothesis. However, this motif has not been observed in other amiloride- sensitive Na+-coupled transporters, including NHE and Na+/ Ca2+ exchangers.

Different amino acid residues on amiloride-sensitive proteins could contribute to the binding of amiloride. For example, the guanidinium group of amiloride might participate in forming hydrogen bonds with residues such as serine or threonine, it might be involved in stacking interactions with planar residues such as tyrosine, or it might be involved in salt bridges or hydrogen bonding with negatively charged groups. Our model of mAb BA7.1 suggests that the guanidinium group participates in stacking interactions. Recent progress on the prediction of secondary and tertiary structures of proteins has re- phrased the problem of structure prediction as one of pattern matching of sequences where, instead of matching one sequence with another, a sequence must somehow be matched with a given topology (Thornton et al., 1991). The concept of matching a sequence to a given topology is at the heart of comparative modeling approaches. Analyses of crystal structures of proteins have long shown that topology is much better conserved than sequence (Thornton et al., 1991). We suggest that antibodies directed against small molecules, such as amiloride, can potentially mimic the most salient features of receptor binding sites and can be used to guide experiments in dissecting binding site relationships among functionally related proteins. In this regard, analysis of other monoclonal anti-amiloride antibodies should further our understanding of the topology of amiloride binding sites.

Acknowledgments-We thank Dr. J. P. Springer and Dr. R. G. Ball for providing report SRrgb0091b detailing the fractional Cartesian coordinates of unprotonated amiloride.

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topology of an hiloride-binding protein*

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