Antiidiotypic Antibody Recognizes an Amiloride Binding Domain
within the
Subunit of the Epithelial Na+ Channel*
Thomas
Kieber-Emmons
,
Chaomei
Lin§¶
,
Mary H.
Foster§, and
Thomas R.
Kleyman§¶**
From the Departments of § Medicine,
Pathology, and ** Physiology, University of
Pennsylvania, and the ¶ Veterans Affairs Medical
Center, Philadelphia, Pennsylvania 19104
 |
ABSTRACT |
We previously raised an antibody (RA6.3) by an
antiidiotypic approach which was designed to be directed against an
amiloride binding domain on the epithelial Na+
channel (ENaC). This antibody mimicked amiloride in that it inhibited transepithelial Na+ transport across A6 cell monolayers.
RA6.3 recognized a 72-kDa polypeptide in A6 epithelia treated with
tunicamycin, consistent with the size of nonglycosylated Xenopus
laevis
ENaC. RA6.3 specifically recognized an amiloride
binding domain within the
-subunit of mouse and bovine ENaC. The
deduced amino acid sequence of RA6.3 was used to generate a
three-dimensional model structure of the antibody. The combining site
of RA6.3 was epitope mapped using a novel computer-based strategy.
Organic residues that potentially interact with the RA6.3 combining
site were identified by data base screening using the program LUDI.
Selected residues docked to the antibody in a manner corresponding to
the ordered linear array of amino acid residues within an amiloride
binding domain on the
-subunit of ENaC. A synthetic peptide spanning
this domain inhibited the binding of RA6.3 to
ENaC. This analysis
provided a novel approach to develop models of antibody-antigen
interaction as well as a molecular perspective of RA6.3 binding to an
amiloride binding domain within
ENaC.
 |
INTRODUCTION |
Epithelial Na+ channels
(ENaCs)1 are expressed in a
variety of tissues, including the distal nephron of the kidney, airway
and alveolar epithelia in the lung, surface cells in the distal colon, urinary bladder epithelia, skin, and ducts within salivary and sweat
glands (1). These transporters facilitate the movement of
Na+ across the apical (or luminal) plasma membrane and have
a critical role in extracellular fluid volume homeostasis, control of
blood pressure, fetal lung maturation, and maintenance of airway fluids (1). ENaCs consist of at least three homologous subunits, termed
-,
-, and
ENaC, and are thought to form a tetrameric complex consisting of 2
-, 1
-, and 1
-subunits (2-5), although one
group has suggested a subunit stoichiometry of 3
-, 3
-, and
3
-subunits (6). cDNAs encoding these Na+ channel
subunits have been isolated and characterized from a variety of
species. Each ENaC subunit has two predicted membrane-spanning domains.
The amino- and carboxyl-terminal regions of the ENaCs are cytoplasmic,
and each subunit has a large ectodomain (7-9). Domains of largely
hydrophobic residues are located immediately following the putative
first membrane-spanning domains and immediately preceding the second
membrane-spanning domains of
-,
- and
rENaC (8, 10, 11). The
hydrophobic domain immediately preceding the second membrane-spanning
region of ENaC (referred to as the H2 domain) may insert in the
membrane and form part of the channel pore (3, 7, 12).
The diuretic drug amiloride is a prototypic inhibitor of epithelial
Na+ channels. Several sites have been identified within the
epithelial Na+ channel which participate in amiloride
binding. Mutagenesis studies suggest that residues preceding the second
membrane-spanning domain (i.e. H2 domain) of
-,
-, and
ENaC and residues within the second membrane-spanning (M2) domain of
ENaC participate in amiloride binding. These data provide strong
evidence that amiloride interacts with residues within or in close
proximity to the channel pore (3, 12, 13). We recently identified a
6-amino acid residue tract within the extracellular domain of the
-subunit of ENaC which participates in amiloride binding (14). The
identification of this 6-residue tract was based on defining the
interaction of an anti-amiloride antibody with amiloride and consequent
sequence homology between the anti-amiloride antibody and a residue
tract within the extracellular domain of the
ENaC (14-16).
Antibodies raised against antihormone (or antiligand) antibodies have
been shown to bind the hormone's (or ligand's) physiologic receptor
(17). These antiidiotypic antibodies, in effect, function as
antireceptor antibodies. We previously generated a monoclonal antiidiotypic antibody (RA6.3) that mimics the effect of amiloride by
inhibiting Na+ transport across A6 cell monolayers (18).
The antibody also inhibits the amiloride-sensitive mechanoelectric
channel of outer hair cells in adult
cochlea.2 The antibody was
used to immunoprecipitate putative Na+ channels from A6
cells and to immunolocalize putative Na+ channels in rabbit
kidney and in a Xenopus epithelial (A6) cell line. However,
the interaction of RA6.3 with cloned ENaC subunits has not been demonstrated.
We now provide evidence that RA6.3 recognizes the
-subunit of ENaC
and is directed against a region within the extracellular domain of
ENaC containing the 6-residue amiloride binding domain we identified
previously (14-16). Analysis of the topology of the antigen combining
site of RA6.3 provided an opportunity to examine potential interactions
of RA6.3 with an amiloride binding domain within
ENaC. We report the
primary sequence of the variable regions of the light (L) and heavy (H)
chains of RA6.3. A molecular structure of RA6.3 was created by homology
modeling, and an epitope map of potential amino acid side chains that
could interact with the RA6.3 combining site (15, 19, 20) was generated
using a structure-based computer screening approach. This epitope map confirmed the potential interaction of an
ENaC amiloride binding domain with RA6.3. The theoretical epitope mapping approach may have
role in examining protein-protein interactions in other systems.
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EXPERIMENTAL PROCEDURES |
Materials--
Electrophoresis reagents were purchased from
Bio-Rad and Tran35S-label from ICN (Costa Mesa, CA). All
other reagents were purchased from Sigma and were reagent grade unless
indicated otherwise.
Cell Culture--
A6 cells were derived from Xenopus
laevis kidney. A subclone of this cell line (2F3) was a gift from
Drs. J. P. Kraehenbuhl and B. C. Rossier (University of Lausanne). A6
cells between passages 92 and 96 were seeded on 4.7-cm2
polycarbonate filters from Costar (Cambridge, MA) at 106
cells/cm2 and maintained for 7-15 days in a humidified
28 °C incubator with 5% CO2 in a medium containing
Leibovitz's medium L-15 (7 parts) and Coon's medium F-12 (3 parts),
modified for amphibian cells by adjusting the final
HCO3
concentration to 25 mM and the final osmolality of 230 mosmol/kg H2O, and supplemented with 5% (v/v) fetal calf serum (21).
A6 cell monolayers were transferred to a modified Ussing chamber and
bathed in a Ringer's saline containing [mM] 100 NaCl, 4 KCl, 2.5 NaHCO3, 1 K2HPO4, 1 CaCl2, 11 glucose, and buffered with HEPES. Electrical
measurements were performed with a modified Ussing chamber and a
DVC-1000 voltage clamp (World Precision Instruments) as described
previously (21, 22). The amiloride-sensitive component of the short
circuit current was determined by adding 10
5
M amiloride to the luminal solution. Data are reported as
mean short circuit current ± S.E.
Metabolic Labeling and Immunoprecipitation--
Cells were
incubated overnight (16 h) with A6 medium supplemented with 300 nM aldosterone without tunicamycin, or with either 0.5 or 5 µg/ml tunicamycin. Cells were washed three times with methionine,
cysteine, and serum-free A6 medium supplemented with 300 nM
aldosterone and 0, 0.5, or 5 µg/ml tunicamycin and then incubated for
15 min with 1 mCi/ml
[35S]methionine/[35S]cysteine added to the
basolateral surface as described previously (23, 24). Cells were then
washed once and incubated for 1 h at 28 °C in A6 medium
supplemented with 2.5 mM methionine, 2.5 mM
cysteine, 300 nM aldosterone, and 0, 0.5, or 5 µg/ml
tunicamycin. Immunoprecipitations were performed as described
previously (18) with a 1:250 dilution of RA6.3 (raised in ascites) or
control ascites (Sigma). The immunoprecipitated proteins were eluted
into a SDS-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (3% (w/v) SDS, 15% (w/v) sucrose and 92.5 mM Tris-HCl, pH
6.9). Samples were incubated with 10 mM dithiothreitol and
boiled for 1 min prior to SDS-PAGE, which was performed as described
previously (18).
In Vitro Translation, Immunoprecipitation, and
Immunoblot--
Mouse and bovine
ENaC were transcribed and
translated in vitro using the TNTTM-coupled
reticulocyte lysate system (Promega, Madison, WI) according to the
manufacturer's instructions. 20 µl of in vitro translated
mENaC was diluted to 0.5 ml of a buffer containing 0.4% (w/v) sodium deoxycholate, 1% (v/v) Nonidet P-40, 50 mM EGTA, 10 mM Tris-HCl, pH 7.4, containing a protease inhibitors
mixture (10 µM phenylmethylsulfonyl fluoride, 1 µM antipain, 1 µM leupeptin, 1 µM pepstatin A). Solubilized proteins were preabsorbed
for 1 h at 4 °C with 50 µl of protein G-agarose, centrifuged
to pellet the protein A-agarose, and then incubated for 16 h with
RA6.3 (1:500 dilution) in the absence or presence of an excess of the peptide DAVREWYRFHYINILSRLSD (20 µg) at 4 °C. Protein G-agarose (50 µl) was then added, and after a 1-h incubation at 4 °C, the agarose beads were washed three times with a buffer containing 150 mM NaCl, 5 mM EGTA, 1% (v/v) Triton X-100, 50 mM Tris, pH 7.4, and protease inhibitors and twice with a
buffer containing 0.1% (w/v) SDS, 2 mM EGTA, 10 mM Tris-HCl, pH 7.4, and protease inhibitors. Precipitated
samples were eluted into SDS-PAGE sample buffer (3% (w/v) SDS, 15%
(w/v) sucrose, and 92.5 mM Tris-HCl, pH 6.9). Samples were
incubated with 10 mM dithiothreitol for 20 min and
subjected to 7.5% SDS-PAGE and autoradiography.
Alternatively, in vitro translated
bENaC (5 µl) was
subjected to 7.5% SDS-PAGE, and proteins were transferred to
nitrocellulose or polyvinylidene difluoride membranes as described
previously (23, 24). Western blot analysis was performed using an
alkaline phosphatase-linked chemiluminescent detection system (Western Light Plus) according to the manufacturer's instructions. In brief, nitrocellulose membranes were incubated in a blocking buffer consisting of 68 mM NaCl, 75 mM NaPO4, pH 7.4 (PBS), supplemented with 0.2% (w/v) purified casein, 0.1% (v/v) Tween
20, for 1 h at room temperature. Membranes were then incubated
with blocking buffer containing either the antibody RA6.3 (1:500
dilution) in the absence or presence of the peptide
DAVREWYRFHYINILSRLSD. Membranes were subsequently washed twice with PBS
supplemented with 0.1% (v/v) Tween 20 and then incubated with a
biotinylated anti-mouse IgG antibody (1:20,000 dilution in blocking
buffer) at room temperature for 30 min. Membranes were washed twice in
PBS and 0.1% Tween 20, incubated with a streptavidin-alkaline phosphatase conjugate (1:20,000 dilution in blocking buffer) at room
temperature for 30 min, and washed again three times with PBS and
Tween. The membranes were washed in assay buffer (0.1 M
diethanolamine, 1 mM MgCl2, pH 10), incubated
for 5 min in a substrate solution (0.25 mM CSPD in assay
buffer) at room temperature, and then exposed to film.
Oligonucleotide Synthesis--
The mouse C
primer was the
15-mer oligodeoxynucleotide 5'-GGCCAGTGGATAGAC-3'. The mouse C
probe
was 5'-TGGGG(CG)TGTTGTTTT-3'. The mouse C
primer was
5'-CTGCTCACTGGATGGTGGGA-3', and the C
probe was
5'-AGATGGATACAGTTGGT-3'. The anchor primer for polymerase chain
reaction was a 33-mer oligodeoxynucleotide
5'-CACTGCAGAAGCTTGGATCCCCCCCCCCCCCCC-3' containing PstI,
HindIII, and BamHI restriction sites. Probes were
5'-end radiolabeled with T4 polynucleotide kinase and
[
-32P]ATP as described previously (25) and purified
over NENSORB-20 cartridges (NEN Life Science Products).
Cloning and Sequencing of the Variable Region and H Chain of
RA6.3--
The nucleotide sequence of the H region of RA6.3 was
obtained using methods described previously (15). Briefly, total RNA was extracted from RA6.3 hybridoma cells cultured in RPMI 1640 medium
supplemented with 10% fetal bovine serum by the guanidinium/CsCl method as described previously. Reverse transcription was performed using RNA as a template with Moloney mouse leukemia virus reverse transcriptase and C
oligonucleotide primer. Escherichia
coli DNA polymerase I (26-28) was then used to synthesize
double-stranded cDNA. T4 DNA polymerase was used to generate blunt
ended cDNA, which was ligated into SmaI-digested and
dephosphorylated M13. E. coli DH5-
F'IQ were transformed
with recombinant phagemids and clones screened with the C
(H chain)
oligonucleotide probe (25). 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.
Cloning and Sequencing of the Variable Region L Chain of
RA6.3--
The method of random extension of cDNA ends was used to
obtain the L chain variable region cDNA. Reverse transcription was performed using RNA from RA6.3 hybridoma cell as a template with Moloney mouse leukemia virus reverse transcriptase and C
oligonucleotide primer at 37 °C for 1 h. An oligo(dG) tail was
added to the cDNA with terminal deoxynucleotide transferase. A
polymerase chain reaction was performed using poly(dC) and the C
3'-primer to amplify oligo(dG)-tailed cDNA. Samples were initially
denatured at 92 °C for 8 min followed by 35 cycles of 92 °C for 1 min, 58 °C for 2 min, and 72 °C for 3 min and a final extension
for 7 min at 72 °C. Products of the correct predicted size were
cloned into a pCR vector (Invitrogen) and sequenced using the C
oligonucleotide as a sequencing primer, as described above.
Model Building and Energy Refinement--
Antibody modeling was
performed as described previously (15, 29-31). For modeling of the H
chain complementarity determining region (CDR) 3 region in particular,
we utilized both a knowledge-based approach to search for suitable
structures that fit a geometry to the base residues of the CDR3 domain
(32-34) and a conformational search procedure (35-38). The latter
involved use of the program AbM (Oxford Molecular Inc.). CDRs and the
framework regions of identified templates were mutated to those of the
respective antibody H and L chains using Insight II (Biosym
Technologies) or automatically assigned by AbM. The program Discover
(version 2.95, Biosym Technologies) was used for conformational
calculations with the supplied consistent valence force field
parameters. The respective modeled structure was energy optimized to
convergence. Molecular dynamics at 300 K and
600 K was used to alleviate any further close contacts
within the antibodies (15, 29-31).
LUDI Search--
In the exploration of the potential epitope
recognized by RA6.3, we made use of the program LUDI (39) (Ligand
Design, Biosym Technologies) to identify amino acid residues that
potentially interact with the RA6.3 combining site as described
previously (20). A LUDI search was performed using standard default
values and fragment library supplied with the program to identify
organic fragments that potentially interact with residues within the
RA6.3 binding site. Amino acid residues with side chains similar in structure to these organic fragments were identified. In evaluating these fragments, we compared amino acid residues identified by LUDI
relative to the VRDWYRFH sequence such that the fragments could occupy
nonredundant sites, be spatially far enough to accommodate the peptide
backbone, and follow the appropriate ordered sequence of amino acid
residues. In effect, one wants to "stitch" the fragments together
to form a peptide that fits within the RA6.3 combining site. To
identify possible fragments that are similar to the VRDWYRFH sequence,
the radius of interaction, which defines the size of spheres in which
LUDI is to fit appropriate fragments, was set as incrementing radii
from 5 to 14 Å. The VRDWYRFH peptide was built using Insight II and
positioned relative to the docked LUDI fragments. The peptide backbone
and side chain torsional angles were rotated until the side chains of
the peptide were approximate to the corresponding LUDI fragments. The
peptide-RA6.3 complex was subjected to energy optimization and
molecular dynamics simulations to relieve steric overlap.
 |
RESULTS |
RA6.3 Recognizes
ENaC--
RA6.3 is a monoclonal antibody
raised by an antiidiotypic approach designed to be directed against an
amiloride binding site within the epithelial Na+ channel
(18). We demonstrated previously that RA6.3 immunoprecipitates a
complex of polypeptides expressed at the apical plasma membrane of A6
epithelia. Cells were treated with tunicamycin to inhibit N-linked glycosylation in order to identify
non-N-linked glycosylated polypeptide(s) recognized by
RA6.3.
A6 cells were incubated overnight (16 h) in the presence (0.5 or 5 µg/ml) or absence of tunicamycin. Treatment of A6 cells with
tunicamycin for 16 h led to a reduction in short circuit current
from 12.4 µA × cm
2 (0 tunicamycin) to 0.7 µA × cm
2 (0.5 µg/ml tunicamycin) and 0.5 µA × cm
2 (5 µg/ml tunicamycin). The A6 monolayers were
metabolically labeled in the absence or presence (0.5 or 5 µg/ml) of
tunicamycin, detergent solubilized, and subjected to
immunoprecipitation with RA6.3. The immunoprecipitate was analyzed by
SDS-PAGE and autoradiography. In the absence of tunicamycin, RA6.3
immunoprecipitated a complex of polypeptides from metabolically labeled
A6 cells, as we have observed previous (18, 40) (Fig.
1A, left panel).
However, in the presence of 0.5 or 5 µg/ml tunicamycin, RA6.3
immunoprecipitated a 72-kDa polypeptide from A6 cells, consistent with
the predicted size of nonglycosylated
xENaC (Fig. 1A,
left panel) (41). This 72-kDa polypeptide was not
immunoprecipitated when control ascites was used for
immunoprecipitation (Fig. 1B). Interestingly, an additional
polypeptide was immunoprecipitated with an apparent molecular mass of
35-45 kDa and is in agreement with the mass of a novel short chain
alcohol dehydrogenase we cloned previously from an A6 cell expression
library based on its binding to RA6.3 (42). Treatment of A6 epithelia
with tunicamycin altered the profile of total labeled cellular proteins
(Fig. 1A, right panel).

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Fig. 1.
RA6.3 recognizes a 72-kDa polypeptide in
tunicamycin-treated A6 epithelia. A6 monolayers were incubated
overnight with or without tunicamycin, metabolically labeled with
[35S]methionine/[35S]cysteine for 15 min,
and then chased for 1 h with an excess of unlabeled
methionine/cysteine. The cells were detergent solubilized, subjected to
immunoprecipitation with RA6.3, and labeled polypeptides were analyzed
by a 5-13% gradient SDS-PAGE and autoradiography (panel A,
left). Alternatively, cell lysate was subjected directly to
SDS-PAGE and autoradiography to examine the profile of total
metabolically labeled proteins (panel A, right).
The concentrations of tunicamycin used (0, 0.5, or 5 µg/ml) are
indicated at the top of the figure. A complex of
polypeptides was recovered after immunoprecipitation with RA6.3 from
metabolically labeled A6 cells that were not exposed to tunicamycin. A
72- and a 35-45-kDa polypeptide were recovered from cells treated with
0.5 or 5 µg/ml tunicamycin. The 72-kDa polypeptide is consistent with
the predicted size of nonglycosylated Xenopus ENaC. A
change in the profile of total labeled A6 cell proteins was detected in
cells treated with tunicamycin compared with control cells not exposed
to tunicamycin (panel A, right). The migration of
molecular mass standards is indicated to the left of the
figure. The 72-kDa polypeptide was not recovered if nonimmune ascites
(panel B, lane 2) was substituted for RA6.3
(panel B, lane 1) for immunoprecipitation.
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To provide additional evidence that RA6.3 recognizes the
-subunit of
ENaC, in vitro transcription/translation of mouse
ENaC was performed in the presence of
[35S]methionine/[35S]cysteine. The
translated product was analyzed by SDS-PAGE and autoradiography (Fig.
2A). A 72-kDa polypeptide was
observed for mouse
ENaC, in agreement with its predicted molecular
mass. Alternatively, the translation product was product subjected to
immunoprecipitation with RA6.3 (1:500 dilution) in the absence or
presence of the peptide DAVREWYRFHYINILSRLSD, corresponding to residues
273-292 of rat
ENaC, residues 273-292 within mouse
ENaC
(with the exception that residue 291 in the mouse sequence is
Pro), and residues 226-245 within bovine
ENaC (with the exception
that residues 243-245 in the bovine sequence are RRQ). RA6.3
specifically immunoprecipitated in vitro translated mouse
ENaC (Fig. 2A). We also observed that RA6.3
specifically recognized in vitro translated bovine
ENaC (Fig. 2B).

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Fig. 2.
RA6.3 recognizes in vitro
translated ENaC. Panel A,
mouse ENaC cDNA was in vitro transcribed and
translated in the presence of
[35S]methionine/[35S]cysteine and subjected
to 7.5% SDS-PAGE (lane 1). Alternatively, mENaC was
subjected to immunoprecipitation with RA6.3 in the absence (lane
2) or presence (lane 3) of the peptide
DAVREWYRFHYINILSRLSD, which contains an amiloride binding domain
(WYRFHY) that we have identified previously (14). RA6.3 specifically
recognized in vitro translated mENaC. This figure is
representative of two separate experiments. Migration of molecular mass
standards is indicated to the left of panel B.
Bovine ENaC cDNA was in vitro transcribed and
translated in the presence of
[35S]methionine/[35S]cysteine as described
under "Experimental Procedures," subjected to 7.5% SDS-PAGE,
transferred to nitrocellulose, and probed with RA6.3 in the absence
(lane 1) or presence (lane 3) of a peptide
corresponding to residues 273-292 of rENaC and containing the tract
WYRFHY. As a control, the blot was probed with mouse ascites
(lane 2). A 75-kDa polypeptide was observed
(arrow). RA6.3 specifically recognized in vitro
translated bENaC. This figure is representative of three separate
experiments. Migration of molecular mass standards is indicated to the
left of the figure.
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Cloning and Sequencing of the L and H Chain cDNA of
RA6.3--
RA6.3 H chain variable region cDNA was synthesized and
introduced into M13. Individual subclones were isolated on the basis of
hybridization to a mouse C
probe. Sequence analysis of the clones
revealed that the H chain is a member of the VH-36-60 gene family,
subgroup I(A). RA6.3 L chain variable region cDNA was obtained by
random extension of cDNA ends. The L chain is encoded by a member
of the V
23 subgroup. The H chain and L chain cDNAs and deduced
amino acid sequences are illustrated in Fig.
3.

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Fig. 3.
cDNA and deduced amino acid sequences of
monoclonal antibody RA6.3 H chain and L chain variable regions.
CDRs are underlined.
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Structural Properties of RA6.3--
Sequence comparisons with
known immunoglobulin crystal structures in the Brookhaven Protein Data
Base provided templates for the variable regions of RA6.3 (Fig.
4). The primary structure of the L chain
of the anti-lysozyme antibody (3HFM) in complex with lysozyme displayed
78% identity with the RA6.3 L chain through the CDR3 region (Fig. 4).
For the H chain of RA6.3, we identified the anti-dinitrophenyl antibody
(1BAF) also in complex with its ligand, as a template displaying 83%
homology with RA6.3 up to CDR3. The H chain of 3HFM displays 81%
homology with RA6.3 (Fig. 4), with a 1-residue deletion in the CDR1
region of 3HFM. Superpositioning these two templates up to CDR3 and
accounting for the 1-residue deletion in 3HFM results in a root mean
square deviation of 0.99 Å, indicating that these two templates are
similar within their CDR1 and CDR2 domains and framework regions. These
two templates for the RA6.3 L and H chain were also identified by the
AbM program.

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Fig. 4.
Sequence comparisons of RA6.3 H chain and L
chain with immunoglobulins of known crystal structure. Numbering
of amino acid residues is according to Kabat et al.
(68).
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To model the CDR3 region of the RA6.3 H chain, we first examined CDR3
folding patterns of several immunoglobulins whose structures have been
determined by crystallography. Based upon the sequence similarities
between 1BAF with RA6.3, we utilized the 1BAF template and searched for
loops in the Brookhaven Protein Data Bank using the program Insight II.
Two potential structures found within antibodies were identified which
differ in conformation but retain the same CDR3 length as RA6.3. These
structures correspond to the
dimer MCG (2MCG) and the antibody
YST9.1 (1MAM) (Fig. 4). Both loop structures were spliced into the 1BAF
template. A dynamics run was performed to relieve any short contacts in the modeled structures. Minimization of the average conformation from
100 structures for each model, derived from a molecular dynamics production run, resulted in average structures that differed in energy
by 20 kcal, with the MCG spliced loop being the energetically preferred
structure. As expected, the major difference in the structures of these
two models was in the conformation of the CDR3 domain of the H chain.
The low energy average conformation generated by the AbM program was
similar to the MCG-derived conformer type. Its CDR2 region was
different from the starting conformation of the 1BAF template (root
mean square 1.2 Å). This structure was 6 kcal more stable than the MCG
spliced structure. Consequently, both model structures (generated by
the knowledge-based approach and by the AbM program) were used for
screening of the LUDI fragments.
Peptide Placement within the RA6.3 Combining Site--
A point
within the middle of the RA6.3 combining site was used to initiate the
LUDI search. 101 small organic molecules were identified based on their
ability to bind to the combining site within the RA6.3 structure
generated by the knowledge-based approach, and 128 small organic
molecules were identified based on binding to the AbM-generated
structure. Most of the organic molecules that bound to the two RA6.3
structures were redundant for the same set of potential hydrogen bond
donors or acceptors. Fig. 5, A
and B, illustrates the placement of small organic fragments within the RA6.3 structure generated by the knowledge-based approach. Organic structures were observed to interact with both H and L chain
amino acids within the various CDRs of RA6.3. A lipophilic group
representative of a valine side chain was bounded by Leu-96 L chain
(CDR3L) and Tyr-50 H chain (CDR2H) of RA6.3. A Trp-like residue was
bounded by Tyr-50 L chain (CDR2L), and Ser-91 L chain (CDR3L), Asn-92 L
chain (CDR3L) of RA6.3. A guanidinium head group formed hydrogen bonds
with Glu-53 L chain within CDR2L of RA6.3. A Phe-like residue was
bounded by Tyr-34 H chain and Tyr-50 H chain. A His-like group also
interacted with Tyr-34 H chain and Tyr-50 H chain.

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Fig. 5.
Docking peptide fragments to the RA6.3 model:
LUDI search. A variety of nonoverlapping molecular species were
identified to fit within the RA6.3 combining site (panels A
and B). Fragments identified by LUDI were compared with the
side chains of amino acid residues within the sequence VRDWYRFH.
Fragments were identified which occupied nonredundant sites and were
spatially close enough to accommodate the peptide backbone (panel
C). We have found that the best match involves the VRDWYRFH
sequence such that the corresponding Val (position 1), Trp (position
4), Arg (position 6), and Phe (position 7) occupied the relative
positions shown in panels C and D. The
interaction of the peptide fragment with RA6.3 results in a favorable
interaction such that the functional groups of the peptide are
spatially similar to the organic fragments identified by the LUDI
search for the structure generated by the knowledge-based approach.
Panel D is a representative model of the VRDWYRFH peptide
bound to RA6.3.
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We examined whether organic molecules identified by LUDI were similar
to the structure of the side chains and spatial order of the sequence
of residues within the amiloride binding domain VRDWYRFH (Fig.
5C). As mentioned earlier, in effect one wants to stitch the
fragments together to form a peptide. We modeled the VRDWYRFH peptide
by two methods. First, individual amino acids were oriented with their
side chains superimposed on the corresponding molecules identified by
LUDI (Fig. 5C). The individual amino acid side chains were
restrained, and then the residues were forced to form amide bonds
(i.e. form the backbone structure). As expected, such an
approach resulted in highly strained conformations. Alternatively, a
peptide was built, and the
,
angles rotated until the respective side chains were in close proximity to the molecules identified by
LUDI. The VRDWYRFH sequence was modeled such that the corresponding Val
(position 1), Trp (position 4), Arg (position 6), and Phe (position 7),
approximated positions identified by the related LUDI fragments. The
positioned peptide fragment-RA6.3 complex was then energy optimized
with a restrained dynamics calculation such that the side chain
positions were held approximate to the spatial positions of the
LUDI-identified molecules. The complex was again energy optimized to
convergence without the imposition of constraints (Fig. 5D).
Deviation of the backbone conformation of RA6.3 bound to peptide
relative to the unbound RA6.3 structure was found to be only 0.36 Å.
This indicates that the placement of the peptide within the antibody
combining site did not dramatically alter the overall conformation of
the RA6.3 structure.
 |
DISCUSSION |
The generation of the monoclonal antibody RA6.3 was designed such
that the antibody was directed against the combining site of
anti-amiloride antibodies (such as BA7.1 (43)) and consequently against
an amiloride binding domain on the epithelial Na+ channel
(18). RA6.3 therefore functions as a molecular mimic of amiloride.
Several lines of evidence support the hypothesis that RA6.3 recognized
an amiloride binding domain within the
-subunit of ENaC. First,
RA6.3 recognized a core, non-N-linked glycosylated polypeptide of 72 kDa in Xenopus A6 cells, consistent with
the predicted core size of Xenopus
ENaC (Fig. 1) (41).
Second, a 20-mer peptide containing the putative amiloride binding
tract WYRFHY (14) inhibited the binding of RA6.3 to in vitro
translated mouse and bovine
ENaC (Fig. 2). RA6.3 did not efficiently
recognize mouse
- or
ENaC (data not shown).
Previous studies have shown that blocking N-linked
glycosylation with tunicamycin inhibits the amiloride-sensitive short
circuit current in A6 epithelia and that this is a result of inhibition of functional Na+ channels (44). Our data are consistent
with this observation in that tunicamycin both inhibits short circuit
current and prevents the assembly of Na+ channels into an
oligomeric complex recognized by RA6.3. In contract, N-linked glycosylation is not required for functional
expression of
ENaC in Xenopus oocytes (9).
Work from several investigators supports the idea that amiloride
interacts within the channel pore (45-50). A hydrophobic region (termed H2) preceding the second transmembrane domain of each subunit
is thought to contribute to the conduction pore because selected
mutations within the region alter cation selectivity or single channel
conductance (3, 12, 13). Furthermore, mutations within these H2 regions
alter amiloride binding characteristics (3, 12, 13). Selected organic
cations other than amiloride, such as 2,4,6-triaminopyrimidine,
triamterene, pentamidine, and trimethoprim, also function as epithelial
Na+ channel inhibitors, although with Ki
values much greater than that of amiloride (51-53). These data suggest
that it is the charged guanidine moiety of amiloride and other organic
cations which is interacting with the channel pore. Amiloride inhibits the Na+ channel with a submicromolar Ki,
and structure-function analyses indicate that the substituted pyrazine
ring of amiloride is required for high affinity interactions with
the channel and likely has a critical role in stabilizing the binding
of amiloride to the channel protein (54, 55).
We propose a model of amiloride binding to the Na+ channel
based on the previous studies examining Na+ channel
blockers and on the recent description of the tertiary structure of the
Streptomyces KcsA K+ channel (56). The
Na+ channel pore may be analogous in structure to the KcsA
K+ channel that consists of a narrow selectivity filter
facing the extracellular space and a wider pore cavity facing the
intracellular space (56). Our proposed model of amiloride binding to
the Na+ channel has two key elements. First, the charged
acylguanidine moiety of amiloride interacts at or near the selectivity
filter to block the channel; the carboxyl-terminal portions of the H2 domains of ENaC form the selectivity filter. Second, the substituted pyrazine ring of amiloride interacts with specific regions within ENaC
ectodomains to stabilize the amiloride interaction with ENaC. We
identified one site within the
-subunit ectodomain (residues 278-283) which participates in amiloride binding (14), based on
sequence homology with an amiloride binding site on the anti-amiloride antibody BA7.1 (15). We reported that
-subunits lacking this 6-residue tract (
ENaC
278-283) confer a loss of sensitivity to submicromolar concentrations of amiloride to Na+ channels
composed solely of
-subunits or to channels composed of
-,
-
and
-subunits (14). Li et al. (57) have also provided evidence suggesting that the ectodomain of
ENaC participates in
amiloride binding. In contrast, we reported that
-subunit channels
formed by the mutant
ENaC H282D were insensitive to submicromolar
concentrations of amiloride, although
H282D,
,
ENaC was
inhibited by amiloride with a Ki similar to wild type
,
,
ENaC (3, 14). Based on these results, we propose that
there are several sites within ENaC ectodomains, including residues
278-283 within
ENaC, which interact with the substituted pyrazine
ring and stabilize the binding of amiloride to the channel.
The antibody RA6.3 was cloned and sequenced in order to build a
structural model of this antibody and to explore potential interactions
with
ENaC. A computer screening approach (LUDI) was used to identify
potential amino acid residues that could theoretically bind to the
RA6.3 combining site. Studies using this computerized screening
strategy have been reported in the discovery of novel enzyme
inhibitors. The use of this approach to design inhibitors of
antibody-related protein-protein interactions remains a largely
unexplored area with great potential (19, 20). A search for potential
amino acid side chain structures that might interact with residues
within the RA6.3 antigen binding site was performed using the program
LUDI (Fig. 5, A and B). Fragments reflective of
amino acid side chains within and flanking the putative amiloride
binding domain (i.e. the tract WYRFHY) were observed to
interact with the antibody in an ordered linear array, providing a
basis to "dock" this peptide sequence to RA6.3 (Fig. 5,
C and D).
Current procedures for predicting ligand-antibody interactions are
limited, mainly because of the conformational flexibility of ligands
and antibodies and the role of solvent in mediating ligand recognition
and binding. Consequently, it was not our intent to elucidate the
binding mode conformation of peptides that interact with RA6.3, but to
validate the potential of the WYRFHY sequence to be recognized by
RA6.3. It has been suggested that proteins may actually interact
through small critical surface-binding epitopes as in the cases of the
human growth hormone (58) and the erythropoietin receptor complexes
(59). Such findings suggest that small binding epitopes may be
sufficient for the effective blockade of large protein-protein interfaces.
In recent years, several reports on the crystal structures of antibody
molecules, with and without bound antigen (for review, see Ref. 60)
have provided insight into this process (61), which is described by the
lock-and-key and induced fit mechanisms (62). Fabs whose structures
have been solved in complex with a peptide have also been solved in
their native or unbound form include B1I32 (63), Fab 50.1 (61), Fab
17/9 (64), 8F5 (65), SD6 (66), and 2H1 (67). In these cases,
conformational changes in the antibody range from side chain
rearrangements and small segmental movements in the CDR loops to a
major rearrangement of the H3 loop in Fab 17/9 (64) as well as
domain-domain rearrangements. The changes observed in the
three-dimensional structure of RA6.3 on binding the 8-mer peptide
sequence (VRDWYRFH) are slight. Hence, binding of this peptide to RA6.3
requires that the peptide antigen assume the proper conformation to fit
in the antibody combining site, which is largely unchanged in this
process. This is most likely the result of modeling RA6.3 from
templates that have undergone transitions upon ligand binding. We have
shown previously that there are conformational similarities among
antibodies that have been solved in complex with ligands (20). In
summary, computer screening provides a novel approach to develop models
of antibody-antigen interaction and allowed us to generate a molecular
perspective of RA6.3 binding to an amiloride binding domain within
ENaC.
 |
ACKNOWLEDGEMENTS |
Bovine
ENaC was a generous gift from
C. M. Fuller and D. J. Benos (University of Alabama at Birmingham).
We thank Shaohu Sheng and Farhad Kosari for helpful discussions
regarding models of amiloride binding to ENaC.
 |
FOOTNOTES |
*
This work was supported in part by a grant from the
Department of Veterans Affairs and the National Institutes of Health
Grant DK50268. This work was performed during the tenure of an
Established Investigatorship Award from the American Heart Association
(T. R. K.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF113618 (H chain) and AF113619 (L chain).
Recipient of a postdoctoral fellowship award from the Cystic
Fibrosis Foundation.

To whom correspondence should be addressed: Medical Research
151, VA Medical Center, University and Woodland Ave., Philadelphia, PA
19104. E-mail: kleyman{at}mail.med.upenn.edu.
2
C. C. Schulte, J. Meyer, D. N. Furness, C. M.
Hackney, T. R. Kleyman, and A. W. Gummer, manuscript in preparation.
 |
ABBREVIATIONS |
The abbreviations used are:
ENaC(s), epithelial
Na+ channel(s);
L chain, light chain;
H chain, heavy chain;
PAGE, polyacrylamide gel electrophoresis;
CSPD, disodium
3-(4-methoxyspiro(1,2-dioxetane-3,2'-(5'-chloro)tricyclo)3.3.1.13,7)decan)4-yl)phenyl
phosphate;
CDR, complementarity determining region.
 |
REFERENCES |
-
Garty, H.,
and Palmer, L. G.
(1997)
Physiol. Rev.
77,
359-396[Abstract/Free Full Text]
-
Firsov, D.,
Gautschi, I.,
Merillat, A. M.,
Rossier, B. C.,
and Schild, L.
(1998)
EMBO J.
17,
344-352[Abstract/Free Full Text]
-
Kosari, F.,
Sheng, S.,
Li, J.,
Mak, D.-O. D.,
Foskett, J. K.,
and Kleyman, T. R.
(1998)
J. Biol. Chem.
273,
13469-13474[Abstract/Free Full Text]
-
Coscoy, S.,
Lingueglia, E.,
Lazdunski, M.,
and Barbry, P.
(1998)
J. Biol. Chem.
273,
8317-8322[Abstract/Free Full Text]
-
Berdiev, B. K.,
Karlson, K. H.,
Loffina, D.,
Halpin, P.,
Stanton, B. A.,
Kleyman, T. R.,
and Ismailov, I. I.
(1998)
Biophys. J.
75,
2292-2301[Abstract/Free Full Text]
-
Snyder, P. M.,
Cheng, C.,
Prince, L. S.,
Rogers, J. C.,
and Welsh, J. M.
(1998)
J. Biol. Chem.
273,
681-684[Abstract/Free Full Text]
-
Renard, S.,
Lingueglia, E.,
Voilley, N.,
Lazdunski, M.,
and Barbry, P.
(1994)
J. Biol. Chem.
269,
12981-12986[Abstract/Free Full Text]
-
Canessa, C. M.,
Merillat, A. M.,
and Rossirr, B. C.
(1994)
Am. J. Physiol.
267,
C1682-C1690[Abstract/Free Full Text]
-
Snyder, P. M.,
McDonald, F. J.,
Stokes, J. B.,
and Welsh, M. J.
(1994)
J. Biol. Chem.
269,
24379-24383[Abstract/Free Full Text]
-
Canessa, C. M.,
Horisberger, J.-D.,
and Rossier, B. C.
(1993)
Nature
361,
467-470[CrossRef][Medline]
[Order article via Infotrieve]
-
Canessa, C. M.,
Schild, L.,
Buell, G.,
Thorens, B.,
Gautschi, I.,
Horisberger, J.-D.,
and Rossier, B. C.
(1994)
Nature
367,
463-467[CrossRef][Medline]
[Order article via Infotrieve]
-
Schild, L.,
Schneeberger, E.,
Gautschi, I.,
and Firsov, D.
(1997)
J. Gen. Physiol.
109,
15-26[Abstract/Free Full Text]
-
Waldmann, R.,
Champigny, G.,
and Lazdunski, M.
(1995)
J. Biol. Chem.
270,
11735-11737[Abstract/Free Full Text]
-
Ismailov, I. I.,
Kieber-Emmons, T.,
Lin, C.,
Berdiev, B. K.,
Schlyonsky, V. G.,
Patton, H. K.,
Fuller, C. M.,
Worrell, R.,
Zuckerman, J. B.,
Sun, W.,
Eaton, D. C.,
Benos, D. J.,
and Kleyman, T. R.
(1997)
J. Biol. Chem.
272,
21075-21083[Abstract/Free Full Text]
-
Lin, C.,
Kieber-Emmons, T.,
Villalobos, A. P.,
Foster, M. H.,
Wahlgren, C.,
and Kleyman, T. R.
(1994)
J. Biol. Chem.
269,
2805-2813[Abstract/Free Full Text]
-
Kieber-Emmons, T.,
Lin, C.,
Prammer, K.,
Villalobos, A.,
Kosari, F.,
and Kleyman, T. R.
(1995)
Kidney Int.
48,
956-964[Medline]
[Order article via Infotrieve]
-
Nisonoff, A.
(1991)
J. Immunol.
147,
2429-2438[Free Full Text]
-
Kleyman, T. R.,
Kraehenbuhl, J. P.,
and Ernst, S. A.
(1991)
J. Biol. Chem.
266,
3907-3915[Abstract/Free Full Text]
-
Li, S.,
Gao, J.,
Satoh, T.,
Friedman, T. M.,
Edling, A. E.,
Koch, U.,
Choksi, S.,
Han, X.,
Korngold, R.,
and Huang, Z.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
73-78[Abstract/Free Full Text]
-
Murali, R.,
and Kieber-Emmons, T.
(1997)
J. Mol. Recognit.
10,
269-276[CrossRef][Medline]
[Order article via Infotrieve]
-
Ling, B. N.,
Zuckerman, J. B.,
Lin, C.,
Harte, B. J.,
McNulty, K. A.,
Smith, P. R.,
Gomez, L. M.,
Worrell, R. T.,
Eaton, D. C.,
and Kleyman, T. R.
(1997)
J. Biol. Chem.
272,
594-600[Abstract/Free Full Text]
-
Chalfant, M. L.,
Coupaye-Gerard, B.,
and Kleyman, T. R.
(1993)
Am. J. Physiol.
264,
C1480-C1488[Abstract/Free Full Text]
-
Coupaye-Gerard, B.,
Bookstein, C.,
Duncan, P.,
Chen, X. Y.,
Smith, P. R.,
Musch, M.,
Ernst, S. A.,
Chang, E. B.,
and Kleyman, T. R.
(1996)
Am. J. Physiol.
271,
C1639-C1645[Abstract/Free Full Text]
-
Coupaye-Gerard, B.,
Zuckerman, J. B.,
Duncan, P.,
Bortnik, A.,
Avery, D. I.,
Ernst, S. A.,
and Kleyman, T. R.
(1997)
Am. J. Physiol.
272,
C1781-C1789[Abstract/Free Full Text]
-
Sambrook, J.,
Fritsch, E. F.,
and Maniatis, T.
(1989)
Molecular Cloning: A Laboratory Manual, 2nd Ed., pp. 11.31-11.33, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
-
Gubler, U.,
and Hoffman, B. J.
(1983)
Gene (Amst.)
25,
263-269[Medline]
[Order article via Infotrieve]
-
Guillaume, T.,
Rubinstein, D. B.,
Young, F.,
Tucker, L.,
Logtenberg, T.,
Schwartz, R. S.,
and Barrett, K. J.
(1990)
J. Immunol.
145,
1934-1945[Abstract/Free Full Text]
-
Levy, S.,
Mendel, E.,
and Kon, S.
(1987)
Gene (Amst.)
54,
167-173[Medline]
[Order article via Infotrieve]
-
Karp, S. L.,
Kieber, E. T.,
Sun, M. J.,
Wolf, G.,
and Neilson, E. G.
(1993)
J. Immunol.
150,
867-879[Abstract/Free Full Text]
-
Lohman, K. L.,
Kieber-Emmons, T.,
and Kennedy, R. C.
(1993)
Mol. Immunol.
30,
1295-1306[Medline]
[Order article via Infotrieve]
-
Tomiyama, Y.,
Brojer, E.,
Ruggeri, Z. M.,
Shattil, S. J.,
Smiltneck, J.,
Gorski, J.,
Kumar, A.,
Kieber-Emmons, T.,
and Kunicki, T. J.
(1992)
J. Biol. Chem.
267,
18085-18092[Abstract/Free Full Text]
-
Fine, R. M.,
Wang, H.,
Shenkin, P. S.,
Yarmush, D. L.,
and Levinthal, C.
(1986)
Proteins
1,
342-362[Medline]
[Order article via Infotrieve]
-
Martin, A. C.,
Cheetham, J. C.,
and Rees, A. R.
(1991)
Methods Enzymol.
203,
121-153[Medline]
[Order article via Infotrieve]
-
Shenkin, P. S.,
Yarmush, D. L.,
Fine, R. M.,
Wang, H. J.,
and Levinthal, C.
(1987)
Biopolymers
26,
2053-2085[Medline]
[Order article via Infotrieve]
-
Novotny, J.,
Bruccoleri, R. E.,
and Haber, E.
(1990)
Proteins
7,
93-98[Medline]
[Order article via Infotrieve]
-
Nell, L. J.,
McCammon, J. A.,
and Subramaniam, S.
(1992)
Biopolymers
32,
11-21[Medline]
[Order article via Infotrieve]
-
Mas, M. T.,
Smith, K. C.,
Yarmush, D. L.,
Aisaka, K.,
and Fine, R. M.
(1992)
Proteins
14,
483-498[Medline]
[Order article via Infotrieve]
-
Bruccoleri, R. E.,
Haber, E.,
and Novotny, J.
(1988)
Nature
335,
564-568[CrossRef][Medline]
[Order article via Infotrieve]
-
Bohm, H. J.
(1992)
J. Comput. Aided Mol. Des.
6,
61-78[Medline]
[Order article via Infotrieve]
-
Kleyman, T. R.,
Coupaye-Gerard, B.,
and Ernst, S. A.
(1992)
J. Biol. Chem.
267,
9622-9628[Abstract/Free Full Text]
-
Puoti, A.,
May, A.,
Canessa, C. M.,
Horisberger, J. D.,
Schild, L.,
and Rossier, B. C.
(1995)
Am. J. Physiol.
269,
C188-C197[Abstract/Free Full Text]
-
Worrell, R. T.,
Lin, C.,
Kleyman, T. R.,
and Eaton, D. C.
(1995)
FASEB J.
9,
309 (abstr.)
-
Kleyman, T. R.,
Kraehenbuhl, J. P.,
Rossier, B. C.,
Cragoe, E. J. J.,
and Warnock, D. G.
(1989)
Am. J. Physiol.
257,
C1135-C1141[Abstract/Free Full Text]
-
Pedemonte, C. H.
(1995)
J. Membr. Biol.
147,
223-231[Medline]
[Order article via Infotrieve]
-
Hamilton, K. L.,
and Eaton, D. C.
(1985)
Am. J. Physiol.
249,
C200-C207[Abstract]
-
Warncke, J.,
and Lindemann, B.
(1985)
J. Membr. Biol.
86,
255-265[Medline]
[Order article via Infotrieve]
-
Gottlieb, G. P.,
Turnheim, K.,
Frizzell, R. A.,
and Schultz, S. G.
(1978)
Biophys. J.
22,
125-129[Abstract]
-
Palmer, L. G.
(1984)
J. Membr. Biol.
80,
153-165[Medline]
[Order article via Infotrieve]
-
Li, J. H.-Y.,
and Lindemann, B.
(1982)
in
Basic Mechanisms in the Action of Lithium (Emrich, H. M., Aldenhoff, J. B., and Lux, H. D., eds), pp. 28-35, Excerpta Medica, Amsterdam
-
Van Driessche, W.,
and Erlij, D.
(1983)
Pfluegers Arch. Eur. J. Physiol.
398,
179-188[Medline]
[Order article via Infotrieve]
-
Balaban, R. S.,
Mandel, L. J.,
and Benos, D. J.
(1979)
J. Membr. Biol.
49,
363-390[Medline]
[Order article via Infotrieve]
-
Choi, M. J.,
Fernandez, P. C.,
Patnaik, A.,
Coupaye-Gerard, B.,
D'Andrea, D.,
Szerlip, H.,
and Kleyman, T. R.
(1993)
N. Engl. J. Med.
328,
703-706[Free Full Text]
-
Kleyman, T. R.,
Roberts, C.,
and Ling, B. N.
(1995)
Ann. Int. Med.
122,
103-106[Abstract/Free Full Text]
-
Kleyman, T. R.,
and Cragoe, E. J., Jr.
(1990)
Methods Enzymol.
191,
739-755[Medline]
[Order article via Infotrieve]
-
Li, J. H.-Y.,
Cragoe, E. J., Jr.,
and Lindemann, B.
(1985)
J. Membr. Biol.
83,
45-56[Medline]
[Order article via Infotrieve]
-
Doyle, D. A.,
Cabral, J. M.,
Pfuetzner, R. A.,
Kuo, A.,
Gulbis, J. M.,
Cohen, S. L.,
Chait, B. T.,
and MacKinnon, R.
(1998)
Science
280,
69-77[Abstract/Free Full Text]
-
Li, X. J.,
Xu, R. H.,
Guggino, W. B.,
and Snyder, S. H.
(1995)
Mol. Pharmacol.
47,
1133-1140[Abstract]
-
Clackson, T.,
and Wells, J. A.
(1995)
Science
267,
383-386[Medline]
[Order article via Infotrieve]
-
Livnah, O.,
Stura, E. A.,
Johnson, D. L.,
Middleton, S. A.,
Mulcahy, L. S.,
Wrighton, N. C.,
Dower, W. J.,
Jolliffe, L. K.,
and Wilson, I. A.
(1996)
Science
273,
464-471[Abstract]
-
Wilson, I. A.,
Ghiara, J. B.,
and Stanfield, R. L.
(1994)
Res. Immunol.
145,
73-78[Medline]
[Order article via Infotrieve]
-
Stanfield, R. L.,
Takimoto, K. M.,
Rini, J. M.,
Profy, A. T.,
and Wilson, I. A.
(1993)
Structure
1,
83-93[Medline]
[Order article via Infotrieve]
-
Davies, D. R.,
and Cohen, G. H.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
7-12[Abstract/Free Full Text]
-
Stanfield, R. L.,
Fieser, T. M.,
Lerner, R. A.,
and Wilson, I. A.
(1990)
Science
248,
712-719[Medline]
[Order article via Infotrieve]
-
Schulze, G. U.,
Rini, J. M.,
and Wilson, I. A.
(1993)
J. Mol. Biol.
234,
1098-1118[CrossRef][Medline]
[Order article via Infotrieve]
-
Tormo, J.,
Blaas, D.,
Parry, N. R.,
Rowlands, D.,
Stuart, D.,
and Fita, I.
(1994)
EMBO J.
13,
2247-2256[Abstract]
-
Verdaguer, N.,
Mateu, M. G.,
Bravo, J.,
Domingo, E.,
and Fita, I.
(1996)
J. Mol. Biol.
256,
364-376[CrossRef][Medline]
[Order article via Infotrieve]
-
Young, A. C.,
Valadon, P.,
Casadevall, A.,
Scharff, M. D.,
and Sacchettini, J. C.
(1997)
J. Mol. Biol.
274,
622-634[CrossRef][Medline]
[Order article via Infotrieve]
-
Kabat, E. A.,
Wu, T. T.,
Reid-Miller, M.,
Perry, H. M.,
and Gottesman, K. S.
(1987)
Sequences of Proteins of Immunologic Interest, United States Department of Health and Human Services, Public Health Service, National Institutes of Health, Bethesda, MD
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.