(Received for publication, October 12, 1994)
From the
In the nicotinic receptor, the quaternary ammonium group of
acetylcholine (ACh) binds to a negative subsite at most 1 nm from a
readily reducible disulfide formed between -subunit residues
Cys
and Cys
. The cross-linker S-(2-[
H]glycylamidoethyl)dithio-2-pyridine
formed a disulfide bond with reduced
Cys
/Cys
and an amide bond with an acidic residue in the
subunit
(Czajkowski, C., and Karlin, A. (1991) J. Biol. Chem. 266,
22603-22612). The fully extended cross-linking moiety
-NHCH
CONHCH
CH
S- is 0.9 nm long.
After the disulfide bond linking
-NHCH
CONHCH
CH
S- to the
subunit was reduced,
-NHCH
CONHCH
CH
SH remained linked to
the
subunit by an amide bond. The
subunit was cleaved at
Met residues, and the radioactive fragments were isolated and sequenced
by automated Edman degradation. Additionally, the isolated radioactive
fragments were further cleaved at Trp residues and sequenced. Peaks of
release of radioactivity were obtained in the sequencing cycles
corresponding to
Asp
,
Asp
, and
Glu
. The mutation of
Asp
to Asn
decreased the affinity of the receptor for ACh 100-fold, whereas the
mutation of either
Asp
,
Glu
, or 8
other acidic residues in the same region of
decreased the
affinity by 3-fold or less (Czajkowski, C., Kaufmann, C., and Karlin,
A.(1993) Proc. Natl. Acad. Sci. U. S. A 90, 6285-6289).
Because
Asp
both contributes to ACh binding and is
suitably close to the binding site disulfide, it is likely to be part
of the ACh-binding site formed in the interface between the
and
the
subunits.
The binding of acetylcholine (ACh) ()by nicotinic
receptors elicits the rapid opening of an ion-conducting channel and
eventually desensitization (Katz and Thesleff, 1957). Even in the
absence of high resolution structures of these receptors, we can begin
to understand their function in terms of their structures through the
identification of the amino acid residues forming the ACh-binding sites
and the ion-conducting pathway (reviewed in Karlin, 1993; Galzi and
Changeux, 1994; Akabas et al., 1994).
The effects of
reduction and the different susceptibilities of the reduced receptor to
affinity labels of different lengths led to the inferences that there
is a readily reducible disulfide in the ACh-binding site and that this
disulfide is within 1 nm of the negative subsite that binds the
quaternary ammonium group of ACh (Karlin, 1969). The cysteines forming
the binding site disulfide were first shown to be in the subunit
(Reiter et al., 1972) and subsequently identified as
Cys
and
Cys
(Kao et al.,
1984; Kao and Karlin, 1986).
Four other -subunit residues, all
aromatic, have also been associated with the ACh-binding sites by
affinity labeling. These are
Tyr
(Galzi et
al., 1990; Cohen et al., 1991),
Trp
(Dennis et al., 1988),
Tyr
(Dennis et al., 1988; Abramson et al., 1989), and
Tyr
(Middleton and Cohen, 1991). Their involvement
in ACh binding was further supported by the functional consequences of
site-directed mutagenesis (Mishina et al., 1985; Tomaselli et al., 1991; Galzi et al., 1991; O'Leary and
White, 1992). The adjacent cysteines and the 4 aromatic residues are
highly conserved among
subunit sequences.
Acidic residues also
contribute to the binding of ACh. The cross-linker S-(2-glycylaminoethyl)dithio-2-pyridine (GCP) specifically
forms a disulfide bond with a sulfhydryl (SH) and in the presence of a
carbodiimide an amide bond with carboxyl groups; the fully extended
cross-linking moiety
-NHCHCONHCH
CH
S- (GC) is 0.9 nm
long. In torpedo ACh receptor, GC, attached by a disulfide bond to
Cys
or
Cys
, formed an amide bond
with aspartate or glutamate residues on the
subunit (Czajkowski
and Karlin, 1991). The cross-linked aspartate or glutamate residues
were located within a segment,
164-224, containing 6
aspartates and 5 glutamates (Fig. 1). To determine the
functional importance of these Asp and Glu residues, we mutated, 1 at a
time, each of the Asp and Glu residues in the homologous segment of the
mouse-muscle
subunit to Asn and Gln (Fig. 1). Each mutant
subunit, together with wild-type
and
subunits, was
expressed in Xenopus oocytes, and the ACh concentration
eliciting half-maximal current (K
) and the K
for ACh block of
-bungarotoxin
binding were determined. By both measures, the mutation of
Asp
to Asn decreased the affinity for ACh 100-fold,
and the mutation of
Glu
to Gln decreased the
affinity for ACh 10-fold; by contrast, the neutralization by mutation
of the other acidic residues decreased the ACh affinity 3-fold or less
(Czajkowski and Karlin, 1993). Thus, we concluded that
Asp
and
Glu
contribute to the binding of ACh. We
did not know, however, whether these residues specifically reacted with
GC and thus how close they were to the binding site disulfide. In this
paper, we demonstrate by peptide mapping and sequencing that in torpedo
ACh receptor
Asp
,
Asp
, and
Glu
are cross-linked by GC to
Cys
or
Cys
and thus are within 0.9 nm of the
binding site disulfide.
Asp
is both at an
appropriate distance and functionally important and thus is likely to
be part of the negative subsite.
Figure 1:
Aligned sequences of torpedo and mouse
subunits from residue 161 to residue 224. In torpedo, this
sequence starts after Met
and ends just before
Pro
, the postulated first residue of the first
membrane-spanning segment. In torpedo
, the sequence Asp
to Lys
includes the third CNBr cleavage fragment
and the extracellular part of the fourth CNBr cleavage fragment. All
the Asp and Glu in the mouse sequence were mutated individually to N and Q as shown below the sequence, and the
mutations that strongly affected ACh binding are marked with an asterisk.
Figure 2:
Cross-linking the receptor with GCP. The
intact receptor complex is represented as a rectangle, within
which are indicated the relevant reacting entities:
COO
is a carboxylate side chain on the
subunit,
SS is the binding site disulfide between
Cys
and
Cys
on the
subunit.
Isolated subunits or their derivatives are not enclosed by a rectangle.
CNBr4 is the CNBr4 fragment of
.
Other abbreviations are DTT, dithiothreitol; H
NRSSP, S-(2-glycylaminoethyl)dithio-2-pyridine) (elsewhere, GCP); NEM, N-ethylmaleimide; EPCD,
1-ethyl-3-(3`-dimethylaminopropyl)carbodiimide; NBS,
5-thio-2-nitrobenzoate; (NBS)
,
5,5`-dithio-(2-nitrobenzoate). In one variant of the procedure, A, H
NRSSP was synthesized with tritiated glycine.
In the other variant, B, H
NRSSP was not
radioactive, the thiol of
CNBr4-COHNRSH was protected with NBS
until the the fragment was isolated, after which the free SH was
restored by reduction and reacted with
[
H]NEM.
Isolated subunit, cross-linked via an amide bond to
[
H]GC was cleaved with CNBr, and the cleavage
fragments were separated by HPLC (Fig. 3). There was a single
major peak of radioactivity centered on the fraction eluting at 63 min (Fig. 3B), which corresponded to a major peak of
peptide, as detected by absorbance at 205 nm (Fig. 3A).
The fractions centered on fraction 63 were pooled and sequenced. The
amino acids released corresponded to the predicted sequences for CNBr 4
(residues 164-257) and to CNBr 3 + 4 (residues
161-257) (data not shown). The latter arises because the cleavage
of the Met-Thr bond by CNBr is incomplete (cf. Czajkowski and
Karlin, 1991). Additionally, a minor sequence was present that
corresponded to CNBr 1 (
1-58). Because we showed previously
(Czajkowski and Karlin, 1991) that all of the radioactivity in this
peak is associated with CNBr 4 and CNBr 3 + 4, we did not need to
consider CNBr 1 in analyzing the release of radioactivity in the
sequencer cycles. The HPLC elution patterns after CNBr cleavage were
the same for
cross-linked with radioactive
[
H]GC, according to the reaction scheme in variation A of Fig. 1, and for
cross-linked with
non-radioactive GC, which was finally radioactively tagged with
[
H]NEM, according to the reaction scheme in variation B of Fig. 1.
Figure 3:
The isolation by HPLC of CNBr cleavage
fragments of covalently linked via an amide bond to radioactive
GC. Starting with 50 nmol of torpedo receptor, the
subunit was
cross-linked to [
H]GC as in Fig. 1, variation A. The
subunit was isolated by electrophoresis
and cleaved with CNBr, and the cleavage products were separated by
HPLC. A, the elution of peptides monitored by A
. The elution rate was 1 ml/min. B,
the elution of tritiated peptide in 1-ml fractions as determined by
scintillation counting of 50-µl aliquots. Input of tritium was
110,000 counts/min; recovery was 98,400 counts/min. The peak eluting
around 63 min contained 36,000 counts/min.
The HPLC fractions containing CNBr 4 and CNBr 3 + 4 either were directly sequenced or were first subjected to Tricine-SDS-PAGE and then sequenced. In the latter case, CNBr 4 and CNBr 3 + 4 were separated from CNBr 1. The patterns of radioactive release were similar in all cases. Peaks of radioactive release were obtained in sequencer cycles 2, 5, 8, and 17 through 22 (Fig. 4).
Figure 4:
Release of radioactive amino acids during
automated Edman degradation of [H]GC-labeled
fragments. The data are the means ± S.E. of nine independent
sequencing experiments. In six experiments, the fractions eluting from
the HPLC in the peak centered on 63 min were combined and sequenced
(see ``Experimental Procedures''). In three experiments, the
HPLC fractions were further purified by Tricine-SDS-PAGE and blotted
onto PVDF paper, and the 15,700-dalton band was sequenced. In all
cases, the radioactivity of the ATZ-derivatized amino acid was
determined and is expressed as a fraction of the total radioactivity
recovered. The radioactivity is that observed, not corrected for the
repetitive yield of the cleavage cycles. Of the radioactivity loaded on
the sequencer, about 30% was released in the sequencer cycles, about
50% remained on the filter and cartridge seal, and about 20% was lost
in the washes.
CNBr 4 and CNBr 3 + 4 contain 2 Trp residues
(Trp and Trp
) (Fig. 1). We used
BNPS-skatole to cleave the mixture of CNBr 4 and CNBr 3 + 4 at
these Trp residues, and we sequenced the mixture of products to
determine the success of the cleavage. The amino acid residues released
corresponded to those of the four expected fragments: the N-terminal
part of CNBr 4, starting at Thr
(SKAT 1), the N-terminal
part of CNBr 3 + 4, starting at Asp
(SKAT 2), the
fragment starting after Trp
at Ile
(SKAT
3), and the fragment starting after Trp
at Glu
(SKAT 4) ( Fig. 1and Table 1). Of the residues
detected, residues in eight cycles were unique for SKAT 1, residues in
five cycles were unique for SKAT 2, residues in six cycles were unique
for SKAT 3, and residues in six cycles were unique for SKAT 4 (Table 1). Thus, each of the four expected sequences were present
in the mixture. In parallel experiments, with CNBr 4 and CNBr 3 +
4 amide coupled to [
H]GC, sequencing of the
products generated by cleavage at Trp yielded peaks of radioactive
release in sequencer cycles 2, 4, and 6 (Fig. 5).
Figure 5:
Release of radioactive amino acids during
automated Edman degradation of [H]GC-labeled CNBr
cleavage fragments also cleaved at Trp residues. The fragments were
separated by HPLC and then by Tricine-SDS-PAGE. The 15,700-dalton band
blotted on PVDF paper was treated with BNPS-skatole
(``Experimental Procedures''), and the resulting cleaved
peptides on the paper were subjected to automated Edman degradation.
The radioactivity released is expressed as the fraction of the total
recovered and is not corrected for repetitive yield. The means ±
S.E. of four independent experiments are shown. In a typical
experiment, fragments from the HPLC containing 15,000 counts/min were
separated by Tricine-SDS-PAGE, 90% of this radioactivity was
electroblotted onto PVDF paper, and 8,700 counts/min were associated
with the 15,700-dalton band (CNBr 4 and CNBr 3 + 4). After
cleavage at Trp and sequencing, 2,900 counts/min were recovered in the
sequencing cycles, and 1,000 counts/min were associated with the blot,
filter, and cartridge seal.
The reaction whereby the subunit was cross-linked to GC
uniquely formed an amide bond between a carboxylate on
and the
Gly-
-amino group of GC. Therefore, in sequential Edman degradation
of labeled CNBr 4 and CNBr 3 + 4, or of their subfragments
generated by cleavage at Trp, release of radioactivity should
correspond to the release of either an Asp or a Glu residue. Sequential
Edman degradation of the mixture of CNBr 4 and CNBr 3 + 4 gave
peaks of radioactivity in cycle 2, uniquely specifying
Asp
, cycles 5 and 8, consistent with either Asp
or Asp
, in addition to Asp
, cycles
17, 18, and 21, uniquely specifying Asp
, and cycle 19 and
22, uniquely specifying Glu
(Fig. 6). Sequential
Edman degradation of the subfragments generated by cleavage at Trp gave
peaks of radioactivity in cycle 2, specifying Asp
, cycle
4, specifying Asp
, and cycle 6, specifying Glu
(Fig. 6).
Figure 6:
The expected amino acid residues released
in each sequencer cycle for the mixture of CNBr 4 and CNBr 3 + 4
and for the BNPS-skatole-cleavage fragments (SKAT1, 2, 3, and 4). The suffix a indicates the amino acids released
without a lag at Pro, and the suffix b indicates the amino
acids released with a lag at Pro. (Under normal sequencing conditions,
cleavage at the Pro carboxyl is incomplete and significant further
cleavage occurs in the next cycle; this causes a lag by one cycle in
the release of residues subsequent to the Pro (Brandt et al.,
1976).) The amino acids in the cycles yielding peaks of radioactivity
are boxed. The Asp and Glu residues are numbered according to
the torpedo sequence as in Fig. 1.
The results of the sequencing of the CNBr
fragments and of their subfragments cleaved at Trp are consistent in
implicating Asp, Asp
, and Glu
as residues that were cross-linked to GC (Fig. 6). The
labeling of Asp
or Asp
, although suggested
by the peaks in cycles 5 and 8 in the sequencing of the CNBr fragments,
was not supported by the sequencing after cleavage at Trp. (The peak in
cycle 5 was at least in part due to Asp
.) Because of the
incomplete cleavage and incomplete recovery of radioactivity from the
sequencer, we can determine with greater certainty which residues were
labeled than which residues were not.
Since the fully extended GC
cross-link is 0.9 nm long, the side chain carboxylate group of the 3
labeled residues in the subunit, Asp
,
Asp
, and Glu
, are not more than 0.9 nm from
Cys
or
Cys
. Of the 3 residues,
only the mutation of
Asp
had a large effect on the
binding of ACh: the mutation to Asn caused an approximately 100-fold
decrease in the affinity for ACh (Czajkowski et al., 1993). We
conclude that
Asp
is likely to contribute directly
to the binding of ACh. Asp
and Glu
are also
within 0.9 nm of
Cys
/Cys
, but mutation
of these residues to Asn and Gln, respectively, caused only a 2.3- and
3.3-fold increase in the K
for ACh.
The
mutation of Glu
to Gln caused a 10-fold decrease in
the affinity of ACh (Czajkowski et al., 1993). The current
experiments provide no evidence, however, that
Glu
is within the binding site.
Glu
was at best 13
cycles out in fragment SKAT 3; given a repetitive yield of about 90%,
about 25% of input labeled
Glu
might have been
released, and the labeling of this residue might have been undetected.
On the other hand, the functional effect of mutating
Glu
to Gln could have been an indirect one mediated through non-local
structural changes.
The cross-linking of Asp
,
Asp
, and
Glu
to
Cys
/Cys
by the hydrophilic GC is
consistent with these residues facing
Cys
/Cys
across a water-filled crevice
between the
and
subunits. We hypothesize that this crevice
between subunits contains the ACh-binding site. The location of these
negatively charged residues at the surface of this crevice is
consistent with the negative electrostatic potential at
Cys
/Cys
(Stauffer and Karlin, 1994).
Only the neutralization by mutation of
Asp
, however,
causes a large decrease in the affinity for ACh (Czajkowski et
al., 1993). The mutation of
Asp
could either
alter the structure of the binding site, or it could eliminate the
electrostatic interaction between the carboxylate of
Asp
and the positively charged quaternary ammonium group of ACh. If
the latter is the case, then
Asp
and
Glu
must not be as close to the quaternary ammonium
group of bound ACh as is
Asp
, although they are also
within 0.9 nm of
Cys
/Cys
. For example,
a molecular model of Asp
-Pro
-Glu
shows that the side chain carboxylates of
Asp
and
Glu
could be 1 nm or more apart and still
face
Cys
/Cys
, 0.9 nm away. Thus, when
ACh is bound, the acetyl group could bind next to
Cys
/Cys
(Karlin, 1969) and the
quaternary ammonium group could bind next to
Asp
but
not next to either
Glu
or
Asp
.
The receptor has two, non-identical ACh-binding sites (Damle and
Karlin, 1978; Neubig and Cohen, 1979; Dowding and Hall, 1987). In the
native muscle-type ACh receptor, with composition
(Reynolds and Karlin, 1978), one site
is formed between the first
and the
subunit, and the other
site is formed between the second
and the
subunit (Kurosaki et al., 1987; Blount and Merlie, 1989; Pedersen and Cohen,
1990; Sine and Claudio, 1991). Consistent with the intersubunit
location of the binding sites, a number of uncharged residues in
and
contribute directly or indirectly to the binding of the
competitive inhibitor d-tubocurarine (Chiara and Cohen, 1992;
Sine, 1993; O'Leary et al., 1994).
In
164-224, only 3 carboxylate residues,
Asp
,
Glu
, and
Glu
, are identically
conserved among all aligned sequences of the
,
, and
subunits (Czajkowski et al., 1993). (The
subunit
substitutes for
in adult muscle ACh receptor (Mishina et
al., 1986).) The conservation of
Asp
is
consistent with its role in the ACh-binding site formed between
and
and with the similar role of the homologous residue in
,
, and
. The mutation to Asn of mouse
Asp
,
which aligns with
Asp
, also causes a
two-order-of-magnitude decrease in the affinity of the receptor for
ACh. (
)The location of the ACh-binding sites in the
interfaces between subunits is consistent with ACh binding inducing a
sliding of one subunit past another (Unwin, 1989), a change that could
be transmitted from the ACh-binding sites on the extracellular side of
the membrane to the channel gate on the cytoplasmic side of the
membrane (Czajkowski et al., 1993; Akabas et al.,
1994).