The Pharmacogenomics Journal (2001) 1, 221–228

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NEWS AND COMMENTARY

The acetylcholine-binding protein: The muscle-type ACh receptor is a het-

ero-pentamer with the composition
␣2␤␥␦.7 The muscle-type receptor has
‘What’s in a name?’ two ACh-binding sites per complex,
which are associated with the two ␣
A Karlin subunits.7 Heterologous expression of
␣ alone, however, did not yield ACh
Center for Molecular Recognition, Departments of Biochemistry and Molecular binding activity. Heterologous co-
Biophysics, Physiology and Cellular Biophysics, and Neurology, Columbia University, expression of ␣ and ␥ or ␣ and ␦, but
New York, NY, USA not ␣ and ␤, did generate ACh-bind-
ing sites.8
Labeling and cross-linking provided
The acetylcholine-binding protein identical to the similarly aligned
evidence that the ACh binding sites
(AChBP) is not an ion-conducting, nic- sequences of the 5HT3, GABAA,
are in the interface between subunits.
otinic ACh receptor by another name. GABAC, and glycine receptor subunits.
(+)-Tubocurarine specifically photolab-
But the recently published high-resol- These receptors are homologous,
eled the aligned pairs in the ␥ and ␦
ution structure of the AChBP1 is richly ligand-gated ion channels, also named
subunits, ␥Trp53 (bpTrp53) and
revealing of the nature of the ligand- the Cys-loop receptors for the epony-
␦Trp55, ␥Tyr111 (bpVal106) and
binding domains and the subunit mous 15-residue loop closed by a dis-
␦Arg113, and ␥Tyr117 (bpLeu112),
interfaces of its more fully articulated ulfide bond3 between two completely
and benzoylbenzoylcholine photolab-
cousins, the nicotinic receptors. conserved cysteinyls. There are 13 larg-
eled the aligned pairs ␥Leu109
The AChBP was detected originally ely conserved intervening residues
(bpArg104) and ␦Leu111.9
in a snail cDNA library. It is a soluble among the Cys-loop receptor subunits,
protein secreted by snail glial cells into but in the AChBP subunit there are A 9-Å bifunctional reagent cross-
cholinergic synapses, where it modu- only 12 intervening residues, which linked reduced ␣Cys192/193 to
lates synaptic transmission by binding are almost all different from the ␦Asp180 (bpAsp161).10 Mutation of
acetylcholine (ACh).2 The AChBP aligned residues in the Cys-loop recep- ␦Asp180 or the aligned ␥Asp174 to Asn
binds agonists and competitive antag- tors. In the AChBP structure, the Cys- decreased the apparent affinities for
onists of the nicotinic ACh receptor, loop is close to the C-terminus, which agonists 100–200 times but the affin-
including ACh, nicotine, epibatidine, in the Cys-loop receptors would con- ities for competitive antagonists only
(+)-tubocurarine, and ␣-bungarotoxin. tinue into the first membrane-span- 10–15 times.11 Mutation of the aligned
The spectrum of affinities most ning segment; hence, in the receptors, ⑀Asp175, however, affects the trans-
resembles that of the homomeric neu- the Cys-loop is close to the mem- duction of agonist binding into chan-
ronal nicotinic receptors composed of brane.1 nel opening (ie gating) rather than
␣7 or ␣9 subunits. By contrast with the Cys-loop binding per se.12
In three dimensions, the AChBP is a region, there is remarkable conser- All of these residues implicated in
cylinder of diameter 80 Å and height vation in the AChBP of the residues the binding function of nicotinic
62 Å.1 Each of the five identical sub- identified by affinity labeling as con- receptors are conserved in the AChBP,
units occupies a sector of the cylinder, tributing to the ACh-receptor binding and all of these conserved residues,
and together the subunits line an axial sites. The initial residues so identified except for bpAsp161, line a cavity that
channel 18 Å in diameter. In face view, were adjacent cysteines in the ␣ sub- undoubtedly contains the ACh bind-
the structure resembles ‘a windmill unit, ␣Cys192 (bpCys187) and ing site.1 The AChBP binding-site resi-
toy’ with five blades. The subunits ␣Cys193 (bpCys188),4 which form a dues aligned with the binding-site resi-
start at their N-termini with a three- highly unusual disulfide bond.3 (The dues in the ␣ subunit are on one side
turn ␣-helix and thereafter form aligned AChBP residues are preceded of the AChBP subunit (the (+) side),
10 ␤-strands and connecting loops. by ‘bp’.) In addition, four aromatic and the residues aligned with the
The ␤ strands are arranged in a residues, ␣Tyr93 (bpTyr89), ␣Trp149 receptor binding-site residues in the ␥
uniquely modified immunoglobulin (bpTrp143), ␣Tyr190 (bpTyr185), and and ␦ subunits are on the opposite (−)
topology. ␣Tyr198 (bpTyr192), were also affin- side of the AChBP subunit. The resi-
The AChBP subunit contains 210 ity labeled.5,6 dues on the (+) side are in loops
amino acids and is 20–24% identical to Although the ACh-receptor ␣ sub- between ␤ strands, while those on the
the aligned sequences of the N-ter- unit plays a principal role in forming (−) side are almost all on ␤ strands.
minal, extracellular halves of nicotinic the receptor ACh-binding sites, These two sets come together in the
ACh receptor subunits and 15–18% neighboring subunits also contribute. interface between neighboring AChBP
 The acetylcholine-binding protein
A Karlin
222

Figure 1 The binding site of AChBP. The residues in or close to the binding site are in ball-and-stick representation. The carbon atoms
of the residues from the (+) side of the subunit interface are colored pink and those from the (−) side are colored green. Nitrogens are
blue, oxygens are red, and sulfurs are yellow. A molecule of tetramethylammonium in CPK representation is placed in roughly the middle
of the aromatic side-chains, avoiding any overlaps with residue atoms. The pictures are in stereo. The view is similar to that in Figure 4A
in Ref 1.

subunits to form a binding site very low affinity for the AChBP, was agonists do so to a greater extent than
(Figure 1). present at 苲100 mM in the mother antagonists. In general, agonists are
The AChBP binding site opens to the liquor. The two potentially protonated smaller than competitive antagonists,
outside of the cylinder, about midway and positively charged nitrogens of which is consistent with the idea that
between its top and its bottom, the piperazine ring are both close to the receptor binding site contracts
defined by the C-terminus. There is no the rings of bpTrp143, within a cage around a bound agonist but less so
opening of the binding site to the axial of six aromatic side chains, consistent around a bulky antagonist. (Even the
channel. Viewed from the top of the with the contribution of cation-␲ binding of competitive antagonists,
cylinder, the (−) side of each AChBP interaction to the binding of quatern- however, leads to some perturbation
binding site is counter-clockwise to ary ammonium ions.14 of the site, which can be sufficient to
the (+) side; therefore, the muscle-type Not only do we not yet know the activate chemically15 or genetically alt-
subunits, previously shown to form a structure of the AChBP binding site ered receptors.16) Affinity labels
circle around the central channel in occupied by an agonist or the possibly attached to reduced ␣Cys192/193 that
the order ␣␥␣␦␤,13 must be in a coun- different structure of the binding site acted as tethered agonists were less
ter-clockwise arrangement as viewed occupied by a competitive inhibitor, than 9 Å long, whereas affinity labels
from the synaptic cleft.1 we do not know how closely these that acted as tethered antagonists were
The question of how ligands are dis- liganded structures resemble the corre- greater than 12 Å long, again consist-
posed in the AChBP binding-site cav- sponding structures in the ACh recep- ent with a contraction of the site
ity is not answered by the present tor. In the ACh receptor, the agonist- around an agonist.15
structure, which was crystallized with- occupied structure and the competi- Another indication of an agonist-
out a cholinomimetic ligand. The crys- tive antagonist occupied structure are induced structural change in the site
tallized AChBP did contain a molecule certainly different. was that the disulfide between
of N-2-hydroxyethylpiperazine-N′-2- The binding of both agonists and ␣Cys192 and ␣Cys193 was much less
ethanesulfonate (HEPES) in the bind- antagonists perturb the structure of susceptible to reduction by dithiothre-
ing site cavity. HEPES, which has a the ACh-receptor binding site but itol in the presence of agonists than in

The Pharmacogenomics Journal

 The acetylcholine-binding protein
A Karlin
223

the presence of competitive antagon- The location of the quaternary DUALITY OF INTEREST
ists.17 The structure of AChBP provides ammonium group within this cage of None declared.
a rationale for this result in that the aromatics is consistent with receptor
disulfide is facing into the crevice at activation by tethered agonists, Correspondence should be sent to
A Karlin, Center for Molecular Recognition,
the tip of a loop that is a loose lid on namely quaternary ammonium moi-
Departments of Biochemistry and Molecular
the binding site cavity (Figure 1). As eties attached to ␣Cys192/193 Biophysics, Physiology and Cellular Biophysics
Brejc points out, the loop would have (bpCys187/188)15 and at the positions and Neurology, Columbia University, New York
to move in order for large antagonists of ␣Tyr93,18 ␣Trp149 (bpTrp143)14 10032, USA.
to enter the site. It is possible that and ␣Tyr198 (bpTyr192).18 In Tel: +1 212 305 5778
when the site is unoccupied and when addition, acetylcholine mustard, in Fax: +1 212 305 5594
it is occupied by antagonist, the loop which the quaternary ammonium E-mail: ak12얀columbia.edu
with the disulfide is mobile; by con- group itself reacts, labeled ␣Tyr93
1 Brejc K et al. Nature 2001; 411: 269–276.
trast, when the agonist occupies the (bpTyr89).19 2 Smit AB et al. Nature 2001; 411: 261–268.
site, the loop might be immobilized, Mutations of ␣Cys192/193,20 3 Kao PN, Karlin A. J Biol Chem 1986; 261:
the binding site capped, and ␣Tyr93,12,21,22 ␣Trp149,21 ␣190,21–24 8085–8088.
the disulfide inaccessible even to a and ␣Tyr19823,24 affected agonist bind- 4 Kao PN et al. J Biol Chem 1984; 259:
11662–11665.
relatively small molecule like dithio- ing or gating and also competitive 5 Galzi J-L et al. J Biol Chem 1990; 265:
threitol. antagonist binding, and together with 10430–10437.
Agonists and competitive antagon- ␦Trp57 undoubtedly form the corre- 6 Middleton RE, Cohen JB. Biochemistry 1991;
ists of the ACh receptor have at least sponding aromatic cage in the ACh 30: 6987–6997.
7 Reynolds JA, Karlin A. Biochemistry 1978; 17:
one quaternary ammonium group or a receptor. By contrast, mutations of 2035–2038.
protonated tertiary ammonium group. ACh receptor residues aligned with 8 Blount P, Merlie JP. Neuron 1989; 3: 349–
The simplest agonist, tetramethylam- bpVal106, bpLeu112, and bpMet114 357.
9 Wang D et al. J Biol Chem 2000; 275:
monium, consists only of a quaternary affected competitive antagonist bind-
28666–28674.
ammonium group (Figure 1). It seems ing but not agonist binding or gating.9 10 Czajkowski C, Karlin A. J Biol Chem 1995;
likely that in the ACh receptor the In the AChBP, these three residues and 270: 3160–3164.
ammonium group binds in the cage of bpArg104 form the top of the binding 11 Martin MD, Karlin A. Biochemistry 1997; 36:
10742–10750.
five aromatic side-chains aligned with site cavity. The aligned residues in the
12 Akk G et al. Biophys J 1999; 76: 207–218.
bpTyr89, bpTrp143, bpTyr185, and ACh receptor likely interact with bulky 13 Karlin A et al. J Biol Chem 1983; 258:
bpTyr192, from the (+) side, and competitive antagonists but not with 6678–6681.
bpTrp53 from the (−) side. A sixth aro- agonists. 14 Zhong W et al. Proc Natl Acad Sci USA 1998;
95: 12088–12093.
matic side-chain in AChBP, bpTyr164, Bumping into the top of the site 15 Karlin A. J Gen Physiol 1969; 54: 245s–264s.
is not conserved in ACh receptor ␥ and might guarantee that a ligand will not 16 Revah F et al. Nature 1991; 353: 846–849.
␦, but two or three negatively charged be an agonist of the native receptor, 17 Damle VN, Karlin A. Biochemistry 1980; 19:
side chains at the aligned position and but not doing so does not guarantee 3924–3932.
18 Sullivan DA, Cohen JB. J Biol Chem 2000;
close by, including the homologs of that it will be an effective agonist. The 275: 12651–12660.
bpAsp161, namely ␥Asp174 and ACh receptors largely ignore choline, 19 Cohen JB et al. J Biol Chem 1991; 266:
␦Asp180, are conserved. These nega- the hydrolysis product of acetyl- 23354–23364.
tively charged residues are the likely choline. 20 Mishina M et al. Nature 1985; 313: 364–
369.
sources of the negative electrostatic The beautiful structure of the AChBP 21 Galzi JL et al. FEBS Lett 1991; 294: 198–202.
potential in the ACh binding site of is a surprise gift to all of us interested 22 Sine SM et al. J Biol Chem 1994; 269:
the receptor,11,12 and their movement in the nicotinic acetylcholine recep- 8808–8816.
23 Tomaselli GF et al. Biophys J 1991; 60:
towards a bound quaternary tors and the other Cys-loop receptors.
721–727.
ammonium group could be part of the It is a gift that we will be unwrapping 24 O’Leary ME, White MM. J Biol Chem 1992;
activation mechanism. for a long time. 267: 8360–8365.

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