-1-
SUPPLEMENTARY MATERIAL
A model for the assembly of nicotinic receptors
based on subunit-subunit interactions
MARCELO O. ORTELLS1, & GEORGINA E. BARRANTES2
Facultad de Medicina, Universidad de Morón and Consejo de Investigaciones
Científicas y Técnicas (CONICET), Machado 914, 4to piso, 1708 Morón,
Argentina
-2MATERIALS AND METHODS
Subunit modeling
The first step was to build an alignment of the receptor and AChBP sequences where we
identified regions capable to be modeled by amino acid replacement (homologous regions),
and those that needed to be reconstructed by loop generation. Loops were built using
templates from known tertiary structures of the Brookhaven databank1 and the appropriate
amino acid substitution. Loop modeling varied among subunits. According to the amino
acid sequence alignment, some subunits needed no loop modeling in some regions whereas
some others did with different sequence lengths. PDB codes of proteins used for loop
templates, their amino acid sequences and related information are available upon request.
Whenever amino acid replacement was needed, a search for the best rotamers was
performed prior to energy minimization. In all simulations, backbone atoms of regions
homologous to the AChBP were kept fixed at their original positions to avoid distortion of
the secondary structure. The exceptions were those amino acids joined to newly formed
loops that were forced to adopt a standard value in their  angles. This was necessary
when the orientation of the inserted loop was such that the  angle between the amino
acids joining AChBP and the loop was not 180o. Minimization of the structure energy was
first performed on the sidechains of mutated residues in homologous regions and on the
whole structure of new loops. We applied a forcing constant of 300 kcal Å-1 to keep
backbone atoms of loops close to their original positions. This was followed by
minimization of first all sidechains in the homologous regions, and finally on all
sidechains, keeping all backbone atoms fixed to their positions. Energy minimization was
performed using molecular mechanics2 in two steps using the program Discover and the
-3CVFF force field3. During the first step of minimization, no Morse functions and no cross
terms were used and the method of steepest descents was employed. The structure was
minimized until the maximum derivative was less than 2.00 kcal Å-1. In a second step, the
model was further minimized until the maximum derivative was less than 0.010 kcal Å-1
but using the full model, with Morse functions and cross terms, and employing the method
of conjugate gradients+.
RESULTS AND DISCUSSION
Tables Is and IIs highlight a common pattern found for both P and H scores, in both
neuronal and muscle dimers. For neuronal combinations, the higher P and H values are
concentrated on dimers involving the minus sides of 2 and (to a lesser extent and with
lower values) 8 subunits. Interestingly, the only two low P values found with the minus
side of 2 are with the 7 and 8 subunits, which can form homomeric receptors. In these
cases their highest P values are found with 8 itself. A similar pattern is found within
muscle dimers. The highest P and H values belong to those dimers involving the minus
side of the 1 subunit and the plus sides of every muscle subunit, including the 1 itself.
The origin of these patterns is not clear and is beyond the scope of this work. However,
a possible explanation is that they are an evolutionary expression of a simplesiomorphy,
that is, the share of a primitive state. In the case of neuronal subunits, it might be derived
from a high hydrophobic and electrostatic potential complementarity in a primordial
heterodimer composed of a primitive 2 subunit and an  neuronal ancestor. As 8 and 2
are somehow similar in this respect, it would be possible that 2 is a highly specialized
derivation of 8 (remember that 8 is lacking in mammals and so it is possible that 2 has
taken its role). A similar process might have occurred in vertebrates muscle but with the 1
-4subunit. According to the phylogeny of muscle subunits5, all muscle subunits are derived
from a common ancestor. Because 1 is the only muscle subunit with the principal part of
the binding site, it is possible that the primitive muscle receptor was
a proto-1 homo-
oligomer with high electrostatic potential and hydrophobic complementarity (present day
1 P=11, H=238).
P and H values for 5HT3 dimeric interactions are not uniformly distributed as well
(Tables II and III). The highest H values are found between 5HT3A and 5HT3B plus sides
and 5HT3B minus side, while the highest P values are between 5HT3A and 5HT3B plus sides
and 5HT3A minus side.
3 cannot form receptors alone with 2-6 subunits
Although it is possible, according to our model, that the plus side of 3 can form dimers
with those  subunits with which it has H values >= 172 (4, 8 and 10) or P values >=
2.5 (4 and 8), a pentamer including 3 is not predicted. With 8 the preferred sequence
of oligomerization includes only the 8 subunit. With 4 and 10 the problem is that the
3 minus side can only form dimers (or trimers) with 2 or 4 (H=190 and 231
respectively; Table II). Thus, only the initial -3+-(4/10)+ dimer is formed because
neither of these subunits are able to join the minus side of 3 or the plus side the 
subunits.
We also tested what receptor configuration would come out when combining 3 with
either 2 or 4, considering it is actually a non-functional  subunit. For the first one, the
model shows that a (3)2(2)3 is to be expected, that is, similar to a normal . In the case
of 4, it is predicted that a nonfunctional, 4 homopentamer is expected. In this case the
-5only possible dimers are 44 and 3-4+. P values for these dimers are respectively -2.74
and -3.56, which are fairly similar. If, as described in the model description, the
concentration of 3 is increased in such a way that the simulated P value of 3-4+ is
greater than the one of 44, the same result as with 23 is obtained.
Validity of the predictions
Are non extracellular regions involved in subunit recognition during assembly?
It was shown that chimeras built with the N-terminal domain of the  subunit fused to
the complementary part of the  subunit could substitute for , but not  during assembly.
On the contrary, the inverse chimera, with the N-terminal region of the  subunit and the
rest from the  subunit (and also with the rest from the  and  subunits), could not
substitute for  or  subunits during assembly and stopped assembly at the dimer stage6.
These results were interpreted as if the chimeras were lacking a recognition site present in
C-terminal half of the  subunit necessary to continue the assembly process. While a
positive result (i.e. the N-/C- chimera) definitively shows that the N-terminal half of the
 subunit is sufficient for a normal assembly, the negative results of the latter chimeras (N/C-,C-,C-) do not necessarily imply the need of the  C-terminal half region for
subunit recognition in the normal protein. A slight deviation in the (otherwise properly
folded) chimera from the normal topology of the transmembrane region might block
assembly beyond the dimer stage. For example, this change can hinder a close approach of
the N-terminal sides with one of the two neighboring subunits, with the result that only
dimers can be formed. In another work using 1 chimeras,7 it was shown that a small
sequence of the cytoplasmic loop in this subunit is required for the final steps of assembly,
-6as at least chimeric 11 trimers could be formed. However, as the authors also
suggested, the required loop may not participate directly in subunit recognition. Moreover,
co-expression of 1, 1 and  subunits yielded significant amounts of pentamers8. This
might indicate that for the chimeric 11 trimer, it is not the loss or lack of formation of a
recognition site for the  or any subunit what prevents further oligomerization (i.e. this
recognition site is not required in a mixture of normal 1, 1, and  subunits to form a full
pentamer). Rather, it could be only a failure that prevents the addition of any subunit, thus
being independent of subunit recognition. What is common to both reports is that the
proper folding of mutated subunits was tested by their ability for toxin binding. However, a
folding sufficient to support toxin binding does not necessarily imply a folding that will
allow pentameric assembly, irrespectively of the problem of subunit recognition.
Assembly process studied using as a model the quaternary structure of AChBP
The assumption that the assembly process can be studied using as a model the
quaternary structure of AChBP is based on the reasoning that no folding event can favor an
energetically poor final quaternary structure. It is known that subunits continue to fold9,10.
However, there is no evidence that these structural changes are involved in subunit
recognition. They might be necessary for assembly, but irrespectively of the type of
subunit involved. For example, when the cys-loop cysteines of the 1 subunit are mutated
to serines, the assembly is blocked at a trimer stage (11). If the homologous cysteines
of 1 are mutated, assembly is blocked at a later stage allowing the formation of a tetramer
(11)10. These data were interpreted in a model where a conformational change in the
1 and 1 cys-loops is necessary for the addition of unassembled  and second 1 subunits
respectively. First, considering the known position of the cys-loop in the AChBP structure,
-7it is very unlikely that it can be involved directly in subunit recognition as it would be in
close contact with the membrane in LGICs, but not at the proper subunit interface. Thus,
this conformational change might be necessary for the process of assembly. Second, the
conformational changes proposed have to occur in subunits that have both interfaces
occupied. Effectively, according to the known subunit arrangement and following the
sequential model of assembly on which this hypothesis is based, the 11 trimer should
exist in that order as both, the  and 1 subunits, are in contact with the 1. Hence, any
changes in the cys-loop of 1 cannot affect directly the recognition site for the  subunit.
Another alternative would be that 1 had one of the interfaces free, but in none of these
cases could the correct arrangements be achieved. The same happens with the
conformational change of the cys-loop in the 1 subunit. At this tetrameric stage, where a
second 1 has to be added (always according to a sequential model of assembly), 1
should be surrounded by an 1 and a  subunit.
REFERENCES
1. Bernstein FC, Koetzle TF, Williams GJB, Meyer EFJ, Brice MD, Rodgers JR, Kennard
O, Shimanouchi T, Tasumi M. 1977. The protein data bank: A computer-based
archival file for macromolecular structures. J Mol Biol 1977;112:535-542.
2. Keserü G, Kolossváry I. 1999. Molecular mechanics and conformational analysis in
drug design. Oxford: Blackwell Science Ltd.; 1999. 168 pp.
3. Discover Molecular Simulations Inc. 2000. San Diego .
4. Gabdoulline RR, Wade RC, Walther D. 2003. MolSurfer: a macromolecular interface
navigator. Nucleic Acids Research 2003;31:3349-3351.
-85 . Le Novere N, Changeux J-P. Molecular evolution of the nicotinic acetylcholinereceptor - an example of multigene family in excitable cells. J Mol Evol 1995;40:155172.
6. Eertmoed, AL, Green WN. Nicotinic receptor assembly requires multiple regions
throughout the  subunit. J Neurosci 1999;19:6298-6308.
7. Xu XM, Hall ZW. A sequence in the main cytoplasmic loop of the  subunit is required
for assembly of mouse muscle nicotinic acetylcholine- receptor. Neuron 1994;13:247255.
8. Nicke A, Rettinger J, Mutschler E, Schmalzing G. Blue native PAGE as useful method
for the analysis of the assembly of distinct combinations of nicotinic acetylcholine
receptors subunits. J Recept Signal Transduct Res 1999;19:493-507.
9. Green WN. Perspective - Ion channel assembly: Creating structures that function. J Gen
Physiol 1999;113:163-169.
10. Green WN, Wanamaker CP. The role of the cysteine loop in acetylcholine receptor
assembly. J Biol Chem 1997;272:20945-20953.
-9Table Is. Summary statistics for neuronal nicotinic values of main Tables II (H scores) and
III (P scores). For each row and column of Tables II and III, the mean, maximum and
minimum H and P values are presented. The last row shows the mean, maximum and
minimum for all neuronal nicotinic H and P values from Tables II and II respectively .
Table IIs. Summary statistics for muscle nicotinic values of main Tables II (H scores) and
III (P scores). For each row and column of Tables II and III, the mean, maximum and
minimum H and P values are presented. The last row shows the mean, maximum and
minimum for all muscle nicotinic H and P values from Tables II and II respectively .
- 10 Tables
Table Is
Subunit
Nic3_hsNic10hs+
Nic2_hs+
Nic3_hsNic6_hsNic5_hsNic3_hs+
Nic4_hs+
Nic5_hs+
Nic2_hsNic8_gg+
Nic9_hs+
Nic7_hsNic6_hs+
Nic9_hsNic7_hs+
Nic4_hsNic2_hs+
Nic10hsNic4_hsNic3_hs+
Nic8_ggNic2_hsNic4_hs+
TOTAL
H
Mean
108
135
138
147
151
151
157
158
161
162
170
170
174
180
185
186
189
190
197
198
199
202
202
222
172
Min Max
76 133
92 176
90 169
103 231
117 211
92 224
76 212
103 226
115 193
107 198
76 256
111 240
142 213
124 230
142 250
115 253
120 297
126 241
140 311
135 280
133 297
167 256
168 253
119 311
76 311
Subunit
Nic6_hsNic9_hsNic5_hsNic8_gg+
Nic5_hs+
Nicb3_hsNic3_hsNic4_hs+
Nic7_hs+
Nic10hsNic4_hsNic2_hs+
Nic2_hs+
Nic3_hs+
Nic4_hs+
Nic10hs+
Nic4_hsNic7_hsNic3_hs+
Nic9_hs+
Nic2_hsNic6_hs+
Nic8_ggNic2_hsTOTAL
P
Mean
-7.3
-5.6
-5.5
-4.5
-3.7
-3.5
-3.5
-3.0
-3.0
-2.6
-2.5
-2.0
-1.7
-1.5
-1.2
-1.0
0.0
0.0
0.3
0.5
1.3
1.7
3.6
6.1
-1.6
Min
-14
-16
-12
-16
-14
-7.2
-9
-8.8
-8.8
-8.4
-6.7
-12
-10
-7.8
-6.2
-9.7
-6.6
-5.2
-8
-9.7
-4.9
-6
-1.8
-2.4
-16
Max
0.4
-0.4
-0.8
4.4
5.3
-0.2
2.4
7.4
6
1
1.7
7.8
3.2
9.4
4
8.1
4
4
5.4
16
5.7
14
7.4
16
16
- 11 -
Table IIs
Subunit
Nic__hsNic__hs+
Nic1_hsNic1_hs+
Nic__hsNic__hsNic__hs+
Nic1_hs+
Nic__hs+
Nic1_hsTOTAL
H
Mean
155
165
182
185
197
224
230
232
264
297
213
Min Max
132 183
132 222
118 245
142 238
152 240
156 272
183 294
161 302
118 427
222 427
118 427
Subunit
Nic1_hsNic__hsNic__hsNic1_hs+
Nic__hsNic__hs+
Nic1_hs+
Nic__hs+
Nic__hs+
Nic1_hsTOTAL
P
Mean
-8.2
-6.6
-5.0
-4.0
-3.2
-2.4
-2.0
0.2
11.1
22.3
0.1
Min
-15
-7.6
-8.6
-15
-4.8
-6.2
-8.6
-7.8
-7.8
4.7
-15
Max
-5.4
-5.2
-0.3
11
-2.1
4.7
15
20
61
61
61