Modelling nicotinic acetylcholine receptors of Caenorhabditis elegans

*HYUN JI KIM, *MARK S. P. SANSOM, VALERIE RAYMOND, EMMANUEL CULETTO and DAVID B. SATTELLE*.

MRC Functional Genetics Unit, Department of Human Anatomy and Genetics, Oxford University, South Parks Road, Oxford OX1 3QX and *Laboratory of Molecular Biophysics, The Rex Richards Building, Department of Biochemistry

*For further information contact David B. Sattelle. E-mail: david.sattelle@anat.ox.ac.uk.

1. Homology modelling based on an invertebrate AChBP has enabledthe generation of the first models for a vertebrate 7 subunit and a C. elegans ACR-16 (7 –like subunit).2. Such studies open the way for parallel in vitro and in silico mutagenesis studies, which together with ligand-docking studies will greatly enhance our understanding of ligand-receptor interactions.

Introduction: Nicotinic Acetylcholine Receptors

1

Crystal structure of an invertebrate acetylcholine binding protein (AChBP)

2

Sequence alignment3 Homology models of chicken 7 and ACR-16 4

(1)Brejc et al (2001) Nature 411, 269-276(2)Mongan et al (2002) Protein Sci. 11, 1162-1171

MRCFGU website, http://www.mrcfgu.ox.ac.uk/index.htm

References

The alignment presented above was generated in two steps. First, Clustal W was used for a starting alignment with various sets of gap open and extension penalties. Secondly, we used Java view for further hand-edited alignment, on the ground of secondary structure information derived from the crystal structure, (1i9b; AChBP) and some experimental data available for this family. The presented alignment is a subset of an original alignment, prepared for the following work of homology modelling. For full details of the C. elegans nAChR gene family see ref 2. The vicinal cysteines shown to be conserved in loop C of the ACh binding site are underlined. We attempted to predict residues that might be involved in ligand binding, through multiple sequence alignment, homology modelling and atomic information based on the identified residues for ligand binding in the AChBP crystal structure. Homology models for CeACR16 and Chick alpha 7 are presented in the following panel.

Nicotinic acetylcholine receptors are pentameric transmembraneproteins that mediate fast cholinergic synaptic transmission. The 7 subunitsof vertebrates and the ACR-16-like subunits of C. elegans form homomeric receptors when heterologously expressed in Xenopus laevis oocytes. The 7 subunits are widely distributed in vertebrate brain and the ACR-16 subunit is expressed in C. elegans neurons.

Conclusions6

5 Ligand-contacting residues for 7 and ACR-16 ACR-16 homomeric receptor gates a cation channel

acr-16::GFP is located inC. elegans neurons

Chick alpha7 C. elegans ACR16

Two adjacent monomers are taken from each pentamer to show the interface between them that contains ligand binding sites. The residues involved in the ligand binding are indicated in the multiple sequence alignment presented above, and they are displayed here in stick format. Structures are centred at the vicinal cysteines (in dark blue) which are highly conserved in the subunit nAChR family members. (Chain A yellow : Chain B light blue)

.

A different presentation of the predicted ligand contacting residues is depicted, based on the multiple sequence alignment shown in panel 3. Two neighbouring monomers are coloured in blue (chain A) and red (chain B) in CeACR16 and chicken alpha 7 as well. In figures a) and b) for CeACR16, ligand contacting residues from chain A are in white, whilst those from chain B are in orange. Residues are shown in ball and stick format and van der Waals radius is shown (dotted) with corresponding atomic colours. In figures c) and d) for chicken 7, ligand contacting residues from chain A are in green, whilst those from chain B are in yellow. Residues are depicted in the same manner as for CeACR16.

40 nA

10 s

ACh

Multiple Sequence alignment of nAchRs.  

AchBP_X-Ray --EAEAYVEFDRADILYNIRQTSRPDVIPTQR-DRPVAVSVSLKFINILEVNEITNEVDV 57CeACR7 FNGFTVEGSKKEAQLYRDLLTNYSYLVRPVRNPKKALTVTMKVFIQQVLLVDAKHQMIEV 78CeACR11 IHSNLCDGSVAETKLFTDLLKGYNPLERPVQNSSQPLEVKIKLFLQQILDVDEKNQIVSV 74CeACR15 IT--QLNGSPAEVRLINDLMSGYVREERPTLDSSKPVVVSLGVFLQQIINLSEKEEQLEV 70CeACR16 ILAAPTLGSLQERRLYEDLMRNYNNLERPVANHSEPVTVHLKVALQQIIDVDEKNQVVYV 71CeACR19 KCSKVIWTGDHERRLYAKLAENYNKLARPVRNESEAVVVLLGMDYQQILDIDEKHQIMNS 79SgalphaL1 LLHHPAAANPDAKRLYDDLLSNYNRLIRPVSNNTDTVLVKLGLRLSQLIDLNLKDQILTT 75ChickA7 GLVRESLQGEFQRKLYKELLKNYNPLERPVANDSQPLTVYFTLSLMQIMDVDEKNQVLTT 74HumanA7 SLLHVSLQGEFQRKLYKELVKNYNPLERPVANDSQPLTVYFSLSLLQIMDVDEKNQVLTT 74 

B B A AchBP_X-Ray VFWQQTTWSDRTLAWNSSHSP--DQVSVPISS--LWVPDLAAYN----AISKPEVLTPQL 109CeACR7 NAWLKYIWTDFRLRWNPLDYENITSVRFQGED-QIWQPDILLYNRYIEDEQESFDITYKT 137CeACR11 NAWLSYTWFDHKLQWEPKKYGGIQDIRFPGSSDHIWKPDVLLYN----SAAEDFDSTFKS 130CeACR15 NAWLKFQWRDENLRWEPTAYENVTDLRHP-PD-ALWTPDILLYN----SVDSEFDSSYKV 124CeACR16 NAWLDYTWNDYNLVWDKAEYGNITDVRFPAG--KIWKPDVLLYN----SVDTNFDSTYQT 125CeACR19 NVWLRMSWTDHYLTWDPSEFGNIKEVRLPINN--IWKPDVLLYN----SVDQQFDSTWPV 133SgalphaL1 NVWLEHEWQDHKFRWDPAEYGGVTELYVPSEH--IWLPDIVLYN----NADGEYVVTTMT 129ChickA7 NIWLQMYWTDHYLQWNVSEYPGVKNVRFPDGL--IWKPDILLYN----SADERFDATFHT 128HumanA7 NIWLQMSWTDHYLQWNVSEYPGVKTVRFPDGQ--IWKPDILLYN----SADERFDATFHT 128

BB B B AAchBP_X-Ray ARVVSD-GEVLYMPSIRQRFSCDVSG-VDTESGATCRIKIGSWTHHSREISVDPTTENSD 167CeACR7 NAVAYSDGVINWIPPGIFKLSCKMDITLFPFDEQICFMKFGSWTYHGFALDLRLDVVKGQ 197CeACR11 NLLTYHTGTVVWIPPGVLKFVCQLDVTWFPFDDQVCEMKFGSWTFHGYAIDLQIDDDTNG 190CeACR15 NLVNYHTGNINWMPPGIFKVSCKLDIYWFPFDEQVCYFKFGSWTYT-------RDKIQLE 177CeACR16 NMIVYSTGLVHWVPPGIFKISCKIDIQWFPFDEQKCFFKFGSWTYDGYKLDLQPATGG– 183CeACR19 NAVVLYTGNVTWIPPAIIRSSCAIDIAYFPFDTQHCTMKFGSWTYSGFFTDLINTTISP- 192SgalphaL1 KAVLHHTGKVVWTPPAIFKSSCEIDVRYFPFDQQTCFMKFGSWTYDGDQIDLKHINQKYD 189ChickA7 NVLVNSSGHCQYLPPGIFKSSCYIDVRWFPFDVQKCNLKFGSWTYGGWSLDLQMQEADIS 188HumanA7 NVLVNSSGHCQYLPPGIFKSSCYIDVRWFPFDVQHCKLKFGSWSYGGWSLDLQMQEADIS 188 

B A AA A AchBP_X-Ray DS--------EYFSQYSRFEILDVTQKKNSVTYSCCPEAYEDVEVSLNFRKKGRSEIL–- 217CeACR7 EP----SADLSTYITNGEWHLLSAPARREEKFYKCCKEPYPTVKFYLHLRRRTFYYVFNV 253CeACR11 TQ----SMDLSTYLVNGEWQVISTNAKRVVSYYKCCPEPYPTVNYYLHIRRRTLYYGFNL 246CeACR15 KG----DFDFSEFIPNGEWIIIDYRTNITVKQYECCPEQYEDITFTLHLRRRTLYYSFNL 233CeACR16 -------FDISEYISNGEWALPLTTVERNEKFYDCCPEPYPDVHFYLHMRRRTLYYGFNL 236CeACR19 ----------ATYKPNGEWELLGLTSQRSIFFYECCPEPYYDVTFTVSIRRRTLYYGFNL 242SgalphaL1 DNKVKVGIDLREYYPSVEWDILGVPAERHEKYYPCCAEPYPDIFFNITLRRKTLFYTVNL 249ChickA7 GY-----------ISNGEWDLVGIPGKRTESFYECCKEPYPDITFTVTMRRRTLYYGLNL 237HumanA7 GY-----------IPNGEWDLVGIPGKRSERFYECCKEPYPDVTFTVTMRRRTLYYGLNL 237   

CC Double Cys A B Residues involved in the ligand-binding site from two interfacing monomers Residues involved in the ligand-binding site from two interfacing monomers

a)

b)

c)

d)

The work of Brejc et al (1) on AChBP from Lymnaea stagnalis, with ~24% identity to the N-terminus of the nAChR subunit, has provided a crystal structure that can serve as a basis for generating homology models of the receptor.