Modelling nicotinic acetylcholine receptors of Caenorhabditis elegans

1. 1 Introduction: Nicotinic Acetylcholine Receptors 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 3 Sequence alignment 4 Homology models of chicken 7 and ACR-16 Multiple Sequence alignment of nAchRs. AchBP_X-Ray --EAEAYVEFDRADILYNIRQTSRPDVIPTQR-DRPVAVSVSLKFINILEVNEITNEVDV 57 CeACR7 FNGFTVEGSKKEAQLYRDLLTNYSYLVRPVRNPKKALTVTMKVFIQQVLLVDAKHQMIEV 78 CeACR11 IHSNLCDGSVAETKLFTDLLKGYNPLERPVQNSSQPLEVKIKLFLQQILDVDEKNQIVSV 74 CeACR15 IT--QLNGSPAEVRLINDLMSGYVREERPTLDSSKPVVVSLGVFLQQIINLSEKEEQLEV 70 CeACR16ILAAPTLGSLQERRLYEDLMRNYNNLERPVANHSEPVTVHLKVALQQIIDVDEKNQVVYV 71 CeACR19 KCSKVIWTGDHERRLYAKLAENYNKLARPVRNESEAVVVLLGMDYQQILDIDEKHQIMNS 79 SgalphaL1 LLHHPAAANPDAKRLYDDLLSNYNRLIRPVSNNTDTVLVKLGLRLSQLIDLNLKDQILTT 75 ChickA7GLVRESLQGEFQRKLYKELLKNYNPLERPVANDSQPLTVYFTLSLMQIMDVDEKNQVLTT 74 HumanA7 SLLHVSLQGEFQRKLYKELVKNYNPLERPVANDSQPLTVYFSLSLLQIMDVDEKNQVLTT 74  BBAAchBP_X-RayVFWQQTTWSDRTLAWNSSHSP--DQVSVPISS--LWVPDLAAYN----AISKPEVLTPQL 109 CeACR7 NAWLKYIWTDFRLRWNPLDYENITSVRFQGED-QIWQPDILLYNRYIEDEQESFDITYKT 137 CeACR11 NAWLSYTWFDHKLQWEPKKYGGIQDIRFPGSSDHIWKPDVLLYN----SAAEDFDSTFKS 130 CeACR15 NAWLKFQWRDENLRWEPTAYENVTDLRHP-PD-ALWTPDILLYN----SVDSEFDSSYKV 124 CeACR16NAWLDYTWNDYNLVWDKAEYGNITDVRFPAG--KIWKPDVLLYN----SVDTNFDSTYQT 125 CeACR19 NVWLRMSWTDHYLTWDPSEFGNIKEVRLPINN--IWKPDVLLYN----SVDQQFDSTWPV 133 SgalphaL1 NVWLEHEWQDHKFRWDPAEYGGVTELYVPSEH--IWLPDIVLYN----NADGEYVVTTMT 129 ChickA7NIWLQMYWTDHYLQWNVSEYPGVKNVRFPDGL--IWKPDILLYN----SADERFDATFHT 128 HumanA7 NIWLQMSWTDHYLQWNVSEYPGVKTVRFPDGQ--IWKPDILLYN----SADERFDATFHT 128 BB B B A AchBP_X-RayARVVSD-GEVLYMPSIRQRFSCDVSG-VDTESGATCRIKIGSWTHHSREISVDPTTENSD 167 CeACR7 NAVAYSDGVINWIPPGIFKLSCKMDITLFPFDEQICFMKFGSWTYHGFALDLRLDVVKGQ 197 CeACR11 NLLTYHTGTVVWIPPGVLKFVCQLDVTWFPFDDQVCEMKFGSWTFHGYAIDLQIDDDTNG 190 CeACR15 NLVNYHTGNINWMPPGIFKVSCKLDIYWFPFDEQVCYFKFGSWTYT-------RDKIQLE 177 CeACR16NMIVYSTGLVHWVPPGIFKISCKIDIQWFPFDEQKCFFKFGSWTYDGYKLDLQPATGG– 183 CeACR19 NAVVLYTGNVTWIPPAIIRSSCAIDIAYFPFDTQHCTMKFGSWTYSGFFTDLINTTISP- 192 SgalphaL1 KAVLHHTGKVVWTPPAIFKSSCEIDVRYFPFDQQTCFMKFGSWTYDGDQIDLKHINQKYD 189 ChickA7NVLVNSSGHCQYLPPGIFKSSCYIDVRWFPFDVQKCNLKFGSWTYGGWSLDLQMQEADIS 188 HumanA7 NVLVNSSGHCQYLPPGIFKSSCYIDVRWFPFDVQHCKLKFGSWSYGGWSLDLQMQEADIS 188 BA AA AAchBP_X-RayDS--------EYFSQYSRFEILDVTQKKNSVTYSCCPEAYEDVEVSLNFRKKGRSEIL–- 217 CeACR7 EP----SADLSTYITNGEWHLLSAPARREEKFYKCCKEPYPTVKFYLHLRRRTFYYVFNV 253 CeACR11 TQ----SMDLSTYLVNGEWQVISTNAKRVVSYYKCCPEPYPTVNYYLHIRRRTLYYGFNL 246 CeACR15 KG----DFDFSEFIPNGEWIIIDYRTNITVKQYECCPEQYEDITFTLHLRRRTLYYSFNL 233 CeACR16 -------FDISEYISNGEWALPLTTVERNEKFYDCCPEPYPDVHFYLHMRRRTLYYGFNL 236 CeACR19 ----------ATYKPNGEWELLGLTSQRSIFFYECCPEPYYDVTFTVSIRRRTLYYGFNL 242 SgalphaL1 DNKVKVGIDLREYYPSVEWDILGVPAERHEKYYPCCAEPYPDIFFNITLRRKTLFYTVNL 249 ChickA7GY-----------ISNGEWDLVGIPGKRTESFYECCKEPYPDITFTVTMRRRTLYYGLNL 237 HumanA7 GY-----------IPNGEWDLVGIPGKRSERFYECCKEPYPDVTFTVTMRRRTLYYGLNL 237 CCDouble Cys ABResidues involved in the ligand-binding site from two interfacing monomers Nicotinic acetylcholine receptors are pentameric transmembrane proteins that mediate fast cholinergic synaptic transmission. The 7 subunits of 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. ACh C. elegans ACR16 Chick alpha7 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) 40 nA 10 s ACR-16 homomeric receptor gates a cation channel 5 Ligand-contacting residues for 7 and ACR-16 acr-16::GFP is located in C. elegans neurons c) a) 2 Crystal structure of an invertebrate acetylcholine binding protein (AChBP) b) d) 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. . 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. 6 Conclusions 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. 1. Homology modelling based on an invertebrate AChBP has enabled the 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 mutagenesisstudies, which together with ligand-docking studies will greatly enhance our understanding of ligand-receptor interactions. References *For further information contact David B. Sattelle. E-mail: david.sattelle@anat.ox.ac.uk. • Brejc et al (2001) Nature 411, 269-276 • Mongan et al (2002) Protein Sci. 11, 1162-1171 MRCFGU website, http://www.mrcfgu.ox.ac.uk/index.htm