The crystal structure of Ac-AChBP in complex with α-conotoxin LvIA reveals the mechanism of its selectivity towards different nAChR subtypes

doi:10.1007/s13238-017-0426-2

Protein Cell

2017, Vol. 8

Issue (9) : 675-685 https://doi.org/10.1007/s13238-017-0426-2

RESEARCH ARTICLE

The crystal structure of Ac-AChBP in complex with α-conotoxin LvIA reveals the mechanism of its selectivity towards different nAChR subtypes

Manyu Xu¹, Xiaopeng Zhu², Jinfang Yu¹, Jinpeng Yu², Sulan Luo²(

), Xinquan Wang¹(

)

¹. The Ministry of Education Key Laboratory of Protein Science, School of Life Sciences, Beijing Advanced Innovation Center for Structural Biology, Collaborative Innovation Center for Biotherapy, Tsinghua University, Beijing 100084, China
². Key Laboratory of Tropical Biological Resources, Ministry of Education, Key Lab for Marine Drugs of Haikou, Hainan University, Haikou 570228, China

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Abstract

The α3* nAChRs, which are considered to be promising drug targets for problems such as pain, addiction, cardiovascular function, cognitive disorders etc., are found throughout the central and peripheral nervous system. The α-conotoxin (α-CTx) LvIA has been identified as the most selective inhibitor of α3β2 nAChRs known to date, and it can distinguish the α3β2 nAChR subtype from the α6/α3β2β3 and α3β4 nAChR subtypes. However, the mechanism of its selectivity towards α3β2, α6/α3β2β3, and α3β4 nAChRs remains elusive. Here we report the co-crystal structure of LvIA in complex with Aplysia californica acetylcholine binding protein (Ac-AChBP) at a resolution of 3.4 Å. Based on the structure of this complex, together with homology modeling based on other nAChR subtypes and binding affinity assays, we conclude that Asp-11 of LvIA plays an important role in the selectivity of LvIA towards α3β2 and α3/α6β2β3 nAChRs by making a salt bridge with Lys-155 of the rat α3 subunit. Asn-9 lies within a hydrophobic pocket that is formed by Met-36, Thr-59, and Phe-119 of the rat β2 subunit in the α3β2 nAChR model, revealing the reason for its more potent selectivity towards the α3β2 nAChR subtype. These results provide molecular insights that can be used to design ligands that selectively target α3β2 nAChRs, with significant implications for the design of new therapeutic α-CTxs.

Keywords base editor high-fidelity mouse embryos proximal-site deamination whole-genome sequencing

Corresponding Author(s): Sulan Luo,Xinquan Wang

Issue Date: 20 September 2017

Cite this article:

Manyu Xu,Xiaopeng Zhu,Jinfang Yu, et al. The crystal structure of Ac-AChBP in complex with α-conotoxin LvIA reveals the mechanism of its selectivity towards different nAChR subtypes[J]. Protein Cell, 2017, 8(9): 675-685.

URL:

https://academic.hep.com.cn/pac/EN/10.1007/s13238-017-0426-2
https://academic.hep.com.cn/pac/EN/Y2017/V8/I9/675

1	AdamsPDet al. (2002) PHENIX: building new software for automated crystallographic structure determination.Acta Crystallogr Sect D-Biol Crystallogr58:1948–1954 https://doi.org/10.1107/S0907444902016657
2	AzamL, McIntoshJM (2009) Alpha-conotoxins as pharmacological probes of nicotinic acetylcholine receptors.Acta Pharmacol Sin30(6):771–783 https://doi.org/10.1038/aps.2009.47
3	BeroukhimR, UnwinN (1995) Three-dimensional location of the main immunogenic region of the acetylcholine receptor.Neuron15(2):323–331 https://doi.org/10.1016/0896-6273(95)90037-3
4	BourneYet al. (2005) Crystal structure of a Cbtx nd antagonistbound s essential interactions between snake α-neurotoxins and nicotinic receptors.The EMBO Journal24(8):1512–1522 https://doi.org/10.1038/sj.emboj.7600620
5	BrejcKet al. (2001) Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors.Nature411(6835):269–276 https://doi.org/10.1038/35077011
6	CecchiniM, ChangeuxJ-P (2015) The nicotinic acetylcholine receptor and its prokaryotic homologues: Structure, conformational transitions & allosteric modulation.Neuropharmacology96 (Part B):137–149 https://doi.org/10.1016/j.neuropharm.2014.12.006
7	CeliePHNet al. (2004) Nicotine and carbamylcholine binding to nicotinic acetylcholine receptors as studied in AChBP crystal structures.Neuron41(6):907–914 https://doi.org/10.1016/S0896-6273(04)00115-1
8	CeliePHNet al. (2005) Crystal structure of nicotinic acetylcholine receptor homolog AChBP in complex with an [alpha]-conotoxin PnIA variant.Nat Struct Mol Biol12(7):582–588 https://doi.org/10.1038/nsmb951
9	DavisIWet al. (2004) MOLPROBITY: structure validation and allatom contact analysis for nucleic acids and their complexes.Nucleic Acids Res32(Web Server issue):W615–W619 https://doi.org/10.1093/nar/gkh398
10	DavisIWet al. (2007) MolProbity: all-atom contacts and structure validation for proteins and nucleic acids.Nucleic Acids Res35 (Web Server issue):W375–W383 https://doi.org/10.1093/nar/gkm216
11	DellisantiCDet al. (2007) Crystal structure of the extracellular domain of nAChR alpha1 bound to alpha-bungarotoxin at 1.94 A resolution.Nat Neurosci10(8):953–962 https://doi.org/10.1038/nn1942
12	DineleyKT, PandyaAA, YakelJL (2015) Nicotinic ACh receptors as therapeutic targets in CNS disorders.Trends Pharmacol Sci36 (2):96–108 https://doi.org/10.1016/j.tips.2014.12.002
13	DutertreSet al. (2007) AChBP-targeted alpha-conotoxin correlates distinct binding orientations with nAChR subtype selectivity.EMBO J26(16):3858–3867 https://doi.org/10.1038/sj.emboj.7601785
14	EmsleyP, CowtanK (2004) Coot: model-building tools for molecular graphics.Acta Crystallogr Sect D-Biol Crystallogr60:2126–2132 https://doi.org/10.1107/S0907444904019158
15	HansenSBet al.(2005) Structures of Aplysia AChBP complexes with nicotinic agonists and antagonists reveal distinctive binding interfaces and conformations.EMBO J24(20):3635–3646 https://doi.org/10.1038/sj.emboj.7600828
16	HendricksonL, GuildfordM, TapperA (2013) Neuronal nicotinic acetylcholine receptors: common molecular substrates of nicotine and alcohol dependence.Front Psychiatry. doi:10.3389/fpsyt.2013.00029 https://doi.org/10.3389/fpsyt.2013.00029
17	HoneAJet al. (2013) Positional scanning mutagenesis of alphaconotoxin PeIA identifies critical residues that confer potency and selectivity for alpha6/alpha3beta2beta3 and alpha3beta2 nicotinic acetylcholine receptors.J Biol Chem288(35):25428–25439 https://doi.org/10.1074/jbc.M113.482059
18	HurstR, RollemaH, BertrandD (2013) Nicotinic acetylcholine receptors: from basic science to therapeutics.Pharmacol Ther137(1):22–54 https://doi.org/10.1016/j.pharmthera.2012.08.012
19	KarlinA (2002) Emerging structure of the nicotinic acetylcholine receptors.Nat Rev Neurosci3(2):102–114 https://doi.org/10.1038/nrn731
20	KouvatsosNet al. (2016) Crystal structure of a human neuronal nAChR extracellular domain in pentameric assembly: Ligandbound alpha2 homopentamer.Proc Natl Acad Sci USA113 (34):9635–9640 https://doi.org/10.1073/pnas.1602619113
21	LaskowskiRAet al. (1993) PROCHECK—a program to check the stereochemical quality of protein structures.J Appl Crystallogr26:283–291 https://doi.org/10.1107/S0021889892009944
22	LavioletteSR, van der KooyD (2004) The neurobiology of nicotine addiction: bridging the gap from molecules to behaviour.Nat Rev Neurosci5(1):55–65 https://doi.org/10.1038/nrn1298
23	Le NovereN, CorringerPJ, ChangeuxJP (2002) The diversity of subunit composition in nAChRs: evolutionary origins, physiologic and pharmacologic consequences.J Neurobiol53(4):447–456 https://doi.org/10.1002/neu.10153
24	LebbeEKet al. (2014) Conotoxins targeting nicotinic acetylcholine receptors: an overview.Mar Drugs12(5):2970–3004 https://doi.org/10.3390/md12052970
25	LiS-Xet al. (2011) Ligand-binding domain of an [alpha]7-nicotinic receptor chimera and its complex with agonist.Nat Neurosci14 (10):1253–1259 https://doi.org/10.1038/nn.2908
26	LinBet al. (2016) From crystal structure of α-conotoxin GIC in complex with Ac-AChBP to molecular determinants of its high selectivity for α3β2 nAChR.Scientific Reports6:22349 https://doi.org/10.1038/srep22349
27	LuoSet al. (2010) Atypical alpha-conotoxin LtIA from Conus litteratus targets a novel microsite of the alpha3beta2 nicotinic receptor.J Biol Chem285(16):12355–12366 https://doi.org/10.1074/jbc.M109.079012
28	LuoSet al.(2014) A novel alpha4/7-conotoxin LvIA from Conus lividus that selectively blocks alpha3beta2 vs. alpha6/alpha3beta2beta3 nicotinic acetylcholine receptors.FASEB J28(4):1842–1853 https://doi.org/10.1096/fj.13-244103
29	McCoyAJet al. (2007) Phaser crystallographic software.J Appl Crystallogr40(Pt 4):658–674 https://doi.org/10.1107/S0021889807021206
30	McDougalOMet al. (2013) pKa determination of histidine residues in alpha-conotoxin MII peptides by 1H NMR and constant pH molecular dynamics simulation.JPhysChemB117(9):2653–2661 https://doi.org/10.1021/jp3117227
31	MirRet al. (2016) Conotoxins: structure, therapeutic potential and pharmacological applications.Curr Pharm Des22(5):582–589 https://doi.org/10.2174/1381612822666151124234715
32	MiyazawaAet al. (1999) Nicotinic acetylcholine receptor at 4.6 Å resolution: transverse tunnels in the channel1.J Mol Biol288 (4):765–786 https://doi.org/10.1006/jmbi.1999.2721
33	Morales-PerezCL, NovielloCM, HibbsRE (2016) X-ray structure of the human alpha4beta2 nicotinic receptor.Nature538 (7625):411–415 https://doi.org/10.1038/nature19785
34	NemeczA, TaylorP (2011) Creating an alpha7 nicotinic acetylcholine recognition domain from the acetylcholine-binding protein: crystallographic and ligand selectivity analyses.J Biol Chem286(49):42555–42565 https://doi.org/10.1074/jbc.M111.286583
35	OrtellsMO, LuntGG (1995) Evolutionary history of the ligand-gated ion-channel superfamily of receptors.Trends Neurosci18 (3):121–127 https://doi.org/10.1016/0166-2236(95)93887-4
36	OtwinowskiZ, MinorW (1997) Processing of X-ray diffraction data collected in oscillation mode.Macromol Crystallogr Pt A276:307–326 https://doi.org/10.1016/S0076-6879(97)76066-X
37	PaoliniM, De BiasiM (2011) Mechanistic insights into nicotine withdrawal.Biochem Pharmacol82(8):996–1007 https://doi.org/10.1016/j.bcp.2011.07.075
38	RucktooaP, SmitAB, SixmaTK (2009) Insight in nAChR subtype selectivity from AChBP crystal structures.Biochem Pharmacol78 (7):777–787 https://doi.org/10.1016/j.bcp.2009.06.098
39	SalasRet al. (2009) Nicotinic receptors in the habenulo-interpeduncular system are necessary for nicotine withdrawal in mice.J Neurosci29(10):3014–3018 https://doi.org/10.1523/JNEUROSCI.4934-08.2009
40	SambasivaraoSVet al. (2014) Cover picture: acetylcholine promotes binding of α-conotoxin MII at α3β2 nicotinic acetylcholine receptors (ChemBioChem 3/2014).ChemBioChem15 (3):413–424 https://doi.org/10.1002/cbic.201300577
41	SmitABet al. (2001) A glia-derived acetylcholine-binding protein that modulates synaptic transmission.Nature411(6835):261–268 https://doi.org/10.1038/35077000
42	TsetlinV, UtkinY, KasheverovI (2009) Polypeptide and peptide toxins, magnifying lenses for binding sites in nicotinic acetylcholine receptors.Biochem Pharmacol78(7):720–731 https://doi.org/10.1016/j.bcp.2009.05.032
43	UlensCet al. (2006) Structural determinants of selective alphaconotoxin binding to a nicotinic acetylcholine receptor homolog AChBP.Proc Natl Acad Sci USA103(10):3615–3620 https://doi.org/10.1073/pnas.0507889103
44	UnwinN (1993) Nicotinic acetylcholine receptor an 9 Å resolution.J Mol Biol229(4):1101–1124 https://doi.org/10.1006/jmbi.1993.1107
45	UnwinN (1995) Acetylcholine receptor channel imaged in the open state.Nature373(6509):37–43 https://doi.org/10.1038/373037a0
46	UnwinN (2005) Refined structure of the nicotinic acetylcholine receptor at 4A resolution.J Mol Biol346(4):967–989 https://doi.org/10.1016/j.jmb.2004.12.031
47	WebbB, SaliA (2014) Comparative protein structure modeling using MODELLER.Curr Protoc Bioinform47:5.6.1–5.6.32 https://doi.org/10.1002/0471250953.bi0506s47
48	ZhangsunDet al. (2015) Key residues in the nicotinic acetylcholine receptor beta2 subunit contribute to alpha-conotoxin LvIA binding.J Biol Chem290(15):9855–9862 https://doi.org/10.1074/jbc.M114.632646
49	ZoliM, PistilloF, GottiC (2015) Diversity of native nicotinic receptor subtypes in mammalian brain.Neuropharmacology96(Pt B):302–311 https://doi.org/10.1016/j.neuropharm.2014.11.003
50	ZouridakisMet al. (2014) Crystal structures of free and antagonistbound states of human alpha9 nicotinic receptor extracellular domain.Nat Struct Mol Biol21(11):976–980 https://doi.org/10.1038/nsmb.2900

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