Analysis On The Subunit-specific Sites And Their Allosteric Modulation Network Of Neuronal NAChR In Vertebrates

The nicotinic acetylcholine receptors?nAChRs?are a family of pentameric ligand gated ion channels,conducting chemical signals into electrical ones when triggered by endogenous neurotransmitter acetylcholine?ACh?or exogenous ligands such as nicotine in a fast mode,and are highly involved in the mediation of neurotransmitters release as well as enzymatic and metabolic processes in neuron cells.The neuronal nAChRs,which are mainly formed by various combinations of the nine subunits identified by now,i.e. the ?2-?7 and ?2-?4 subunits,are widely distributed across both the central nervous system and the peripheral nervous system within vertebrate brains,including humans.Beside their important roles in neuronal signal transmissions,the neuronal nAChRs have also been proved to be responsible for smoking addiction and many other major neuronal diseases.Though sharing similar architectures of the protein sequence and molecular structure,each of the nine subunits has formed its individual characteristics during evolution,leading to the diversity of molecular dynamical,biophysical and pharmacological properties of the neuronal nAChR subtypes.By systematically studying the differences among their protein sequences,molecular structures and functions can we not only provide basic information to figure out the various performances of different nAChR subtypes,but also help to understand their relationships with various neuronal diseases such as smoking addiction,and to develop more efficient and targeted drugs.Experimental researches on the biophysical features of the nAChRs have been challenged by the complexities of their multiform compositions,stoichiometries and molecular structures.While on the other hand,increased amount of data concerning the protein sequences and structures have been unraveled by the great developments of genomics and structural biology,which make the application of bioinformatical methods a valuable approach to identify functional divergent sites among different nAChR subunits,analyze on their structure-function relationships and their capacities being targeted by subtype-specific allosteric modulators.Based on the biological molecular evolutionary theory,we identified the functional divergences among the nine neuronal nAChR subunits from 12 representative model vertebrate species,which possess the most intact and high quality sequences,by applying a series of statistical algorithms and models including the evolutionary trace,the gamma distribution model,the two-state probability model,the maximum likelihood method and so on.Then we screened a profile of sites which are highly related to these functional divergences according to their posterior probability scores calculated by the empirical Bayes method and Hidden Markov model.Thirdly we proposed three models to optimize the site profile by ranking residues according to their combined probability scores related to functional divergence to discriminate a given subunit from other background ones.Considering on the independencies of the sites within a protein molecule,we also provided weighted co-evolution analysis to identify energy coupling residues for coordinated allosteric modulations.In our results,we found out that:?1?residues which are highly conserved across the entire family preferred to locate at the principal face of the orthosteric ligand binding domain?LBD?,the“energy coupling pathway”between the LBD and the channel gating(ECP_L-T),implying their important contributions to basic functions.Compared with these highly conserved sites which clustered within the inner space of the protein 3D structure,the functional divergences related residues are more likely to distribute on the surface,indicating a responding with new features to the environmental stimulations.?2?The ?2-?3 linker and the ?4-?5 linker seem to play as“energy coupling pathways”between the principal face and the complementary face of the LBD.While residues within the ?2-?3 linker are more likely to experience site-specific functional constraint shifts among subunits and modulation of sites nearby would possibly alter the ligand binding parameters in a subunit-selective manner,residues within the ?4-?5 linker are more likely to experience gene cluster-specific functional divergences and conformational changes of this region may affect both the channel gating and the ligand binding ability.?3?The gene cluster-specific functional divergences caused by the second duplication event shown in our phylogenetic tree,which separate the branches of ?2 and ?4 subunits with those of the ?2-?6 and ?3 subunits,are related highly with radical substitutions of sites within the ECP_L-T,suggesting the value of this domain to subunit-specific allosteric modulations.?4?The weighted models?M1 and M2?we proposed in our study have shown greater improvements than the M0 model in ranking the functional specific site profile of a target subunit especially when it has high sequence similarities with the background ones.And the choice of the prerequisite sites would influence the results of M2 directly.?5?There exist three networks of sites within the nAChR subunit,responsible for the coupling of energies from the principal face of LBD to the channel gating,or from the complementary face of LBD to the channel gating,or of the two faces of LBD separately.The combined consideration of a residue its possible involvements within the divergent functions among subunits,and its coupling relationships with the other sites would draw us a more refined picture of its value in nAChR subtype-specific modulations.