Effects of C-Terminal Carboxylation on α-Conotoxin LsIA Interactions with Human α7 Nicotinic Acetylcholine Receptor: Molecular Simulation Studies

Abstract

α-Conotoxins selectively bind to nicotinic acetylcholine receptors (nAChRs), which are therapeutic targets due to their important role in signaling transmission in excitable cells. A previous experimental study has demonstrated that carboxylation of the C-terminal of α-conotoxin LsIA reduces its potency to inhibit human α7 nAChR relative to naturally amidated LsIA. However, little is known about the contribution of conformational changes in the receptor and interactions, induced by C-terminal amidation/carboxylation of conotoxins, to selective binding to nAChRs, since most conotoxins and some disulfide-rich peptides from other conotoxin subfamilies possess a naturally amidated C-terminal. In this study, we employ homology modeling and molecular dynamics (MD) simulations to propose the determinants for differential interactions between amidated and carboxylated LsIAs with α7 nAChR. Our findings indicate an overall increased number of contacts favored by binding of amidated LsIA versus its carboxylated counterpart. Toxin-receptor pairwise interactions, which may play a role in enhancing the potency of the former, include ARG10-TRP77, LEU141 and CYS17-GLN79 via persistent hydrogen bonds and cation-π interactions, which are weakened in the carboxylated form due to a strong intramolecular salt-bridge formed by ARG10 and carboxylated C-terminus. The binding of amidated LsIA also induces enhanced movements in loop C and the juxtamembrane Cys-loop that are closely associated with receptor function. Additionally, the impacts of binding of LsIA on the overall structure and inter-subunit contacts were examined using inter-residue network analysis, suggesting a clockwise tilting of the α7 C and F loops upon binding to carboxylated LsIA, which is absent for amidated LsIA binding. The predicted molecular mechanism of LsIA binding to the α7 receptor may provide new insights into the important role of the C-terminal in the binding potency of conotoxins at neuronal nAChRs for pharmacological purposes.

Interactions of the α3β2 Nicotinic Acetylcholine Receptor Interfaces with α-Conotoxin LsIA and its Carboxylated C-terminus Analogue: Molecular Dynamics Simulations

“…The enhanced contacts of the LsIA# may be induced by the potential intra-molecular salt bridges or hydrogen bonds established by the C-T (C17) and the R10 ( Figure S5), which enhanced the rigidity of the residues in loop2, namely V11, N13, P14, N15, I16 and C-T, rather than R10. This increased rigidity was also suggested in a previous experimental study using an electrophysiological recording approach and predicted in our computational study on LsIA/α7 nAChR systems [9,61]. Therefore, the C-terminal carboxylation not only directly results in the enhanced receptor interactions by the C-T, but also improves the receptor interactions by P14 of the LsIA#.…”

“…The geometry of LsIA#-C17 confers strong hydrogen bonds or salt-bridges established by the carboxyl group of C17 with β2(−)-K187. In our previous study, similar hydrogen bonds between LsIA#-C17 and (−)Q79 of human α7 nAChR were determined [61]. Interestingly, salt bridges/hydrogen bonds formed by other α-conotoxins and the receptor are involved in anchoring the inhibitors to nAChRs.…”

“…This may, in part, be due to LsIA# being more able to effectively draw together the subunits. Additionally, α3(−)-I144 is also homologous with (−)L141 on α7 subunit of human α7 nAChR, which was determined to form an enhanced interaction with LsIA-R10 of LsIA versus the carboxylated analogue [61]. In summary, the hydrophobic residues, isoleucine and leucine, at this homologous position, may contribute to the enhanced interactions by LsIA# at α7 and α3β2 nAChRs versus the wild type.…”

“…Similar π−π interactions were found between α-conotoxin MII-H9 and H12 and (−)F119 at α3(+)β2(−) interface and the (−)W55 at β2(+)α3(−) interfaces of rat α3β2 nAChR by Sambasivarao and colleagues [20]. Other cation-π and π−π interactions were determined in the F9 of AuIB with W59 and K61 on β4(−) subunit of α3β4, and LsIA-R10 with (−)W77 of human α7 nAChR, respectively [36,61].…”

“…The comparative model of LsIA-and LsIA#-α3β2 nAChR complexes were built according to the process described in our previous study [61]. Briefly, the sequence of LsIA bound to the extracellular domain (ECD) of rat α3 subunit (UniProtKB-P04757) and rat β2 subunit (UniProtKB-P12390) was aligned to that of α-conotoxin PnIA[A10K, D14K] bound to Aplysia californica acetylcholine binding protein (Ac-AChBP) (PDB ID: 2BR8) [35], with the known crystal structure.…”