•Structural description of ivermectin binding to GlyRα3 at high-resolution
•Binding of ivermectin expands the ion channel pore
•Structural information could provide insights into the discovery of new analgesic
Ivermectin acts as a positive allosteric modulator of several Cys-loop receptors including the glutamate-gated chloride channels (GluCls), γ-aminobutyric acid receptors (GABAARs), glycine receptors (GlyRs), and neuronal α7-nicotinic receptors (α7 nAChRs). The crystal structure of Caenorhabditis elegans GluCl complexed with ivermectin revealed the details of its ivermectin binding site. Although the electron microscopy structure of zebrafish GlyRα1 complexed with ivermectin demonstrated a similar binding orientation, detailed structural information on the ivermectin binding and pore opening for Cys-loop receptors in vertebrates has been elusive. Here we present the crystal structures of human GlyRα3 in complex with ivermectin at 2.85 and 3.08 Å resolution. Our structures allow us to explore in detail the molecular recognition of ivermectin by GlyRs, GABAARs, and α7 nAChRs. Comparisons with previous structures reveal how the ivermectin binding expands the ion channel pore. Our results hold promise in structure-based design of GlyR modulators for the treatment of neuropathic pain.
Results and Discussion
Crystallization and Architecture
The same crystallography construct GlyRα3cryst used for previous structural studies of GlyRα3 with antagonist strychnine (Huang et al., 2015 ) and potentiator AM-3607 (Huang et al., 2016 ) is applied here for structural studies of GlyRα3 with ivermectin. Attempts to obtain the crystal structure of GlyRα3cryst in complex with ivermectin were not successful, as those crystals only diffracted to 4.6 Å. However, we were able to determine the crystal structure of the quaternary complex of GlyRα3cryst with AM-3607, glycine, and ivermectin at 2.85 Å resolution (Table 1 and Figure 1). The structure of the GlyRα3cryst/AM-3607/glycine/ivermectin complex is very similar to that of the GlyRα3cryst/AM-3607/glycine complex with root-mean-square deviation (RMSD) of 0.42 Å for all Cα atoms between the two structures. We also mutated residue Asn38 to Gln to eliminate the only glycosylation site on the protein and determined the crystal structure of the GlyRα3crystN38Q/AM-3607/glycine/ivermectin complex at a resolution of 3.08 Å from a different crystal form (Table 1). The GlyRα3cryst/AM-3607N38Q/glycine/ivermectin complex is very similar to the GlyRα3cryst/AM-3607/glycine/ivermectin complex with an RMSD of 0.29 Å for all Cα atoms. Comparison with the cryo-EM structures of GlyRα1 revealed that these two crystal structures of glycine/AM-3607/ivermectin-bound GlyRα3 are also similar to the cryo-EM structure of glycine/ivermectin-bound GlyRα1 (Du et al., 2015 ), with an RMSD of 0.91 Å for all Cα atoms.
Ivermectin Binding Site
As observed in the 3.8 Å resolution cryo-EM structure of glycine/ivermectin-bound GlyRα1 (Du et al., 2015 ), ivermectin in our GlyRα3 structures binds to the TMD of GlyRα3 in a cleft at the interface of adjacent subunits, proximal to the extracellular side of the membrane bilayer (Figures 2A, 2B , and S1). Wedged between the M3 helix from the (+) subunit and the M1 helix from the (−) subunit, ivermectin inserts deeply into the subunit interface and makes important contacts with the M2(+) helix and the M2-M3 loop(+). Unlike the cryo-EM structure of glycine/ivermectin-bound GlyRα1 (Du et al., 2015 ), the ivermectin binding site was revealed in greater detail in our crystal structures of GlyRα3. The benzofuran head of ivermectin is orientated toward the pore, such that the C5-hydroxyl of ivermectin forms two hydrogen bonds with the hydroxyl side chain of Ser267 from M2(+) and the main chain oxygen of Gln226 from M1(−), while the C7-hydroxyl of ivermectin forms a hydrogen bond with the backbone carbonyl of Ile225 from M1(−). The spiroketal is orientated toward the cytoplasm and the disaccharide remains outside the binding cleft in contact with the surrounding lipids and the protein surface. In addition to the above-mentioned three hydrogen bonds, there are extensive hydrophobic interactions between ivermectin and the M1(−), M2(+), and M3(+) helices as well as the M2-M3 loop(+). For example, the side chain of Pro230 from the M1(−) helix is stacked against the benzofuran moiety of ivermectin while the side chain of Ala288 from M3(+) is involved in a number of hydrophobic interactions with C14-methyl, spiroketal C18, and C10 of ivermectin. All the residues critical for ivermectin binding are identical among α subunits of GlyRs (Figure S2), which explains why ivermectin activates GlyRs.
The analgesic effects of Δ9-tetrahydrocannabinol (THC), the major psychoactive component in cannabis, are mediated through the potentiation of GlyRs, particularly GlyRα3 (Hejazi et al., 2006 , Xiong et al., 2011 ). The THC binding site in GlyRα3 was identified by mutagenesis and NMR analysis to be also located in the TMD, with Ser296 of M3 as being critical for THC potentiation. Ser296 is only about 7 Å from the ivermectin binding site in our GlyRα3 crystal structures and it is possible that ivermectin-based CNS-penetrant analogs could have similar analgesic effects. Our crystal structures of GlyRα3 in complex with ivermectin provide a valuable structural basis for screening and designing novel pain therapeutics that act on the TMD of GlyRα3.