Site-specific unglycosylation to improve crystallization of the metabotropic glutamate receptor 3 extracellular domain

Metabotropic glutamate receptors (mGluRs) are involved in the regulation of many physiological and pathological processes in the central nervous system. The extracellular domain (ECD) of mGluR subtype 3 (mGluR3) was produced using the baculovirus expression system and purified from the culture medium. However, the recombinant protein showed heterogeneity in molecular weight on SDS–PAGE analysis. It was found that the unglycosylation of Asn414 significantly reduced the heterogeneity. Consequently, three site-specifically unglycosylated mutant proteins of mGluR3 ECD, replacing Asn414 only or replacing Asn414 in combination with other glycosylation sites, were successfully crystallized in the presence of L-glutamate. Among them, crystals of the N414/439Q mutant diffracted X-rays to 2.35 Å resolution using synchrotron radiation. The crystal belonged to the monoclinic space group P2₁, with unit-cell parameters a = 84.0, b = 97.5, c = 108.1 Å, β = 93.0°. Assuming the presence of two protomers per crystallographic asymmetric unit, the Matthews coefficient V_M was calculated to be 3.5 ų Da⁻¹ and the solvent content was 65%.