Open AccessSite‐specific unglycosylation to improve crystallization of the metabotropic glutamate receptor 3 extracellular domain
Author(s) -
Muto Takanori,
Tsuchiya Daisuke,
Morikawa Kosuke,
Jingami Hisato
Publication year - 2009
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309109002267
Subject(s) - metabotropic glutamate receptor , monoclinic crystal system , metabotropic glutamate receptor 3 , glutamate receptor , extracellular , metabotropic glutamate receptor 2 , mutant , recombinant dna , chemistry , metabotropic receptor , crystallography , biology , biochemistry , receptor , crystal structure , gene
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 P 2 1 , 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 Å 3 Da −1 and the solvent content was 65%.