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 A resolution using synchrotron radiation. The crystal belonged to the monoclinic space group P2(1), with unit-cell parameters a = 84.0, b = 97.5, c = 108.1 A, beta = 93.0 degrees . Assuming the presence of two protomers per crystallographic asymmetric unit, the Matthews coefficient V(M) was calculated to be 3.5 A(3) Da(-1) and the solvent content was 65%.
Muto
Takanori
T
Biomolecular Engineering Research Institute, Suita, Osaka, Japan.
Tsuchiya
Daisuke
D
Morikawa
Kosuke
K
Jingami
Hisato
H
D000818:Animals; D015153:Blotting, Western; D002850:Chromatography, Gel; D003460:Crystallization; D018360:Crystallography, X-Ray; D006031:Glycosylation; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry; D017434:Protein Structure, Tertiary; D051381:Rats; D018094:Receptors, Metabotropic Glutamate / Q000737:chemistry*
D050505:Mutant Proteins; D018094:Receptors, Metabotropic Glutamate; C102718:metabotropic glutamate receptor 3