TY - JOUR
T1 - L-Asp is a useful tool in the purification of the ionotropic glutamate receptor A2 ligand-binding domain
AU - Krintel, Christian
AU - Frydenvang, Karla
AU - Ceravalls de Rabassa, Anna
AU - Kaern, Anne Mette
AU - Gajhede, Michael
AU - Pickering, Darryl S
AU - Kastrup, Jette S
N1 - This article is protected by copyright. All rights reserved.
PY - 2014/5
Y1 - 2014/5
N2 - In purification of the ionotropic glutamate receptor A2 (GluA2) ligand-binding domain (LBD), l-Glu-supplemented buffers have previously been used for protein stabilization during the procedure. This sometimes hampers structural studies of low-affinity ligands, because l-Glu is difficult to displace, despite extensive dialysis. Here, we show that l-Asp binds to full-length GluA2 with low affinity (Ki = 0.63 mm) and to the GluA2 LBD with even lower affinity (Ki = 2.6 mm), and we use differential scanning fluorimetry to show that l-Asp is able to stabilize the isolated GluA2 LBD. We also show that l-Asp can replace l-Glu during purification, providing both equal yields and purity of the resulting protein sample. Furthermore, we solved three structures of the GluA2 LBD in the presence of 7.5, 50 and 250 mm l-Asp. Surprisingly, with 7.5 mm l-Asp, the GluA2 LBD crystallized as a mixed dimer, with l-Glu being present in one subunit, and neither l-Asp nor l-Glu being present in the other subunit. Thus, residual l-Glu is retained from the expression medium. On the other hand, only l-Asp was found at the binding site when 50 or 250 mm l-Asp was used for crystallization. The binding mode observed for l-Asp at the GluA2 LBD is very similar to that described for l-Glu. Taking our findings together, we have shown that l-Asp can be used instead of l-Glu for ligand-dependent stabilization of the GluA2 LBD during purification. This will enable structural studies of low-affinity ligands for lead optimization in structure-based drug design. Database Structural data are available in the Protein Data Bank under accession numbers 4O3B (7.5 mm l-Asp), 4O3C (50 mm l-Asp), and 4O3A (250 mm l-Asp) Structured digital abstract GluA2 and GluA2 bind by x-ray crystallography (View interaction) Here, we use differential scanning fluorimetry and X-ray crystallography to show that the low affinity agonist l-Asp can stabilize the isolated ionotropic glutamate receptor A2 ligand-binding domain by binding in the glutamate binding pocket. We also show that l-Asp can replace l-Glu during purification, providing both equal yields and purity of the resulting protein preparation.
AB - In purification of the ionotropic glutamate receptor A2 (GluA2) ligand-binding domain (LBD), l-Glu-supplemented buffers have previously been used for protein stabilization during the procedure. This sometimes hampers structural studies of low-affinity ligands, because l-Glu is difficult to displace, despite extensive dialysis. Here, we show that l-Asp binds to full-length GluA2 with low affinity (Ki = 0.63 mm) and to the GluA2 LBD with even lower affinity (Ki = 2.6 mm), and we use differential scanning fluorimetry to show that l-Asp is able to stabilize the isolated GluA2 LBD. We also show that l-Asp can replace l-Glu during purification, providing both equal yields and purity of the resulting protein sample. Furthermore, we solved three structures of the GluA2 LBD in the presence of 7.5, 50 and 250 mm l-Asp. Surprisingly, with 7.5 mm l-Asp, the GluA2 LBD crystallized as a mixed dimer, with l-Glu being present in one subunit, and neither l-Asp nor l-Glu being present in the other subunit. Thus, residual l-Glu is retained from the expression medium. On the other hand, only l-Asp was found at the binding site when 50 or 250 mm l-Asp was used for crystallization. The binding mode observed for l-Asp at the GluA2 LBD is very similar to that described for l-Glu. Taking our findings together, we have shown that l-Asp can be used instead of l-Glu for ligand-dependent stabilization of the GluA2 LBD during purification. This will enable structural studies of low-affinity ligands for lead optimization in structure-based drug design. Database Structural data are available in the Protein Data Bank under accession numbers 4O3B (7.5 mm l-Asp), 4O3C (50 mm l-Asp), and 4O3A (250 mm l-Asp) Structured digital abstract GluA2 and GluA2 bind by x-ray crystallography (View interaction) Here, we use differential scanning fluorimetry and X-ray crystallography to show that the low affinity agonist l-Asp can stabilize the isolated ionotropic glutamate receptor A2 ligand-binding domain by binding in the glutamate binding pocket. We also show that l-Asp can replace l-Glu during purification, providing both equal yields and purity of the resulting protein preparation.
U2 - 10.1111/febs.12795
DO - 10.1111/febs.12795
M3 - Journal article
C2 - 24673938
SN - 1742-464X
SP - 2422
EP - 2430
JO - F E B S Journal
JF - F E B S Journal
ER -