4O3A

Crystal structure of the glua2 ligand-binding domain in complex with L-aspartate at 1.80 a resolution

Summary for 4O3A

• Entry DOI: 10.2210/pdb4o3a/pdb
• Related: 1FTJ 1FTO 4O3B 4O3C
• Descriptor: Glutamate receptor 2, GLYCEROL, ASPARTIC ACID, ... (8 entities in total)
• Functional Keywords: ampa receptor ligand-binding domain, membrane protein-agonist complex, membrane protein/agonist
• Biological source: Rattus norvegicus (Rat); More
• Cellular location: Cell membrane ; Multi-pass membrane protein : P19491
• Total number of polymer chains: 3
• Total formula weight: 90808.54

Authors

Krintel, C.,Frydenvang, F.,Gajhede, M.,Kastrup, J.S. (deposition date: 2013-12-18, release date: 2014-04-16, Last modification date: 2024-10-16)

Primary citation

Krintel, C.,Frydenvang, K.,Ceravalls de Rabassa, A.,Kaern, A.M.,Gajhede, M.,Pickering, D.S.,Kastrup, J.S.
L-Asp is a useful tool in the purification of the ionotropic glutamate receptor A2 ligand-binding domain.
Febs J., 281:2422-2430, 2014

PubMed Abstract: 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.

PubMed: 24673938
DOI: 10.1111/febs.12795

Experimental method

X-RAY DIFFRACTION (1.8 Å)