Toward the Structural Characterization of the Gabapentin Binding site

Abstract

Gabapentinoids are prescription drugs used to relieve neuropathic pain, epilepsy and anxiety. One member of this family, gabapentin has been shown to bind with nanomolar affinity to the CaVα2δ subunit of pre‐synaptic voltage‐gated calcium channels. We undertook a study to characterize the structural determinants of the gabapentin binding site on the extracellular N‐terminal domain of CaVα2δ. The nucleotide sequence of a domain of 246 residues, including the putative binding site, was inserted in a pET28 vector. The protein, expressed in BL21(DE3) bacteria, was found to be mostly located in inclusion bodies and had to be solubilized in 8 M urea. Primary sequence of the purified protein was confirmed by MALDI mass spectrometry and protein refolding was promoted by removing urea through overnight dialysis. The recovery of the native‐like structure was validated using size exclusion chromatography, circular dichroism, and small‐angle X‐ray scattering. The protein was found to be stable at 4°C for 2 weeks. Interaction between the 246‐residue domain (2 µM) and gabapentin (2 µM, 20 µM and 40 µM) was examined using differential scanning fluorimetry. Preliminary data show that gabapentin does not significantly affect the stability of this domain. We have nonetheless undertaken crystallization trials with and without gabapentin to obtain a high‐resolution 3D structure of this domain. Other structural domains of CaVα2 are concurrently in the process of purification using similar approaches. Altogether our studies will ultimately shed light on the molecular mechanism underlying the effect of gabapentin in pain management.