Gabapentinoid drugs for pain and anxiety act on the Ca_Vα₂δ-1 and Ca_Vα₂δ-2 subunits of high-voltage-activated calcium channels (Ca_V1s and Ca_V2s). Here we present the cryo-EM structure of the gabapentin-bound brain and cardiac Ca_V1.2/Ca_Vβ₃/Ca_Vα₂δ-1 channel. The data reveal a binding pocket in the Ca_Vα₂δ-1 dCache1 domain that completely encapsulates gabapentin and define Ca_Vα₂δ isoform sequence variations that explain the gabapentin binding selectivity of Ca_Vα₂δ-1 and Ca_Vα₂δ-2.

HapR is designated as a high cell density quorum sensing master regulatory protein of Vibrio cholerae. It is a member of the TetR family protein and functions both as an activator and a repressor by directly communicating with cognate promoters, thus controlling the expression of a plethora of genes in a density-dependent manner. Molecular insights reveal the domain architecture and further unveil the significance of a cross talk between the DNA binding domain and the dimerization domain for the functionality of the wild-type protein. The DNA binding domain is made up of three α-helices, where a helix-turn-helix motif spans between the helices α2 and α3. The essentiality of the glycine-rich linker linking helices α1 and α2 came into prominence while unraveling the molecular basis of a natural non-functional variant of HapR. Subsequently, the importance of linker length was demonstrated. The present study, involving a series of biochemical analyses coupled with molecular dynamics simulation, has illustrated the indispensability of a critical arginine within the linker at position 37 contributing to HapR-DNA binding activity.

Voltage-gated ion channels (VGICs) comprise multiple structural units, the assembly of which is required for function^1,2. Structural understanding of how VGIC subunits assemble and whether chaperone proteins are required is lacking. High-voltage-activated calcium channels (Ca_Vs)^3,4 are paradigmatic multisubunit VGICs whose function and trafficking are powerfully shaped by interactions between pore-forming Ca_V1 or Ca_V2 Ca_Vα₁ (ref. ³), and the auxiliary Ca_Vβ⁵ and Ca_Vα₂δ subunits^6,7. Here we present cryo-electron microscopy structures of human brain and cardiac Ca_V1.2 bound with Ca_Vβ₃ to a chaperone-the endoplasmic reticulum membrane protein complex (EMC)^8,9-and of the assembled Ca_V1.2-Ca_Vβ₃-Ca_Vα₂δ-1 channel. These structures provide a view of an EMC-client complex and define EMC sites-the transmembrane (TM) and cytoplasmic (Cyto) docks; interaction between these sites and the client channel causes partial extraction of a pore subunit and splays open the Ca_Vα₂δ-interaction site. The structures identify the Ca_Vα₂δ-binding site for gabapentinoid anti-pain and anti-anxiety drugs⁶, show that EMC and Ca_Vα₂δ interactions with the channel are mutually exclusive, and indicate that EMC-to-Ca_Vα₂δ hand-off involves a divalent ion-dependent step and Ca_V1.2 element ordering. Disruption of the EMC-Ca_V complex compromises Ca_V function, suggesting that the EMC functions as a channel holdase that facilitates channel assembly. Together, the structures reveal a Ca_V assembly intermediate and EMC client-binding sites that could have wide-ranging implications for the biogenesis of VGICs and other membrane proteins.