Figure 2: Disulphide crosslinking between LF and DF constrains the relative motion of LF and DF domains.

(a) A superposition of the closed (grey, PDB ID code 3H9V) and open (pink, PDB ID code 4DW1) structures of zfP2X4. Blue arrows denote movements of LF and DF domains from the closed state to the ATP-bound open state. Red arrows indicate the relative movements of C_α atoms of ^zfV291 and ^zfS214 from the closed state to the open state. (b) Interdomain/intersbunit disulphide bond between ^rV288C and ^rT211C in the homooligomeric rat P2X4 (rP2X4) receptor. Wild-type rP2X4, ^rV288C, ^rT211C and ^rV288C/T211C subunits (each carrying a C-terminal EE epitope) were transiently expressed in HEK-293 cells. Cells were lysed in buffers with or without β-ME (1%, 10 mM) as indicated. Protein samples were separated on SDS-PAGE gels and detected by western blotting via an anti-EE antibody. The band indicated by arrowheads corresponds to the expected size of the monomeric, dimer and trimetric rP2X4 subunits, respectively; protein molecular weight markers are indicated on right. These results were observed in at least three independent experiments for each receptor. (c) The locations of ^zfV291 and ^zfS214 in the resting (upper) and open (down) structures of zfP2X4. Red dashed line indicates the measured C_β–C_β distance between ^zfV291 and ^zfS214. (d) Zoom-in view of constructed disulfide-bridge model of zfP2X4^V291C/S214C based on the closed structure showing details of C_β–C_β distance (red dashed line) and the dihedral angle measured between C_β–S_γ–S_γ–C_β atoms from the ^zfC291 and ^zfC214.