Figure 2.

The grid-like noncovalently interacting mesh network along the PE-dependent gating pathway of the rTRPV2 channel in closed state 3 at pH8 and 4 °C. (A) The thermoring structure based on cryo-EM data of a single subunit of the closed state 3 of rTRPV2 in DMNG/MSP2N2 nanodisc at pH8 and 4 °C (PDB ID, 8EKR). The pore domain, the S4–S5 linker, the TRP domain, and the pre-S1 domain are indicated by black arrows, except for the VSLD. Salt bridges, π interactions, and H-bonds between paired amino acid side chains along the PE-dependent partial gating pathway from W351 to E685 are denoted in purple, green, and orange, respectively. The specific grid sizes necessary to regulate the least-stable noncovalent interactions in the grids are indicated with black numbers. The R369-E469 H-bond in the biggest Grid₁₂ is emphasized in yellow. The total grid sizes and the total grid size-controlled noncovalent interactions along the whole PE-dependent gating pathway from F330 to W715 are displayed in cyan and black circles, respectively. (B) The structure of the biggest Grid₁₂ with a 12-residue size to regulate the R369-E469 H-bond at the pre-S1/VSLD interface. (C) The structure of the putatively thermosensitive Grid₆ with a 6-residue size to regulate the F344-R702 and W351-R684 bridges at the interface between C and N-termini. (D) The sequence of the biggest Grid₁₂ to control the highlighted R369-E469 H-bond in the blue box. In contrast, the sequence of the putative biggest Grid₂₀ in the rTRPV2/V1(357–434) chimeric channel to control the R409-D469 and R459-E397 bridges for a T_m of 34 °C. Additionaly, sequence alignment of the putatively thermosensitive Grid₆ is shown between rTRPV2 and hTRPV2.