2.1 The Non-covalent Interactions Form Multiple Hairpin Topological Loops along the Gating Pathway of Oxidized mTRPV3 at 42⁰C
C612 and C619 in the outer pore of mTRPV3 can form a disulfide bond upon oxidization . In the closed state at 42⁰C, the diversity of non-covalent interactions between amino acid side chains in oxidized mTRPV3 can produce multiple hairpin topological loops along the gating pathway from D396 in the pre-S1 domain to K505 in the TRP domain. First, salt bridges are present between several charged pairs. For example, R416 on pre-S1 and D519 in the S2-S3 linker, K432 on pre-S1 and E704 in the TRP domain or D396 on pre-S1, D586 and K589 on S5, E631 and K634 on the pore helix, E682 on S6 and K686 in the TRP domain, E687 and K690, E689 and R693, R698 and E702 in the TRP domain (Figure 1).
Second, p-p interactions appear between several pairing aromatic residues. They include: F441 on S1 and W433 on pre-S1 or Y565 on S4, F445 and F449 on S1 or Y565 on S4, Y448 on S1 and F526 on S3 or Y565 on S4, W493 and F489 on S2 or F447 or Y451 on S1, F526 on S3 and Y564 on S4, Y540 on S3 and Y547 on S4, Y564 and Y565 on S4, F590 and Y594 on S5, Y622 in the first pore loop and F654 on S6. In addition, R696 in the TRP domain forms a cation-p interaction with W433 on pre-S1 while F590 on S5 and L673 on S6 form a CH-p interaction (Figure 1).
Third, H-bonds emerge between different hydrophilic residues. These H-bonding pairs are T397 and K432 on pre-S1 or E704 in the TRP domain, Q529 on S3 and Y448 or Y451 on S1, T566 on S4 and S576 in the S4-S5 linker, R567 on S4 and T699 in the TRP domain, Q570 in the S4-S5 linker and E689 in the TRP domain, D586 on S5 and T680 on S6, Y594 on S5 and Y661 on S6, E679 and N683 on S6. In addition, the vanilloid site phosphatidylcholine (PC) also relates W521 on S3 to Q580 in the S4-S5 linker via H-bonds. Of special note, there are several H-bonds between E610 and K614 in the first pore loop, S621 in the first pore loop and Q646 in the second pore loop, S624 and D627 in the first pore loop, T636 on the pore helix and Y594 on S5 or Y661 on S6, N647 in the second pore loop and E610 or K614 in the first pore loop (Figure 1).
Taken together, one largest hairpin with a 23-residue loop is present to control the D519-R416 salt bridge in the VSLD/pre-S1 interface. It starts with R416 to W433, F441, Y565, F445, F449, Y448 and ends with F526. The total numbers of all non-covalent interactions and hairpin sizes along the gating pathway from D396 to K705 are 44 and 117, respectively (Figure 1).
2.2. The Largest Hairpin Melts to Initiate a Temperature Threshold 42⁰C for Oxidized mTRPV3 Activation
In the heat-activated sensitized state at 42⁰C, oxidized mTRPV3 has several changes. In the pore domain, the H-bonds between N647 and E610 or K614, S621 and Q646, S624 and D627, E679 and N683 are broken while the S620-Q646 H-bond emerges. In the S4-S5 linker/TRP interface, the Q570-E689 H-bond is disconnected. In the TRP domain and its interfaces with the pore domain and the pre-S1 domain, the E682-K686 and E689-R693 salt bridges are replaced with the N683-K686 and E689-R693 H-bonds, respectively. In addition, the T397-K432/E704 H-bonds are disrupted while the E405-K705 salt bridge and the E423-T427 and E418-R690 H-bonds are present. In the VSLD and its interface with the pre-S1 domain, the p-p interactions shift from F445-Y565 and W493-Y451 to F526-Y565 and F489-F450, respectively, with the broken W493-F489 p-p interaction. The D519-R416 salt bridge is substituted by the S511-N412 and R509-N410 H-bonds. Of special interest, in the VSLD/S4-S5 linker interface, the T566-S576 H-bond is broken. However, D519 forms a salt bridge with R567 when a vanilloid site PC in the closed state disappears  (Figure 2).
Together, after the largest hairpin with a 23-residue loop in the VSLD/pre-S1 interface melts, two largest hairpins follow. One has a 9-residue loop in the pore domain starting with S620 and through Y622 and F654 and ending with Q646. The other contains an 8-residue loop from D519 to F526, Y565 and R567 in the VSLD/S4-S5 linker interface. The total numbers of all non-covalent interactions and hairpin sizes along the gating pathway from D396 to K705 become 41 and 87, respectively (Figure 2).
2.3. Two Smaller Hairpins in the S4-S5 Linker/VSLD Interface Are Required to Open Oxidized mTRPV3
When oxidized mTRPV3 is finally open from the sensitized state, there are small changes in the outer pore loop. The E610-K614/D615 H-bonds are replaced with the K616-D618 salt bridge. The D586-K589 salt bridge is disconnected while the S624-D627 H-bond is restored. In the meanwhile, a CH-p interaction is present between Q646 and F654, producing the smallest hairpin with a 1-residue loop from S620 to Y622 and F654 and Q646. Thus, the largest hairpin appears in the pore domain with a 10-residue loop from D586 to F590 and L673 and T580 and back to D586 (Figure 3).
In stark contrast, in the TRP domain and its interface with the pre-S1 domain, the E687-R690 and E405-K705 salt bridges and the T397-E704 H-bond are broken. In the meantime, the E689-R693 H-bond becomes a salt bridge and T411 forms a new H-bond with R416. In the VSLD/pre-S1 interface, the N410-R509 and N412-S511 H-bonds are replaced with the T411-S515 and N412-Q514 ones. In the VSLD, the p-p interaction shifts from F450-F489 to Y451-W493. The H477-Y540 H-bond and the H471-Y547 p-p interaction also produce a new small hairpin with a 5-residue loop. When the T566-S576 H-bond is reinstated in the S4-S5 linker/VSLD interface, the resultant second largest hairpin with a 9-residue loop, together with an 8-residue loop from D519 to F526 and Y564 and Y565 and R567 and then back to D519, may be required for mTRPV3 opening. By all accounts, in the open state, the total numbers of all non-covalent interactions and hairpin sizes along the gating pathway from D396 to K705 become 41 and 71, respectively (Figure 3).
2.4. Reduced mTRPV3 Has the Different Largest Hairpin in the Outer Pore
In the closed state without the C612-C619 disulfide bond, reduced mTRPV3 is different than oxidized one. In the pore domain, the D586-T680, E610-K614, S621-Q646, S624-D627 and N647-E610/K614 H-bonds are disrupted together with the F590-L673 and Y622-F654 CH/p-p interactions and the E631-K634 salt bridge. In the meanwhile, the S613-N647 and E631-N643 H-bonds and the F625-V629 and N647-F654 CH-p interactions are formed. By all accounts, the largest hairpin with a 17-residue loop in the pore domain is born from S613 to F625, V629, E631, N643, N647, and then back to S613 (Figure 4).
However, the largest hairpin in the VSLD/pre-S1 interface, which is present in the oxidized mTRPV3 (Figure 1), is unavailable because the D519-R416 salt bridge has been replaced with the new Q514-T411/N412 H-bonds to produce the smallest hairpin with no residue in the loop. When the Y451-W493 p-p interaction is disconnected with the formation of the K500-E702 salt bridge, a smaller hairpin with a 9-residue loop is present from W493 to K500, E702, R698, R696, W433, F441, Y565, Y448, F447 and then back to W493. In the S4-S5 linker/TRP interface, the R567-T699 H-bond is substituted by the R567-Q695 H-bond. In the S4-S5 linker/pore domain interface, the D586-T680 H-bond is replaced with the K581-E687 salt bridge. In the TRP domain and its interface with the pre-S1 domain, the E687-R690 salt bridge is substituted by the 693-H426 H-bond. In the closed state, reduced mTRPV3’s total numbers of all non-covalent interactions and hairpin sizes along the gating pathway from D396 to K705 are 40 and 96, respectively (Figure 4).
2.5 2-APB Can Open Reduced mTRPV3 at Low Temperature without Significant Changes in the Largest Hairpin Size and the Total Hairpin Sizes along the Gating Pathway
When one 2-APB agonist binds between F526 and S444 and the other between W433 and W692 via CH/p-p interactions (Figure 5), reduced mTRPV3 is open with several changes. In the VSLD, the Y565-F445/Y448 and Y540-Y547 p-p interactions are replaced with the F447-F489 and H471-Y540/Y547 ones and the S511-S515 H-bond. In the VSLD/TRP interface, the K500-E702 salt bridge is disconnected. In the S4-S5 linker/VSLD interface, the T566-S576 H-bond appears again. In the S4-S5 linker/TRP interface, Q570 also forms an H-bond with R693. In the TRP domain, the E679-N683 H-bond is disrupted. When these conformational changes are extended to the outer pore loop, the S613-N647 H-bond is broken. In the meanwhile, the E610-S624 H-bond, the E631-K634 salt bridge and the L605-F654 and F625-V629 CH-p interactions are born. However, the largest hairpin size still has 17 residues in the loop from Y594 to L605, F654, Y661 and then back to Y594. In addition to the smaller hairpin with a 9-residue loop from T566 to S576, another with no residue in the loop from Q570 to E689 and R693 and then back to Q570 in the S4-S5 linker/TRP interface may be required to stabilize the 2-APB efficacy. In any way, the total numbers of all non-covalent interactions and hairpin sizes along the gating pathway from D396 to K705 become 44 and 99, respectively (Figures 4-5).