Structural Analysis of TRPC5 and TRPC6
In order to understand the mechanisms of Clemizole binding at the molecular level, we analysed the structures of both TRPC5 and TRPC6 proteins. The electron microscopy solved structure of human TRPC5 in complex with clemizole (PDB: 7D4P) and Cryo-EM structure of human TRPC6 in complex with an antagonist AM-1473 (PDB: 6UZA) were used. As four clemizoles were bound to each tetrameric TRPC5 structure, we truncated the latter and retained the chain A of the TRPC5 structure. Similarly, the tetrameric structure of TRPC6 was truncated to Chain A to facilitate analyses.
We first investigated the key interaction sites involved in Clemizole binding to TRPC5. This included the key residues making direct interactions with TRPC5 as well as those present within a 5Å distance of bound Clemizole, and hence might be responsible for long-range interactions. Next, structure-based and sequence-based alignments (Fig.1) of TRPC5 and TRPC6 were performed to identify the identical, similar, or different residues in both channels. Out of a total of 19 amino acids located in a 5 Å vicinity of TRPC5-bound clemizole, 10 residues were different from those in TRPC6, Table 1. Among these, 5 residues were those that appeared to interact directly with Clemizole in the PDB structure of TRPC5.
Docking
We analysed the docking efficiency of Clemizole with TRPC5 and TRPC6. We first validated the accuracy of our docking protocol. For that, we extracted Clemizole from the available TRPC5 structure and redocked it back to the receptor to identify whether it would attain the same docked conformation as the originally bound clemizole. The redocked conformation was almost superimposable with the originally determined confirmation identified by Cryo-electron microscopy, Fig 2. We then used the same docking approach to dock Clemizole on the TRPC6 protein in order to determine the bound conformation, binding energy, as well as the molecular interactions. While the score for the top docked conformation of Clemizole in TRPC5 was -10, it was -8.2 for TRPC6. We also compared the docked conformations of Clemizole in both structures, Fig.3, which too were different from each other in the three-dimensional space of the respective binding sites. We next analysed the interactions of Clemizole in the TRPC6 binding site and compared them with those in TRPC5, Fig 4. We found that Clemizole made fewer interactions with the binding site residues of TRPC6 as compared to those in TRPC5. This led us to further investigate the residues in the binding sites of both receptors more closely.
In silico mutagenesis
After comparing the complexes of Clemizole with both TRPC5 and TRPC6, it was found that, although the two proteins have few similar binding site residues, many important Clemizole binding residues are quite different, and this might be the reason for its differential binding. To check this, we performed in silico mutagenesis of the variable residues in both structures. For example, Thr371 in TRPC5 had been changed to Ala which is the corresponding residue of Thr371 in TRPC6. Similarly, we mutated Phe414, Gly417, Asn443, Tyr446, Leu493, Ser495, Leu496 and Pro659 to Met, Ala, Leu, Phe, Ile, Tyr, Ile, and Val respectively. Furthermore, we mutated the corresponding residues in TRPC6 to those as in TRPC5. For example, Phe450 has been changed to Tyr, Met505 to Phe, Ala508 to Gly, Leu 534 to Asn, Phe537 to Tyr, Ile610 to Leu, Tyr 612 to Ser, Iso613 to Leu and Val762 to Pro.
We first mutated each of the five directly interacting residues in TRPC5 to those present in TRPC6. This was done individually as well as for all 5 together, Table 2. There was not much difference in the binding energy after these mutations either single or all 5, we extended the in silico mutagenesis to the remining 5 residues which are different in both, along with mutations in all 10. While the docking energy of Clemizole after individual mutations was not deviated much, we found that the binding energy was reduced to that as in TRPC6 in all 10 mutations, Table2. This may shed light on possible explanations for the low affinity of Clemizole towards TRPC6.
We next mutated the corresponding five residues in TRPP6 which were involved in direct binding with Clemizole in TRPC5. We found that the change in docking energy was negligible in individual mutations and increased slightly only in case of mutations of all 5 residues. Further, after mutating the rest of 5 mutations, again there was negligible change in docking energy, except Tyr to Ser mutation at the 612 amino acid position, which interestingly decreased instead of an expected increase. Surprisingly, even in the case of all 10 mutations, there was no change in overall docking energy, Table 3. This led us to investigate that there might be more to inhibitor binding than just the variability of amino acids in the binding sites of the two.
ASA and RASA calculation
We further led our studies to investigate the ASA, which is the accessible surface area of each amino acid and the RASA, which is the accessibility of each residue calculated by dividing the accessible surface area by the maximum accessible surface area of each amino acid type. RASA equal to 1 means that the residue is fully exposed, whereas RASA equal to 0 means that the residue is fully buried. A threshold of 0.2 is routinely used to distinguish exposed from buried residues.
The ASA and RASA were first calculated for residues within a 5Å radius of the clemizole binding in both TRPC5 and TRPC6 structures. We observed that there was a large difference in these values amongst the two binding sites, Table 4a & 4b. Ala447, Gly504, Phe537, Ser606 in TRPC6 have ASA and RASA values of 0 which indicates no accessibility of these residues for any ligand while in the case of TRPC5, only Gly413 has ASA and RASA values of 0. Interestingly, the 5 residues in direct interactions with Clemizole in the TRPC5 binding site have ASA and RASA values greater than the corresponding residues in TRPC6. For example, TRPC5 Tyr374 has a higher value of 31.447 and 0.130 for ASA and RASA respectively as compared to the Phe450 in TRPC6 whose ASA and RASA values are only 14.367 and 0.063 respectively. This is almost half of the accessibility as compared to TRPC5. Phe414, Tyr446, Leu496 similarly have accessibility values more than their corresponding residues in TRPC6. However, Pro659 residue of TRPC5 and its corresponding residue Val762 have comparable values. Although there are few residues in TRPC6 like Ala508 and Val762 which exhibit higher accessibility values than their corresponding residues in TRPC5, although the difference is not large. Another important and critical residue in TRPC5 binding site, Met442 too has correspondingly higher ASA and RASA as compared to its homolog Met533 in TRPC6. Next, we checked the accessibility values in the bound forms of both structures, Table 5a&b. As expected, there was no change in the accessibility values of TRPC6 residues Ala447, Gly504, Phe537, Ser606, after ligand binding. Their RASA and ASA were 0 before ligand binding which remained so after docking Clemizole as well. Phe453 too remained highly inaccessible for ligand binding as the values do not change at all between the unbound and the bound forms. Same is true for residues Ala508, Met533, Leu534, Ile610, and Asn765 whose ASA and RASA values do not alter at all after ligand binding. This shows that the ligand does not engage with these residues, possibly accounting for lower in silico binding and efficacy in vitro. Contrary to that of TRPC6, the binding site residues of TRPC5 are actively engaged by the ligand in the docked state. As observed in Table 4a and 5a, it is clear that Tyr374, Phe414, Gly417, Glu418, Leu496, and Arg492 could be involved in direct and close binding with the ligand as their ASA and RASA decreases considerably after ligand binding. Further, residues like Met442, Phe377, Asp439, and Ser489 that have higher accessibility values in the unbound form become totally occluded and have 0 accessibility values post ligand binding.
We propose that the difference in the ASA and RASA of residues in the unbound states of TRPC5 and TRPC6 might be the main reason for differential binding of clemizole. As can we see, Table 4&5, the ASA and RASA of the key residues are far apart for both. The accessibility values of bound versus unbound structures of both TRPC5 and TRPC6 further support this hypothesis. The residues with higher values in the unbound form in TRPC5 have very little or zero values of ASA and RASA after Clemizole binding. While those in TRPC6, which already had lower accessibility values to start with, have either unchanged or decreased values with the exception of one or two residues like Val762 and His446.
The overall ASA and RASA for both TRPC5 and TRPC6 in the bound and unbound forms is given in Supplementary Tables 1-4.