Figure 5: (A) Z-score of betweeness centrality (Tau_glyc-orange & Tau_plane-blue), indicating Phe378, Lys353 are functionally important resides which helps in initiating folding process in glycosylated tau and (B) Z-score of closeness centrality showing Phe378, Lys347&Lys370 are the residues which helps stabilize the glycosylated tau energy minimum structure.
In Tau_glyc (orange) we can see that residue Phe378 and Lys353 have the two highest BC values, while Tau_plane (blue) has one peak BC value corresponding to Thr361. Structural analysis of Tau_glyc confirmation revealed that Phe378 is having a strong H bond with Thr377, both of which are involved in beta sheet formation at the region. Phe378 is also involved in a weak hydrogen bond with Asp345, aromatic hydrophobic interactions with Leu344 and Val363. Lys-353 is involved in weak hydrogen bond formation between Lys370 and Ile371. In Tau_plane Thr361 was found to have a strong hydrogen bond between Phe378 and Leu376. Thr377 was also one of the two residues forming the beta bridge, the other one being Thr377 with which Thr361 is having a weak hydrogen bond.
Closeness centrality is a measure of the ability of a node in a network to spread information very efficiently. It is considered as the inverse of the average shortest path length. It provides global information, indicating how many steps are needed to reach every other node in the network [64]. Dokholyan and coworkers have demonstrated that the proteins in post transition state (i.e., those confirmations which are more likely to form native structures or close to native structures) have low average shortest path length [65]. Since CC is inverse of average shortest path length, The residues which have high CC tend to be important as they help stabilize the structure, and usually lie in the protein core and remain compact [66].
CC analysis of Tau_glyc (Figure 5 B) showed that in Tau_glyc the residues having the highest CC are Phe378, Lys347 &Lys370, while in Tau_plane Thr361, Ile360 & Asn359. In Tau_glyc Phe378 is involved in beta sheet formation as explained earlier, while Lys347 is also involved in the second beta sheet formation having a strong hydrogen bond between Thr377. It also has a weak hydrogen bond with Leu376. Lys370 is involved in an isolated beta bridge having a weak hydrogen bond with Lys353. In Tau_plane (blue) we have Thr361 with an isolated beta bridge, having interactions as discussed earlier. Ilel360 has a strong hydrogen bond with Leu357 & weak interaction with Leu376. Asn359 is involved in strong hydrogen bonds between Arg349, Ser356 & Leu344. The CC graph revealed that residues which showed high CC are those which were involved in secondary structure formation and helps in protein stabilization.
The above two centrality measures suggest that when tau protein is not glycosylated the central residues or the functionally important residues are those present in the middle region of the protein, close to Asn359 (N-glycosylation site), but these residues do not induce much folding or aggregation. while the tau is glycosylated the centrality of residues shifted away from Asn359. BC analysis showed that in the N glycosylated folded state, Phe378 & Lys353 are the functionally important residues, suggesting their role in initiating the folding process in transition state ensemble or stabilization of the native structure. Comparison of RIN analysis results to Cryo Electron microscopic (EM) structure of PHF tau showed that the residues having high BC, i.e., Phe378 is part of the β8 and Lys353 is part of β6 region of PHF tau [67]. Also, residues having high CC values, i.e., Phe378&Lys370, part of the β8 region, Lys347 part of the β5 region of PHF tau. This indicates that the functionally important residues as indicated by the RIN analysis are actually key players in the beta core region of tau as predicted by the Cryo-EM structure of tau.