Figure 4.
Analysis of hydrogen bonds/salt bridges at the domain interfaces.(A) Details of the hydrogen bonds/salt bridges at the domain interface detected by HBPLUS. D61–R283 interaction (red box) could not be detected by HBPLUS, but it was found by the hydrogen bond/salt bridge analysis after the MD simulation shown in B. (B–F) Analysis of probabilities of forming hydrogen bonds/salt bridges during the MD simulations in the selected mutants. Hydrogen bonds/salt bridges between AC and BC domains are considerably strong. However, hydrogen bonds/salt bridges between AB domains are relatively weak. Compared with the R402C/H mutants, the distribution of hydrogen bond/salt bridge probabilities of C36R and G249R was similar to that of WT. (G) A B(E283)–A(D61)–C(R402)–A(E40)–C(S406) network (B–A–C–A–C network) formed around R402. R402 is thought to play an important role in the connection of A, B, and C domains. R402 plays a key role in the B–A–C–A–C network. (H) The NTD in the WT after 50 ns of the MD simulation. Thick and thin lines indicate strong and weak hydrogen bonds/salt bridges, respectively. Springs indicate the hydrogen bonds/salt bridges playing important roles in the rotation of the BC domains in the R402C/H mutants, but they are weak. The interactions belonging to the B–A–C–A–C network are shown in black, and others are shown in blue. The interactions between AC domains and BC domains are considerably strong; meanwhile, the interaction between the AB domains is weak. R402 plays an important role to maintain a tight connection between the A domain and the C domain through the B–A–C–A–C network. (I) Superimposition of the R402C/H mutant (violet) on the NTD in the WT (light blue). The hydrogen bonds/salt bridges between the AB domains and BC domains are maintained similar to that in the WT. However, the interaction between the AC domains becomes drastically weak because two of four important interactions are completely lost. Two interactions (E40–S406 and R45–D447) bend, and the BC domains move together. Although the B domain moves in accordance with the movement of the C domain, two interactions (D61–R283 and D18–S207) between the AB domains bend. Thus, the BC domains together rotate clockwise by 13.5 ± 0.7 degrees (R402C) or 14.3 ± 1.2 degrees (R402H) with respect to the A domain. The rotation axis is indicated by a black line. Values are shown as mean ± SEM. *P < 0.0001 by Student's t test compared with the WT.

Analysis of hydrogen bonds/salt bridges at the domain interfaces. (A) Details of the hydrogen bonds/salt bridges at the domain interface detected by HBPLUS. D61–R283 interaction (red box) could not be detected by HBPLUS, but it was found by the hydrogen bond/salt bridge analysis after the MD simulation shown in B. (B–F) Analysis of probabilities of forming hydrogen bonds/salt bridges during the MD simulations in the selected mutants. Hydrogen bonds/salt bridges between AC and BC domains are considerably strong. However, hydrogen bonds/salt bridges between AB domains are relatively weak. Compared with the R402C/H mutants, the distribution of hydrogen bond/salt bridge probabilities of C36R and G249R was similar to that of WT. (G) A B(E283)–A(D61)–C(R402)–A(E40)–C(S406) network (B–A–C–A–C network) formed around R402. R402 is thought to play an important role in the connection of A, B, and C domains. R402 plays a key role in the B–A–C–A–C network. (H) The NTD in the WT after 50 ns of the MD simulation. Thick and thin lines indicate strong and weak hydrogen bonds/salt bridges, respectively. Springs indicate the hydrogen bonds/salt bridges playing important roles in the rotation of the BC domains in the R402C/H mutants, but they are weak. The interactions belonging to the B–A–C–A–C network are shown in black, and others are shown in blue. The interactions between AC domains and BC domains are considerably strong; meanwhile, the interaction between the AB domains is weak. R402 plays an important role to maintain a tight connection between the A domain and the C domain through the B–A–C–A–C network. (I) Superimposition of the R402C/H mutant (violet) on the NTD in the WT (light blue). The hydrogen bonds/salt bridges between the AB domains and BC domains are maintained similar to that in the WT. However, the interaction between the AC domains becomes drastically weak because two of four important interactions are completely lost. Two interactions (E40–S406 and R45–D447) bend, and the BC domains move together. Although the B domain moves in accordance with the movement of the C domain, two interactions (D61–R283 and D18–S207) between the AB domains bend. Thus, the BC domains together rotate clockwise by 13.5 ± 0.7 degrees (R402C) or 14.3 ± 1.2 degrees (R402H) with respect to the A domain. The rotation axis is indicated by a black line. Values are shown as mean ± SEM. *P < 0.0001 by Student's t test compared with the WT.

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