Mechanistic Evidence Summary
Integrated Interpretation
The current simulations support a distributed and multiresidue pH-response model rather than a mechanism controlled by one residue alone. The strongest convergence is observed for:
- B293 alternative polar microenvironments
- B324 pKa-associated local remodeling
- Increased Module B compactness and internal contacts at pH 6.8
- Reorganization of histidine-rich dynamic and communication networks
The simulations support a candidate distributed pH-switch mechanism in which local electrostatic changes involving B293, B324, and additional histidines are coupled to broader changes in Module B compactness and intermodule communication.
Main Observations
Moderate-to-strong Evidence
- B324 local electrostatic remodeling
- Distributed histidine-network reorganization
- Multiresidue pH-response model
Moderate Evidence
- B293 alternative microenvironment switching
- Module B compactness at pH 6.8
Moderate Directional Evidence
Global pH-dependent A–B PB-MMPBSA endpoint energetics:
PB-MMPBSA yielded a mean ΔTOTAL of +61.43 kcal/mol at pH 6.8 and +71.15 kcal/mol at pH 7.4, corresponding to a pH 6.8 minus pH 7.4 difference of -9.72 kcal/mol. The lower pH result was driven by more favorable gas-phase electrostatics, largely offset by less favorable polar solvation.
These calculations exclude configurational entropy and derive from one trajectory per pH. Therefore, the difference should be described as directional endpoint-energy evidence rather than replicate-level statistical validation.
Limitations
Most analyses originate from one 300 ns trajectory per pH condition. Consequently, agreement between analyses is mechanistically informative but does not constitute fully independent replicate-level statistical validation.