Large-scale conformational transition from open to closed state of adenylate kinase (ADK) is essential for its catalytic cycle. Apo-ADK undergoes conformational transition in a way that closely resembles an open-to-closed conformational transition. Here, equilibrium simulations, free energy simulations, and quantum mechanics/molecular mechanics (QM/MM) calculations in combination with several bioinformatics approaches have been used to explore the molecular origin of this confOrmational transition in apo-ADK. In addition to its conventional open state, Escherichia coli apo-ADK adopts conformations that resemble a closed-like intermediate, the "half-open-half-closed" (HOHC) state, and a pi-cation interaction can account for the stability of this HOHC state. Energetics and the electronic properties of this pi-cation interaction have been explored using "QM/MvI: calculations. Upon rescinding the pi-cation interaction, the conformational landscape of the ape-ADK changes completely. The apo-ADK population is shifted completely toward the open state. This pi-cation interaction is highly conservedin bacterial ADK; the cationic guanidinium moiety of a conserved ARG interacts with the delocalized pi-electroia cloud of-either PHE or TYR. Interestingly, this study demonstrates the-Modulation of a principal. protein-dyiianneS by a conserved specific pi-cation interaction across different organisms.