Protein kinase CK2 is a novel potential target for cancer treatment. The tricyclic quinoline compound CX-4945 (R2 = COOH) is the first bioavailable CK2 inhibitor used in human clinical trials for advanced solid tumors. CX-4945 analogs with non-R2 carboxylate function were demonstrated to be approximately 5000-fold less potent than compound 12 (R2 = COOH) in vitro. Molecular docking and molecular dynamics simulations were employed to elucidate the structural mechanisms through which the R2 non-ionizable and R3 carboxylic acid substituents influence binding affinity. Results show that the structure of CK2α and the orientation of ligands changed to different degrees in non-R2 carboxylate function systems. The inappropriate electrostatic interactions between the non-R2 carboxylate group and the positive region lead to improper protein–ligand recognition, which is followed by the reorientation of tricyclic skeletons. For CK2α, the affected positions are distributed over the glycine-rich loop (G-loop), C-loop, and the β4/β5 loop. The allosteric mechanisms between the deviated ligands and the changed regions are proposed. Detailed energy calculation and residue-based energy decomposition indicate the energetic influences on the contributions of the critical residues. These results are in accordance with one another and could provide rational clues to the design of more potent CK2 inhibitors.