Molecular Dynamics of the Interaction Between Apis mellifera Melittin and Na⁺,K⁺-ATPase in the E1 Conformation

Melittin, a 26-amino-acid peptide and the main component of Apis mellifera apitoxin, inhibits Na⁺,K⁺-ATPase. Previous studies found that crude Apis mellifera venom inhibited Na⁺,K⁺-ATPase activity with an IC₅₀ of 12.39 µg/mL. Molecular docking analyses showed melittin interacts with the enzyme's cytoplasmic domains in distinct conformations, with particular emphasis on the TGES motif in the actuator domain (A domain). However, docking provides only a static pose and no information on the temporal persistence of these contacts. In the present study, we subjected the best docking pose for the E1 conformation to a 100 ns molecular dynamics simulation in explicit solvent using the CHARMM36m force field. Melittin remained associated with the cytoplasmic face of the enzyme throughout the entire simulation, with no evidence of dissociation, and retained its conformation as two α-helical segments connected by the Thr11–Gly12 hinge. The interaction occurred through two simultaneous binding sites: the loop containing the TGES motif (Ser216–Glu223), which was persistently occupied by the melittin cationic residues Lys21, Arg22, and Gln25; and the Gln281–His293 region, together with Glu362, which was intermittently occupied by the amino-terminal region of the melittin. Furthermore, analysis of the four enzyme conformations showed that melittin increased the eigenvalue in all cases, from 5.9% to 16.5%, indicating increased conformational rigidity and a higher energetic cost for the domain movements that drive the catalytic cycle. Accordingly, the predicted binding affinity remained comparable across the four conformations, ranging from −8.4 to −9.1 kcal·mol⁻¹. An electrostatic anchoring switch was also observed at approximately 36 ns, during which the Lys7–Glu362 (melittin-enzyme) salt bridge lost stability, while melittin amino-terminal contacts and the Arg22–Glu221 (melittin-enzyme) interaction became established. Thus, these results support the proposed inhibition mechanism, which involves blocking the TGES motif, and suggest that melittin’s affinity for the catalytic region in the E1 conformation is mainly sustained by its cationic carboxy-terminal segment.