Antimicrobial peptides (AMPs) disrupt the integrity of the bacterial membrane, ultimately leading to their death. In counter-defense, pathogens are reported to have developed systems such as the sensitivity to antimicrobial peptides (Sap) transport system that evade the action of AMPs and sequester essential micronutrients. However, recent contrasting reports cloud the functional prospects of the Sap system. Hence, this study aimed to characterize the Escherichia coli Sap (EcSap) transport system using biophysical techniques. The results obtained from various approaches suggested the binding of heme to the substrate-binding component (EcSapA) of the EcSap system. Further, this study suggests the interaction of EcSapA with the AMP protamine. In summary, the findings of this study suggest the dual ligand-binding ability of EcSapA. Impact statement The present study reports the functional prospects of the enigmatic substrate-binding protein SapA of E. coli. This analysis highlights the essentiality of the intra-protein disulfide bonds in maintaining the structural integrity of EcSapA. Further, biophysical studies of EcSapA highlight its dual ligand binding propensity, earmarking it as a drug target.