Biophysical characterization of the zinc-binding protein AdcA from Enterococcus faecium - Poster presentation

Antimicrobial resistance represents one of the most serious threats to global health, dramatically reducing the effectiveness of available treatments against bacterial infections. Enterococcus faecium, a member of the ESKAPE group of multidrug-resistant pathogens, is frequently responsible for hard-to-treat nosocomial infections. The identification of new therapeutic targets is therefore a major priority in microbiological and biochemical research.A promising strategy to counteract such infections involves interfering with essential bacterial survival processes such as metal ion acquisition. Zinc is a crucial micronutrient required for many enzymatic and structural functions, and its availability is strongly restricted by the host during infection. To overcome zinc limitation, several pathogenic bacteria have evolved high-affinity transport systems that ensure efficient zinc uptake.In Gram-positive bacteria, the AdcABC complex plays a key role in this process, consisting of a zinc-binding lipoprotein (AdcA), a membrane permease, and an ATPase component. In E. faecium, AdcA is believed to be essential for growth under zinc-limited conditions and therefore represents a promising target for antimicrobial drug development.In this study, the zinc-binding protein AdcA from E.faecium was subjected to detailed biophysical characterization. Bioinformatic analysis revealed a modular structure with an N-terminal ZnuA-like domain and a C-terminal ZinT-like domain connected by a long flexible loop. Both domains contain histidine clusters potentially involved in zinc coordination. A three-dimensional homology model, based on the corresponding Streptococcus pneumoniae protein, showed high structural conservation, supporting a similar functional mechanism.The recombinant AdcA protein was successfully expressed and purified, and its secondary structure, thermal stability, and folding behavior were analyzed using circular dichroism (CD) spectroscopy and differential scanning calorimetry (DSC).Furthermore, the interaction between AdcA and Zn²⁺ was investigated by isothermal titration calorimetry (ITC). Overall, these findings provide valuable insight into the structural and functional features of AdcA and its role in zinc homeostasis in E. faecium. The results highlight AdcA as a potential molecular target for the development of new antimicrobial strategies aimed at disrupting metal acquisition in multidrug-resistant pathogens.