Zinc is the second most abundant transition metal ion in humans and is increasingly being recognised as a crucial antimicrobial to control bacterial infections.1 It is established that host Zn2+ availability changes dramatically during infection2 and recent research has shown that Zn2+ concentrations are elevated at sites of pneumococcal infection3,4, while studies at a cellular level have revealed the use of Zn2+ by phagocytic cells to kill bacteria.5,6 Despite these observations, the molecular details of how zinc mediates toxicity towards pathogenic bacteria remain to be elucidated. Characterised bacterial metal efflux proteins, that confer heavy metal resistance, include those belonging to the Cation Diffusion Facilitator (CDF) family of integral membrane protein transporters. However, structural and functional information on this class of proteins is limited, with only a single structure of the YiiP protein from E. coli [PDB code 3H90]7 so far elucidated.
In this work, we have targeted the protein CzcD, which associated with S. pneumoniae virulance and survival in the host. CzcD is a member of CDF family, with only ca 20% sequence identity to E. coli homologue, YiiP. We have succcesfully cloned, purified and characterised S. pneumoniae CzcD in detergent solution. Our protocol produces milligram quantities of pure recombinant CzcD sufficient for structural investigations. Metal binding assays on pure CzcD show the isolated protein is functional. This poster will present our progress toward the structural and functional characterisation of Streptococcus pneumoniae CzcD.