Streptococcus pneumoniae is a significant global pathogen, responsible for over one million deaths annually. As a host-adapted organism, the pneumococcus must acquire all nutrients directly from the host environment. Manganese is one such nutrient and is essential for virulence and viability. Consequently, the manganese uptake pathway, PsaBCA, has been investigated for therapeutic development in numerous studies.
PsaBCA is a Type II ABC permease comprised of a manganese binding protein, PsaA, and transmembrane transporter, PsaBC. While the structure of PsaA has been previously determined, the structural and functional elements of PsaBC have remained undefined.
To investigate this, PsaBCA was purified and reconstituted into liposomes. Intriguingly, functional studies revealed that despite the essentiality of manganese for S. pneumoniae, transporter activity (measured by ATP hydrolysis) was relatively low and uncoupled from the presence of the substrate. To gain insight into the structural features mediating this activity, we solved the crystal structure of PsaBC to 2.9 Å in an open-inward conformation (Neville et al., 2021 Science Advances). Structural analyses revealed substantial differences between PsaBC and previously reported Type II transporters, particularly the nucleotide binding domains, likely responsible for the slow ATP turnover.
Given the relatively low and uncoupled activity of the transporter, we sought to determine how PsaBC achieves efficacious manganese transport. Molecular dynamics revealed the primary conformation of the transporter was effectively closed to the extra-cytoplasmic environment, with specific extracellular gating residues acting to exclude water molecules and prevent reflux of manganese during transport. Below these residues, we observed a novel metal coordination site, not previously described for Type II ABC transporters. Through mutagenesis, we showed that these residues were essential for the unidirectional translocation of manganese and therefore, the viability of S. pneumoniae. Subsequent bioinformatics revealed that these unique structural features were conserved not only in prokaryotes but also represented throughout all kingdoms of life.
Collectively, our results define the structure of PsaBC, and reveal conserved features required for divalent cation transport.