Structural and functional investigation of the essential secondary active transporter LicB from the human pathogen S. pneumoniae

Streptococcus pneumoniae is a human pathogen that can cause mild symptoms but also exhibits the potential to cause severe infections in the lungs or the brain. As a Gram-positive bacteria it has teichoic acids attached to its surface, where there are two types which are either embedded in the membrane or bound to the peptidoglycan. The synthesis of those teichoic acids is unique for S. pneumoniae, as it shares the same biosynthetic pathway for both types. Another unique feature is their modification with phosphorylcholine. The attachment of phosphorylcholine happens at the inner leaflet during the synthesis of the nascent teichoic acid chain and only those modified teichoic acids are exported to the surface. Choline is the substrate for the phosphorylcholine moieties and is essential for the survival of S. pneumoniae. The substrate cannot be synthesized by the bacteria and can only be imported by a secondary active transporter denoted as LicB. This transporter belongs to the drug/metabolite transporter superfamily, which is comprised of transporters with ten transmembrane helices of two inverted repeats arising from internal gene duplication. The structure and function of LicB has not been described before and known structures of other members of the drug/metabolite transporter superfamily are sparse and mostly describe only a small part of the families.
This study describes the functional and structural features of the pneumococcal choline importer LicB. LicB exhibits promiscuous transport behavior, where it shows to transport not only choline but also arsenocholine and acetylcholine. The half maximal effective concentrations have been determined by solid supported membrane electrophysiology to 47 ± 15 μM for choline, 170 ± 9 μM for arsenocholine and 740 ± 84 μM for acetylcholine. Radiolabeled acetylcholine was supplemented in choline reduced media to grow S. pneumoniae cells and to subsequently extract the teichoic acids. The extracted teichoic acids exhibited a signal which provides evidence of the import and catabolization of acetylcholine as an alternative choline source. Proton-coupling as the driving force for the alternating access mechanism during the transport cycle has been confirmed with a fluorescence-based assay. Additionally, a library of synthetic nanobodies, selected against LicB, was characterized based on their inhibitory potential resulting in the determination of several unique inhibitors. In this case solid supported membrane electrophysiology has proven to be a robust and fast technique to screen inhibitory nanobodies and presents an application for other potential drug targets. The structure of the transporter LicB was solved in the substrate bound occluded state at a resolution of 3.8 Å via x-ray crystallography and in the outward facing state, reconstituted into nanodiscs and bound to a synthetic nanobody at a resolution of 3.75 Å via cryo-EM. The transporter plays a crucial role for the survival of the human pathogen and the knowledge about the structure, the function and the identification of inhibitory synthetic nanobodies can help to provide a platform for antimicrobial drug development and for novel alternatives to combat pathogens.