Staphylococcus epidermidis (Se) is a highly abundant gram-positive bacterium predominantly found on human skin. It poses significant threat to immunocompromised patients due to its ability to form biofilms on medical devices. In this study, we determined and refined the first structure of a penicillin-binding protein (PBP) from SePBP3 to a resolution of 2.5 Å. The apo form analysis revealed a shift in the head sub-domain (HSD) relative to the homologous structure in Staphylococcus aureus (Sa). The discovery led us to conduct an analysis of SePBP3’s flexibility applying also X-ray solution scattering. Additional molecular dynamics simulations revealed a rigid transpeptidase domain paired with a flexible pedestal domain, displaying an open and closed interface between the N-terminal anchor domain and the HSD. Furthermore, we solved and refined the structure of SePBP3 in complex with the β-lactam antibiotic cefotaxime and the boron-based antibiotic vaborbactam to 2.51 and 2.3 Å resolution, respectively. Both ligands demonstrated high binding affinity, as confirmed by ITC measurements. Since Staphylococcus epidermidis is a potential major contributor to nosocomial infections, the new structural insights into a highly affine PBP capable of binding various classes of antibiotics provide valuable information for future drug design investigations.

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous health problems and economical challenges for mankind. To date, no effective drug is available to directly treat the disease and prevent virus spreading. In a search for a drug against COVID-19, we have performed a massive X-ray crystallographic screen of repurposing drug libraries containing 5953 individual compounds against the SARS-CoV-2 main protease (Mpro), which is a potent drug target as it is essential for the virus replication. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds binding to Mpro. In subsequent cell-based viral reduction assays, one peptidomimetic and five non-peptidic compounds showed antiviral activity at non-toxic concentrations. Interestingly, two compounds bind outside the active site to the native dimer interface in close proximity to the S1 binding pocket. Another compound binds in a cleft between the catalytic and dimerization domain of Mpro. Neither binding site is related to the enzymatic active site and both represent attractive targets for drug development against SARS-CoV-2. This X-ray screening approach thus has the potential to help deliver an approved drug on an accelerated time-scale for this and future pandemics.