Discovery of Five SOS2 Fragment Hits with Binding Modes Determined by SOS2 X-Ray Cocrystallography.

SOS1 and SOS2 are guanine nucleotide exchange factors that mediate RTK-stimulated RAS activation. Selective SOS1:KRAS PPI inhibitors are currently under clinical investigation, whereas there are no reports to date of SOS2:KRAS PPI inhibitors. SOS2 activity is implicated in MAPK rebound when divergent SOS1 mutant cell lines are treated with the SOS1 inhibitor BI-3406; therefore, SOS2:KRAS inhibitors are of therapeutic interest. In this report, we detail a fragment-based screening strategy to identify X-ray cocrystal structures of five diverse fragment hits bound to SOS2.

Site-Specific Immobilization of the Peptidoglycan Synthase PBP1B on a Surface Plasmon Resonance Chip Surface.

Surface plasmon resonance (SPR) is one of the most powerful label-free methods to determine the kinetic parameters of molecular interactions in real time and in a highly sensitive way. Penicillin-binding proteins (PBPs) are peptidoglycan synthesis enzymes present in most bacteria. Established protocols to analyze interactions of PBPs by SPR involve immobilization to an ampicillin-coated chip surface (a ß-lactam antibiotic mimicking its substrate), thereby forming a covalent complex with the PBPs transpeptidase (TP) active site. However, PBP interactions measured with a substrate-bound TP domain potentially affect interactions near the TPase active site. Furthermore, in vivo PBPs are anchored in the inner membrane by an N-terminal transmembrane helix, and hence immobilization at the C-terminal TPase domain gives an orientation contrary to the in vivo situation. We designed a new procedure: immobilization of PBP by copper-free click chemistry at an azide incorporated in the N terminus. In a proof-of-principle study, we immobilized Escherichia coli PBP1B on an SPR chip surface and used this for the analysis of the well-characterized interaction of PBP1B with LpoB. The site-specific incorporation of the azide affords control over protein orientation, thereby resulting in a homogeneous immobilization on the chip surface. This method can be used to study topology-dependent interactions of any (membrane) protein.

Activities and regulation of peptidoglycan synthases.

Peptidoglycan (PG) is an essential component in the cell wall of nearly all bacteria, forming a continuous, mesh-like structure, called the sacculus, around the cytoplasmic membrane to protect the cell from bursting by its turgor. Although PG synthases, the penicillin-binding proteins (PBPs), have been studied for 70 years, useful in vitro assays for measuring their activities were established only recently, and these provided the first insights into the regulation of these enzymes. Here, we review the current knowledge on the glycosyltransferase and transpeptidase activities of PG synthases. We provide new data showing that the bifunctional PBP1A and PBP1B from Escherichia coli are active upon reconstitution into the membrane environment of proteoliposomes, and that these enzymes also exhibit DD-carboxypeptidase activity in certain conditions. Both novel features are relevant for their functioning within the cell. We also review recent data on the impact of protein-protein interactions and other factors on the activities of PBPs. As an example, we demonstrate a synergistic effect of multiple protein-protein interactions on the glycosyltransferase activity of PBP1B, by its cognate lipoprotein activator LpoB and the essential cell division protein FtsN.

Coordination of peptidoglycan synthesis and outer membrane constriction during Escherichia coli cell division.

To maintain cellular structure and integrity during division, Gram-negative bacteria must carefully coordinate constriction of a tripartite cell envelope of inner membrane, peptidoglycan (PG), and outer membrane (OM). It has remained enigmatic how this is accomplished. Here, we show that envelope machines facilitating septal PG synthesis (PBP1B-LpoB complex) and OM constriction (Tol system) are physically and functionally coordinated via YbgF, renamed CpoB (Coordinator of PG synthesis and OM constriction, associated with PBP1B). CpoB localizes to the septum concurrent with PBP1B-LpoB and Tol at the onset of constriction, interacts with both complexes, and regulates PBP1B activity in response to Tol energy state. This coordination links PG synthesis with OM invagination and imparts a unique mode of bifunctional PG synthase regulation by selectively modulating PBP1B cross-linking activity. Coordination of the PBP1B and Tol machines by CpoB contributes to effective PBP1B function in vivo and maintenance of cell envelope integrity during division.