MS08-P01 Structure of an essential inner membrane protein-LPS complex Thomas Clairfeuille (Department of Structural Biology, Genentech, South San Francisco, United States of America) Kerry Buchholz (Department of Infectious diseases, Genentech, South San Francisco, United States of America) Jian Payandeh (Department of Structural Biology, Genentech, South San Francisco, United States of America) Steven Rutherford (Department of Infectious Diseases, Genentech, South San Francisco, United States of America)email: clairfet@gene.comMulti-drug resistant Gram-negative bacteria (GNB) represent a global health threat and urgent unmet medical need.  A defining feature of GNB is their outer membrane (OM), where lipopolysaccharide (LPS) establishes a permeability barrier that notoriously frustrates most antibiotic discovery efforts.  LPS is also a potent stimulator of the innate immune system and responsible for a growing number of deaths, where ~5 million childhood deaths are attributed to bacterial sepsis every year.  Despite the clear biomedical and pharmacological relevance of LPS, the essential mechanisms that coordinate LPS and phospholipid transport to the OM remain unknown. During efforts to identify new targets for the treatment of GNB, we became focused on a multi-pass integral inner membrane protein of unknown function that we call PlbA. Here, we find that PlbA is essential to establish infection in Escherichia coli, and that PlbA depletion produces defects in the OM barrier.  High-resolution X-ray crystallographic structural analyses using traditional and XFEL diffraction methods of PlbA identifies a superfamily of transmembrane proteins that catalyze diverse modifications in the envelopes of Gram-positive and Gram-negative bacteria.  However, the unique architecture of PlbA reveals a defunct active site.  Instead, an LPS molecule is selectively coordinated to an unanticipated LPS-binding motif found along the periplasmic membrane leaflet of PlbA, where core-lipid A binding is achieved through an intricate network of backbone and water mediated interactions. Notably, this PlbA-LPS interaction complex is unprecedented when compared to known structures of selective LPS binding proteins, including the LPS transporter MsbA and the immune Toll-like receptor TLR4. Introduction of disruptive single point mutations at the PlbA-LPS interface leads to defects in the OM barrier.  Overall, this work reveals unprecedented insight into the structural basis of LPS perception within the inner membrane of GNB and implicates PlbA as an LPS-sensor that helps coordinate LPS and PL transport to the OM.

Keywords: lipopolysaccharide, outer membrane, lipid transport