Knowles, T. J. | Search bioRxiv

bioRxiv2020

Structure-function analyses of dual-BON domain protein DolP identifies phospholipid binding as a new mechanism for protein localisation

The Gram-negative outer membrane envelops the bacterium and functions as a permeability barrier against antibiotics, detergents and environmental stresses. Some virulence factors serve to maintain the integrity of the outer membrane, including DolP (formerly YraP) a protein of unresolved structure and function. Here we reveal DolP is a lipoprotein functionally conserved among Gram-negative bacteria and that loss of DolP increases membrane fluidity. We present the NMR solution structure for DolP, which is composed of two BON domains that form an interconnected opposing pair. The C-terminal BON domain binds to anionic phospholipids through an extensive membrane:protein interface providing evidence of subcellular localization of these phospholipids within the outer membrane. This interaction is essential for DolP function and is required for sub-cellular localization of the protein to the cell division site. The structure of DolP provides a new target for developing therapies that disrupt the integrity of the bacterial cell envelope.

biochemistry↗

bioRxiv2020

MlaFEDB displays flippase activity to promote phospholipid transport towards the outer membrane of Gram-negative bacteria

MlaFEDB is a Gram-negative inner membrane protein complex involved in the inter membrane trafficking of phospholipids. Originally proposed to transport phospholipids in a retrograde direction, recent evidence suggests MlaFEDB may actually export phospholipids from the inner membrane to the periplasmic carrier protein, MlaC, potentially suggesting a role in either anterograde trafficking of phospholipids to the outer membrane or bidirectional phospholipid movement. MlaFEDB is part of the ABC transporter superfamily of proteins and has been shown to hydrolyse ATP through the cytoplasmic facing MlaF component. However, the movement of PLs from FEDB to MlaC has been shown to occur in an ATP independent fashion hence the role of ATP hydrolysis within this complex remains unclear. In this study we sought to elucidate the role of ATP and provide evidence to suggest MlaFEDB has flippase activity, utilising ATP hydrolysis to translocate phospholipids from the outer to the inner leaflet of the IM. We also show that in the absence of ATP MlaFEDB mediates the loading of MlaC with phospholipids directly from the inner leaflet only. Our data provides a novel role for MlaFEDB and presents a link between Mla driven phospholipid transport and ATP hydrolysis.

molecular biology↗