Holdbrook, D. A. | bioRxiv Science

bioRxiv2017

Structural basis for endotoxin neutralization and anti-inflammatory activity of thrombin-derived C-terminal peptides

Thrombin-derived C-terminal peptides (TCP) of about 2 kDa are present in wounds, where they exert anti-endotoxic functions. In an effort to elucidate the structural and molecular aspects of these functions, we here employ a combination of nuclear magnetic resonance spectroscopy (NMR), ellipsometry, fluorescence spectroscopy, circular dichroism (CD) measurements, and in silico multiscale modeling to define interactions and the bound conformation of a TCP generated by neutrophil elastase, HVF18 (HVFRLKKWIQKVIDQFGE) in complex with bacterial lipopolysaccharide (LPS). In contrast to the disordered state of HVF18 in aqueous solution, its binding to LPS leads to a structural transition, wherein the N- terminus of the peptide forms a unique {beta}-turn whilst the C-terminus becomes helical. In silico modelling and simulations demonstrated that HVF18, as well as related peptides, target the LPS-binding site of CD14, and this interaction was experimentally supported using microscale thermophoresis. Collectively, the results demonstrate the role of structural transitions in LPS complex formation as well as in CD 14 interaction, and provide a molecular explanation for the previously observed therapeutic effects of TCPs in experimental models of bacterial sepsis and endotoxin shock.\n\nSignificanceThrombin-derived C-terminal peptides (TCPs) of various sizes are present in human wounds, where they bind bacteria as well as \"free\" lipopolysaccharide (LPS), and thereby reduce inflammation. In this work, employing a combination of cellular, biophysical and structural studies, combined with in silico multiscale modeling, we present the molecular structure of a TCP in association with LPS, and define a previously undisclosed interaction between TCPs and CD14. Further, we show that TCPs exhibit relatively weak but specific affinities, all in the M range, to both LPS and CD14. These novel structural insights into the function of this class of host-defense molecules will facilitate rational design of novel \"dual function\" anti-infectives, which target both bacteria and inflammatory signaling.

biophysics↗

bioRxiv2017

Molecular basis for the maintenance of lipid asymmetry in the outer membrane of Escherichia coli

A distinctive feature of the Gram-negative bacterial cell envelope is the asymmetric outer membrane (OM), where lipopolysaccharides (LPS) and phospholipids (PLs) reside in the outer and inner leaflets, respectively. This unique lipid asymmetry renders the OM impermeable to external insults. In Escherichia coli, the OmpC-MlaA complex is believed to maintain lipid asymmetry by removing mislocalized PLs from the outer leaflet of the OM. How it performs this function is unknown. Here, we define the molecular architecture of the OmpC-MlaA complex to gain insights into its role in PL transport. We establish that MlaA sits entirely within the bilayer in complex with OmpC and provides a hydrophilic channel possibly for PL translocation across the OM. Furthermore, we show that flexibility in a hairpin loop adjacent to the channel modulates MlaA activity. Finally, we demonstrate that OmpC plays an active role in maintaining OM lipid asymmetry together with MlaA. Our work offers glimpses into how the OmpC-MlaA complex transports PLs across the OM and has important implications for future antimicrobial drug development.

biochemistry↗