Dynamic interplay between a TonB-dependent heme transporter and a TonB protein in a membrane environment.

The envelope of Gram-negative bacteria is composed of two membranes separated by the periplasmic space. This organization imposes geometrical and distance constraints that are key for the mechanism of action of multicomponent systems spanning the envelope. However, consideration of all three compartments by experimental approaches is still elusive. Here, we have used the state-of-the-art molecular dynamics simulation in an Escherichia coli envelope model to obtain a dynamic view of molecular interactions between the outer membrane heme transporter HasR and the inner membrane TonB-like protein HasB. Their interaction allows the transfer of the inner membrane proton-motive force derived energy to the transporter for heme internalization. The simulations that incorporate both membranes show the key role of periplasmic domains of both proteins and their dynamics in complex formation and stability. They revealed a previously unidentified network of HasR-HasB protein-protein interactions in the periplasm. Experimental validation (mutations, in vivo phenotypic and biophysical assays) provides support for the simulation-predicted interactions. Based on structural and sequence conservation, the network of interaction revealed in this study is expected to occur in other nutrient import systems. IMPORTANCE: Gram-negative bacteria import scarce nutrients such as metals and vitamins by an energized mechanism involving a multicomponent protein system that spans the cell envelope. It consists of an outer membrane TonB-dependent transporter (TBDT) and a TonB complex in the inner membrane that provides the proton motive force energy for the nutrient entry. Despite the intense research efforts focused on this system (a) from structural and fundamental microbiology perspectives and (b) for the interest in the development of new antibacterial strategies, the molecular mechanism of the system is not at all well understood. The lack of understanding comes from incomplete structural data and the experimental difficulties of studying an inherently flexible multicomponent complex that resides within the heterogeneous environment of the double membrane bacterial cell envelope. To address these challenges and obtain a comprehensive view of the molecular interactions at atomic level, here, we have used the combined power of advanced molecular simulations and complementary microbiology and biochemical experiments. Our results represent a significant step forward in understanding the structural and molecular bases of this vital mechanism.

Structure and lipid dynamics in the maintenance of lipid asymmetry inner membrane complex of A. baumannii.

Multi-resistant bacteria are a major threat in modern medicine. The gram-negative coccobacillus Acinetobacter baumannii currently leads the WHO list of pathogens in critical need for new therapeutic development. The maintenance of lipid asymmetry (MLA) protein complex is one of the core machineries that transport lipids from/to the outer membrane in gram-negative bacteria. It also contributes to broad-range antibiotic resistance in several pathogens, most prominently in A. baumannii. Nonetheless, the molecular details of its role in lipid transport has remained largely elusive. Here, we report the cryo-EM maps of the core MLA complex, MlaBDEF, from the pathogen A. baumannii, in the apo-, ATP- and ADP-bound states, revealing multiple lipid binding sites in the cytosolic and periplasmic side of the complex. Molecular dynamics simulations suggest their potential trajectory across the membrane. Collectively with the recently-reported structures of the E. coli orthologue, this data also allows us to propose a molecular mechanism of lipid transport by the MLA system.

Uptake of monoaromatic hydrocarbons during biodegradation by FadL channel-mediated lateral diffusion.

In modern societies, biodegradation of hydrophobic pollutants generated by industry is important for environmental and human health. In Gram-negative bacteria, biodegradation depends on facilitated diffusion of the pollutant substrates into the cell, mediated by specialised outer membrane (OM) channels. Here we show, via a combined experimental and computational approach, that the uptake of monoaromatic hydrocarbons such as toluene in Pseudomonas putida F1 (PpF1) occurs via lateral diffusion through FadL channels. Contrary to classical diffusion channels via which polar substrates move directly into the periplasmic space, PpF1 TodX and CymD direct their hydrophobic substrates into the OM via a lateral opening in the channel wall, bypassing the polar barrier formed by the lipopolysaccharide leaflet on the cell surface. Our study suggests that lateral diffusion of hydrophobic molecules is the modus operandi of all FadL channels, with potential implications for diverse areas such as biodegradation, quorum sensing and gut biology.

Probing the binding affinities of imipenem and ertapenem for outer membrane carboxylate channel D1 (OccD1) from P. aeruginosa: simulation studies.

Pseudomonas aeruginosa is an important nosocomial human pathogen. The major difficulty in the fight against this pathogen is the relative impermeability of its outer membrane (OM). Only specific substrates can penetrate through the OM of P. aeruginosa via substrate-specific porins, so this has become one of the most problematic drug-resistant pathogens. Carbapenems are the most effective drugs for treating P. aeruginosa infections. One such carbapenem that is applied in cases of P. aeruginosa infection is imipenem (IMI), which uses outer membrane carboxylate channel D1 (OccD1) as a point of entry into the pathogen. Unlike IMI, ertapenem (ERTA, another carbapenem) shows only weak activity towards P. aeruginosa, as it is blocked from penetrating through the OM. However, it is currently unclear as to why IMI is allowed to pass through the OM while ERTA is not. Therefore, we conducted molecular dynamics (MD) simulations to elucidate the behavior of these drugs inside OccD1 as compared to the ligand-free state. We discovered another possible binding site in the constriction region close to the side-pore opening. Both drugs employ the core lactam part to tether themselves to the binding site, whereas the tail governs the direction of permeation. L132 and F133 appear to be involved in interactions that are key to core attachment. At least four hydrogen bonds are required for drug binding. The direction of motion of L2 also plays a role: inward flipping traps IMI in the constriction area, while a shift of L2 towards the membrane brings ERTA into contact with more water, which prompts the expulsion of ERTA to the mouth of the channel protein. The opening of L2 seems to facilitate the rejection of ERTA.