Uncovering the folding mechanism of pertactin: A comparative study of isolated and vectorial folding.

Autotransporters are a large family of virulence factors found in Gram-negative bacteria that play important roles in their pathogenesis. The passenger domain of autotransporters is almost always composed of a large ß-helix, with only a small portion of it being relevant to its virulence function. This has led to the hypothesis that the folding of the ß-helical structure aids the secretion of the passenger domain across the Gram-negative outer membrane. In this study, we used molecular dynamics simulations and enhanced sampling methods to investigate the stability and folding of the passenger domain of pertactin, an autotransporter from Bordetella pertussis. Specifically, we employed steered molecular dynamics to simulate the unfolding of the entire passenger domain as well as self-learning adaptive umbrella sampling to compare the energetics of folding rungs of the ß-helix independently ("isolated folding") versus folding rungs on top of a previously folded rung ("vectorial folding"). Our results showed that vectorial folding is highly favorable compared with isolated folding; moreover, our simulations showed that the C-terminal rung of the ß-helix is the most resistant to unfolding, in agreement with previous studies that found the C-terminal half of the passenger domain to be more stable than the N-terminal one. Overall, this study provides new insights into the folding process of an autotransporter passenger domain and its potential role in secretion across the outer membrane.

Lpp positions peptidoglycan at the AcrA-TolC interface in the AcrAB-TolC multidrug efflux pump.

The multidrug efflux pumps of Gram-negative bacteria are a class of complexes that span the periplasm, coupling both the inner and outer membranes to expel toxic molecules. The best-characterized example of these tripartite pumps is the AcrAB-TolC complex of Escherichia coli. However, how the complex interacts with the peptidoglycan (PG) cell wall, which is anchored to the outer membrane (OM) by Braun's lipoprotein (Lpp), is still largely unknown. In this work, we present molecular dynamics simulations of a complete, atomistic model of the AcrAB-TolC complex with the inner membrane, OM, and PG layers all present. We find that the PG localizes to the junction of AcrA and TolC, in agreement with recent cryo-tomography data. Free-energy calculations reveal that the positioning of PG is determined by the length and conformation of multiple Lpp copies anchoring it to the OM. The distance between the PG and OM measured in cryo-electron microscopy images of wild-type E. coli also agrees with the simulation-derived spacing. Sequence analysis of AcrA suggests a conserved role for interactions with PG in the assembly and stabilization of efflux pumps, one that may extend to other trans-envelope complexes as well.

Folding free energy landscapes of ß-sheets with non-polarizable and polarizable CHARMM force fields.

Molecular dynamics (MD) simulations of peptides and proteins offer atomic-level detail into many biological processes, although the degree of insight depends on the accuracy of the force fields used to represent them. Protein folding is a key example in which the accurate reproduction of folded-state conformations of proteins and kinetics of the folding processes in simulation is a longstanding goal. Although there have been a number of recent successes, challenges remain in capturing the full complexity of folding for even secondary-structure elements. In the present work, we have used all-atom MD simulations to study the folding properties of one such element, the C-terminal ß-hairpin of the B1 domain of streptococcal protein G (GB1). Using replica-exchange umbrella sampling simulations, we examined the folding free energy of two fixed-charge CHARMM force fields, CHARMM36 and CHARMM22*, as well as a polarizable force field, the CHARMM Drude-2013 model, which has previously been shown to improve the folding properties of α-helical peptides. The CHARMM22* and Drude-2013 models are in rough agreement with experimental studies of GB1 folding, while CHARMM36 overstabilizes the ß-hairpin. Additional free-energy calculations show that small adjustments to the atomic polarizabilities in the Drude-2013 model can improve both the backbone solubility and folding properties of GB1 without significantly affecting the model's ability to properly fold α-helices. We also identify a non-native salt bridge in the ß-turn region that overstabilizes the ß-hairpin in the C36 model. Finally, we demonstrate that tryptophan fluorescence is insufficient for capturing the full ß-hairpin folding pathway.

Conformational Dynamics of AcrA Govern Multidrug Efflux Pump Assembly.

Multidrug efflux pumps of pathogenic, Gram-negative bacteria comprise an innate resistance mechanism and are key contributors to the emerging global pandemic of antibiotic resistance. Several increasingly detailed cryo-electron microscopy maps have been resolved of an entire efflux pump complex, AcrAB-TolC, resulting in atomistic structural models. Using a recent model, we have carried out nearly 40 µs of molecular dynamics simulations to study one of the key components of the protein complex AcrA, the membrane fusion protein that connects the inner-membrane-bound AcrB to the outer-membrane-bound TolC. We determined a three-dimensional potential of mean force (PMF) for AcrA, which displays two main conformational basins representing assembly competent and incompetent states. Corresponding experiments show that stabilizing mutations at an interdomain interface shift the dynamic equilibrium between these states to the incompetent one, disrupting pump assembly and function and resensitizing bacteria to existing antibiotics. The modulation of AcrA dynamics through pharmacological intervention therefore presents a promising route for the development of new antibiotics.