Mechanistic studies of the biogenesis and folding of outer membrane proteins in vitro and in vivo: what have we learned to date?

Research into the mechanisms by which proteins fold into their native structures has been on-going since the work of Anfinsen in the 1960s. Since that time, the folding mechanisms of small, water-soluble proteins have been well characterised. By contrast, progress in understanding the biogenesis and folding mechanisms of integral membrane proteins has lagged significantly because of the need to create a membrane mimetic environment for folding studies in vitro and the difficulties in finding suitable conditions in which reversible folding can be achieved. Improved knowledge of the factors that promote membrane protein folding and disfavour aggregation now allows studies of folding into lipid bilayers in vitro to be performed. Consequently, mechanistic details and structural information about membrane protein folding are now emerging at an ever increasing pace. Using the panoply of methods developed for studies of the folding of water-soluble proteins. This review summarises current knowledge of the mechanisms of outer membrane protein biogenesis and folding into lipid bilayers in vivo and in vitro and discusses the experimental techniques utilised to gain this information. The emerging knowledge is beginning to allow comparisons to be made between the folding of membrane proteins with current understanding of the mechanisms of folding of water-soluble proteins

Modulation of conformational space and dynamics of unfolded outer membrane proteins by periplasmic chaperones

Beta-barrel outer membrane proteins (OMPs) present on the outer membrane of Gram-negative bacteria are vital to cell survival. Their biogenesis is a challenging process which is tightly regulated by protein-chaperone interactions at various stages. Upon secretion from the inner membrane, OMPs are solubilized by periplasmic chaperones seventeen kilodalton protein (Skp) and survival factor A (SurA) and maintained in a folding competent state until they reach the outer membrane. As periplasm has an energy deficient environment, thermodynamics plays an important role in fine tuning these chaperone-OMP interactions. Thus, a complete understanding of such associations necessitates an investigation into both structural and thermodynamic aspects of the underlying intercommunication. Yet, they have been difficult to discern because of the conformational heterogeneity of the bound substrates, fast chain dynamics and the aggregation prone nature of OMPs. This demands for use of single molecule spectroscopy techniques, specifically, single molecule Förster resonance energy transfer (smFRET). In this thesis, upon leveraging the conformational and temporal resolution offered by smFRET, an exciting insight is obtained into the mechanistic and functional features of unfolded and Skp/SurA - bound states of two differently sized OMPs: OmpX (8 β-strands) and outer membrane phospholipase A (OmpLA – 12 β-strands). First, it was elucidated that the unfolded states of both the proteins exhibit slow interconversion within their sub-populations. Remarkably, upon complexing with chaperones, irrespective of the chosen OMP, the bound substrates expanded with localised chain reconfiguration on a sub-millisecond timescale. Yet, due to the different interaction mechanisms employed by Skp (encapsulation) and SurA (multivalent binding), their clients were found to be characterised by distinct conformational ensembles. Importantly, the extracted thermodynamic parameters of change in enthalpy and entropy exemplified the mechanistically dissimilar functionalities of the two chaperones. Furthermore, both Skp and SurA were found to be capable of disintegrating aggregated OMPs rather cooperatively, highlighting their multifaceted chaperone activity. This work is of significant fundamental value towards understanding the ubiquitous chaperone-protein interactions and opens up the possibility to design drugs targeting the chaperone-OMP complex itself, one step ahead of the OMP assembly on the outer membrane

Tripartite efflux pumps of the RND superfamily: what did we learn from computational studies?

Bacterial resistance to antibiotics has been long recognized as a priority to address for human health. Among all micro-organisms, the so-called multi -drug resistant (MDR) bacteria, which are resistant to most, if not all drugs in our current arsenal, are particularly worrisome. The World Health Organization has prioritized the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species) pathogens, which include four Gram-negative bacterial species. In these bacteria, active extrusion of antimicrobial compounds out of the cell by means of 'molecular guns' known as efflux pumps is a main determinant of MDR phenotypes. The resistance-nodulation- cell division (RND) superfamily of efflux pumps connecting the inner and outer membrane in Gram-negative bacteria is crucial to the onset of MDR and virulence, as well as biofilm formation. Thus, understanding the molecular basis of the interaction of antibiotics and inhibitors with these pumps is key to the design of more effective therapeutics. With the aim to contribute to this challenge, and complement and inspire experimental research, in silico studies on RND efflux pumps have flourished in recent decades. Here, we review a selection of such investigations addressing the main determinants behind the polyspecificity of these pumps, the mechanisms of substrate recognition, transport and inhibition, as well as the relevance of their assembly for proper functioning, and the role of protein-lipid interactions. The journey will end with a perspective on the role of computer simulations in addressing the challenges posed by these beautifully complex machineries and in supporting the fight against the spread of MDR bacteria

Trends of the major porin gene (ompF) evolution

OmpF is one of the major general porins of Enterobacteriaceae that belongs to the first line of bacterial defense and interactions with the biotic as well as abiotic environments. Porins are surface exposed and their structures strongly reflect the history of multiple interactions with the environmental challenges. Unfortunately, little is known on diversity of porin genes of Enterobacteriaceae and the genus Yersinia especially. We analyzed the sequences of the ompF gene from 73 Yersinia strains covering 14 known species. The phylogenetic analysis placed most of the Yersinia strains in the same line assigned by 16S rDNA-gyrB tree. Very high congruence in the tree topologies was observed for Y. enterocolitica, Y. kristensenii, Y. ruckeri, indicating that intragenic recombination in these species had no effect on the ompF gene. A significant level of intra- and interspecies recombination was found for Y. aleksiciae, Y. intermedia and Y. mollaretii. Our analysis shows that the ompF gene of Yersinia has evolved with nonrandom mutational rate under purifying selection. However, several surface loops in the OmpF porin contain positively selected sites, which very likely reflect adaptive diversification Yersinia to their ecological niches. To our knowledge, this is a first investigation of diversity of the porin gene covering the whole genus of the family Enterobacteriaceae. This study demonstrates that recombination and positive selection both contribute to evolution of ompF, but the relative contribution of these evolutionary forces are different among Yersinia species

Computational Studies on Pharmaceutical Targets in Human Diseases

Bacterial multidrug resistance (i.e. the ability of some bacterial species to survive in presence of various drugs) has become a primary challenge at a global level. Due to various factors, such as the overuse of antibiotics in human activities like health care and farming or inadequate diagnostic, many bacteria have indeed evolved acquiring novel and highly efficient resistance mechanisms. Some species, in particular, have become resistant to almost all in-use drugs. Among the several mechanisms of resistance, efflux pumps of the RND superfamily (resistance-nodulation-cell division) play a major role. These complexes span the cell wall and are able to expel a wide range of noxious compounds, including antibiotics of many different classes. In order to reinvigorate the action of these drugs, a viable route is to hinder their transport out of the cell through co-administration of efflux pumps inhibitors (EPIs). At present several EPIs have been identified, but none of them is usable in clinical therapies due to adverse effects. Moreover, several questions are still open regarding the mode of action of known EPIs as well as the functioning mechanism of RND efflux pumps. Further research in this field is thus needed. In order to characterize the mode of action of several EPIs of this pump, we applied computational techniques such as molecular docking and molecular dynamics (MD) simulations. Specifically, we focused on the EPIs: (i) amitriptyline and chlorpromazine, repurposed drugs which were proven to act as inhibitors against AcrB; (ii) PAβN, a known inhibitor of the pump whose mode of action is not fully understood. This thesis focuses on the inhibition of the AcrB efflux pump, the best known representative of the RND superfamily. High-resolution structural data are indeed available for this protein (specifically, for its Escherichia coli orthologue). Moreover, a fluoroquinolone resistant variant of this pump has been detected in clinical environments. With regard to amitriptyline and chlorpromazine, our in silico investigations revealed that both compounds tend to occupy a known binding pocket of AcrB. Their binding mode presents considerable similarities with that of several substrates and other EPIs of the pump, indicating that amitriptyline and chlorpromazine may inhibit the AcrB pump through competitive binding. In the case of PAβN, MD simulations were compared with experimental data from hydrogen-deuterium exchange mass spectrometry. From these analyses, it emerged that PAβN can significantly restrain the conformational dynamics of AcrB and its fluoroquinolone resistant variant. This EPI, therefore, may act by preventing conformational changes that are functional for AcrB. Importantly, our MD simulations revealed that PAβN and the antibiotic ciprofloxacin can simultaneously occupy the same binding pocket, suggesting that the EPI does not act by competitive binding. Further computational analyses were conducted on structural models of Salmonella Typhimurium AcrB. Experimental structural data on this wt protein are indeed missing, while the structure of its fluoroquinolone resistant variant has recently been solved through cryo-electron microscopy (cryo-EM). In order to assess the structural differences between the two proteins, we derived their structural models through homology modelling and MD simulations (modeling of the fluoroquinolone resistant variant was integrated with cryo-EM data). Structural analyses were then performed, with focus on the binding pockets of the protein. Considerable differences were detected regarding the volume as well as the hydration properties of the pockets. Although not strictly related to EPI development, this information may be valuable for the design of novel drugs and/or inhibitors of AcrB from Salmonella

Towards Understanding How Membrane Proteins Approach And Fold Into Membranes

Membrane proteins must fold into phospholipid bilayers to function. Some membrane proteins are cotranslationally inserted into the membrane, while others rely on chaperone networks to solubilize and traffic unfolded membrane proteins to the membrane. In my thesis work I have studied both facets of membrane protein folding: chaperone interactions and insertion into the membrane. The first part of my thesis investigates the intrinsic conformational properties and unfolded outer membrane protein (uOMP) binding of the main chaperone in OMP biogenesis pathway in E. coli: SurA. We found that SurA is monomeric and exists in three major conformations in solution. Next, we mapped the uOMP binding site on SurA, finding that the least intrinsically populated conformation is the chaperone-active state. Using a plethora of experimental data as restraints, we constructed a model of the SurA-uOMP complex in which the uOMP is greatly expanded by SurA. In the second part of my thesis I examined the effect of the local environment imposed by both the protein and bilayer on individual side chain transfer free energies. I found that the transfer free energies for most side chains are only slightly altered by changes in neighboring side chains and packing. By relating the local composition of the membrane to nonpolar side chain transfer free energies, I discovered a linear correlation between the nonpolar solvation parameter and local concentration of water in the bilayer. Together these studies highlight the structural and thermodynamic parameters that drive efficient membrane protein biogenesis and folding

Structure and mechanism of bacterial tripartite efflux pumps.

Efflux pumps are membrane proteins which contribute to multi-drug resistance. In Gram-negative bacteria, some of these pumps form complex tripartite assemblies in association with an outer membrane channel and a periplasmic membrane fusion protein. These tripartite machineries span both membranes and the periplasmic space, and they extrude from the bacterium chemically diverse toxic substrates. In this chapter, we summarise current understanding of the structural architecture, functionality, and regulation of tripartite multi-drug efflux assemblies.ER

In silico investigation and surmounting of lipopolysaccharide barrier in Gram-negative bacteria: how far has molecular dynamics come?

Lipopolysaccharide (LPS), a main component of the outer membrane of Gram-negative bacteria, has crucial implications on both antibiotic resistance and the overstimulation of the host innate immune system. Fighting against these global concerns calls for the molecular understanding of the barrier function and immunostimulatory ability of LPS. Molecular dynamics (MD) simulations have become an invaluable tool for uncovering important findings in LPS research. While the reach of MD simulations for investigating the immunostimulatory ability of LPS has been already outlined, little attention has been paid to the role of MD simulations for exploring its barrier function and synthesis. Herein, we give an overview about the impact of MD simulations on gaining insight into the shield role and synthesis pathway of LPS, which have attracted considerable attention to discover molecules able to surmount antibiotic resistance, either circumventing LPS defenses or disrupting its synthesis. We specifically focus on the enhanced sampling and free energy calculation methods that have been combined with MD simulations to address such research. We also highlight the use of special-purpose MD supercomputers, the importance of appropriate LPS and ions parameterization to obtain reliable results, and the complementary views that MD and wet-lab experiments provide. Thereby, this work, which covers the last five years of research, apart from outlining the phenomena and strategies that are being explored, evidences the valuable insights that are gained by MD, which may be useful to advance antibiotic design, and what the prospects of this in silico method could be in LPS research.Financial support from the Spanish Ministry of Science, Innovation and Universities under the project RTI2018-093310-B-I00 is gratefully acknowledged