The mechanism of an electrostatic nanofilter: overcoming entropy with electrostatics.

General porins are nature's sieving machinery in the outer membrane of Gram-negative bacteria. Their unique hourglass-shaped architecture is highly conserved among different bacterial membrane proteins and other biological channels. These biological nanopores have been designed to protect the interior of the bacterial cell from leakage of toxic compounds while selectively allowing the entry of the molecules needed for cell growth and function. The mechanism of transport through porins is of utmost and direct interest for drug discovery, extending toward nanotechnology applications for blue energy, separations, and sequencing. Here we present a theoretical framework for analysing the filter of general porins in relation to translocating molecules with the aid of enhanced molecular simulations quantitatively. Using different electrostatic probes in the form of a series of related molecules, we describe the nature of this filter and how to finely tune permeability by exploiting electrostatic interactions between the pore and the translocating molecule. Eventually, we show how enhanced simulations constitute today a valid tool for characterising the mechanism and quantifying energetically the transport of molecules through nanopores.

Structure of the connexin-43 gap junction channel in a putative closed state.

Gap junction channels (GJCs) mediate intercellular communication by connecting two neighbouring cells and enabling direct exchange of ions and small molecules. Cell coupling via connexin-43 (Cx43) GJCs is important in a wide range of cellular processes in health and disease (Churko and Laird, 2013; Liang et al., 2020; Poelzing and Rosenbaum, 2004), yet the structural basis of Cx43 function and regulation has not been determined until now. Here, we describe the structure of a human Cx43 GJC solved by cryo-EM and single particle analysis at 2.26 Å resolution. The pore region of Cx43 GJC features several lipid-like densities per Cx43 monomer, located close to a putative lateral access site at the monomer boundary. We found a previously undescribed conformation on the cytosolic side of the pore, formed by the N-terminal domain and the transmembrane helix 2 of Cx43 and stabilized by a small molecule. Structures of the Cx43 GJC and hemichannels (HCs) in nanodiscs reveal a similar gate arrangement. The features of the Cx43 GJC and HC cryo-EM maps and the channel properties revealed by molecular dynamics simulations suggest that the captured states of Cx43 are consistent with a closed state.

How the physical properties of bacterial porins match environmental conditions.

Transmembrane ß-barrel proteins are key systems for transport phenomena in biology. Based on their broad substrate specificity, they represent good candidates for present and future technological applications, such as DNA/RNA and protein sequencing, sensing of biomedical analytes, and production of blue energy. For a better understanding of the process at the molecular level, we applied parallel tempering simulations in the WTE ensemble to compare two ß-barrel porins from Escherichia coli, OmpF and OmpC. Our analysis showed a different behavior of the two highly homologous porins, where subtle amino acid substitutions can modulate critical properties of mass transport. Interestingly, the differences can be mapped to the respective environmental conditions under which the two porins are expressed. Apart from reporting on the advantages of the enhanced sampling methods in assessing the molecular properties of nanopores, our comparative analysis provided new and key results to better understand biological function and technical applications. Eventually, we showed how results from molecular simulations align well with experimental single-channel measurements, thus demonstrating the mature evolution of numerical methodologies for predicting properties in this field crucial for future biomedical applications.

The Role of Host Cell Glycans on Virus Infectivity: The SARS-CoV-2 Case.

Glycans are ubiquitously expressed sugars, coating the cell and protein surfaces. They are found on many proteins as either short and branched chains or long chains sticking out from special membrane proteins, known as proteoglycans. This sugar cushion, the glycocalyx, modulates specific interactions and protects the cell. Here it is shown that both the expression of proteoglycans and the glycans expressed on the surface of both the host and virus proteins have a critical role in modulating viral attachment to the cell. A mathematical model using SARS-Cov-2 as an archetypical virus to study the glycan role during infection is proposed. It is shown that this occurs via a tug-of-war of forces. On one side, the multivalent molecular recognition that viral proteins have toward specific host glycans and receptors. On the other side, the glycan steric repulsion that a virus must overcome to approach such specific receptors. By balancing both interactions, viral tropism can be predicted. In other words, the authors can map out the cells susceptible to virus infection in terms of receptors and proteoglycans compositions.

A Multiscale Study of Phosphorylcholine Driven Cellular Phenotypic Targeting.

Phenotypic targeting requires the ability of the drug delivery system to discriminate over cell populations expressing a particular receptor combination. Such selectivity control can be achieved using multiplexed-multivalent carriers often decorated with multiple ligands. Here, we demonstrate that the promiscuity of a single ligand can be leveraged to create multiplexed-multivalent carriers achieving phenotypic targeting. We show how the cellular uptake of poly(2-(methacryloyloxy)ethyl phosphorylcholine)-poly(2-(diisopropylamino)ethyl methacry-late) (PMPC-PDPA) polymersomes varies depending on the receptor expression among different cells. We investigate the PMPC-PDPA polymersome insertion at the single chain/receptor level using all-atom molecular modeling. We propose a theoretical statistical mechanics-based model for polymersome-cell association that explicitly considers the interaction of the polymersome with the cell glycocalyx shedding light on its effect on the polymersome binding. We validate our model experimentally and show that the binding energy is a nonlinear function, allowing us to tune the interaction by varying the radius and degree of polymerization. Finally, we show that PMPC-PDPA polymersomes can be used to target monocytes in vivo due to their promiscuous interaction with SRB1, CD36, and CD81.

Curved membrane structures induced by native lipids in giant vesicles.

Native lipids in cell-membrane support crucial functions like intercell communication via their ability to deform into curved membrane structures. Cell membrane mimicking Giant unilamellar vesicles (GUV) is imperative in understanding native lipid's role in membrane transformation however remains challenging to assemble. We construct two giant vesicle models mimicking bacterial inner-membrane (IM) and outer-membrane (OM) under physiological conditions using single-step gel-assisted lipid swelling. IM vesicles composed of native bacterial lipids undergo small-scale membrane remodeling into bud and short-nanotube structures. In contrast, OM vesicles asymmetrically assembled from Lipopolysaccharide (LPS) and bacterial lipids underwent global membrane deformation under controlled osmotic stress. Remarkably, highly-curved structures mimicking cell-membrane architectures, including daughter vesicle networks interconnected by necks and nano-tubes ranging from micro to nanoscale, are generated in OM vesicles at osmotic stress comparable to that applied in IM vesicles. Further, we provide a quantitative description of the membrane structures by experimentally determining membrane elastic parameters, i.e., neck curvature and bending rigidity. We can conclude that a larger spontaneous curvature estimated from the neck curvature and softer membranes in OM vesicles is responsible for large-scale deformation compared to IM vesicles. Our findings will help comprehend the shape dynamics of complex native bacterial lipid membranes.

A different perspective for nonphotochemical quenching in plant antenna complexes.

Light-harvesting complexes of plants exert a dual function of light-harvesting (LH) and photoprotection through processes collectively called nonphotochemical quenching (NPQ). While LH processes are relatively well characterized, those involved in NPQ are less understood. Here, we characterize the quenching mechanisms of CP29, a minor LHC of plants, through the integration of two complementary enhanced-sampling techniques, dimensionality reduction schemes, electronic calculations and the analysis of cryo-EM data in the light of the predicted conformational ensemble. Our study reveals that the switch between LH and quenching state is more complex than previously thought. Several conformations of the lumenal side of the protein occur and differently affect the pigments' relative geometries and interactions. Moreover, we show that a quenching mechanism localized on a single chlorophyll-carotenoid pair is not sufficient but many chlorophylls are simultaneously involved. In such a diffuse mechanism, short-range interactions between each carotenoid and different chlorophylls combined with a protein-mediated tuning of the carotenoid excitation energies have to be considered in addition to the commonly suggested Coulomb interactions.

Concurrent mutations in RNA-dependent RNA polymerase and spike protein emerged as the epidemiologically most successful SARS-CoV-2 variant.

The D614G mutation in the Spike protein of the SARS-CoV-2 has effectively replaced the early pandemic-causing variant. Using pseudotyped lentivectors, we confirmed that the aspartate replacement by glycine in position 614 is markedly more infectious. Molecular modelling suggests that the G614 mutation facilitates transition towards an open state of the Spike protein. To explain the epidemiological success of D614G, we analysed the evolution of 27,086 high-quality SARS-CoV-2 genome sequences from GISAID. We observed striking coevolution of D614G with the P323L mutation in the viral polymerase. Importantly, the exclusive presence of G614 or L323 did not become epidemiologically relevant. In contrast, the combination of the two mutations gave rise to a viral G/L variant that has all but replaced the initial D/P variant. Our results suggest that the P323L mutation, located in the interface domain of the RNA-dependent RNA polymerase, is a necessary alteration that led to the epidemiological success of the present variant of SARS-CoV-2. However, we did not observe a significant correlation between reported COVID-19 mortality in different countries and the prevalence of the Wuhan versus G/L variant. Nevertheless, when comparing the speed of emergence and the ultimate predominance in individual countries, it is clear that the G/L variant displays major epidemiological supremacy over the original variant.

The Influence of Permeability through Bacterial Porins in Whole-Cell Compound Accumulation.

The lack of new drugs for Gram-negative pathogens is a global threat to modern medicine. The complexity of their cell envelope, with an additional outer membrane, hinders internal accumulation and thus, the access of molecules to their targets. Our limited understanding of the molecular basis for compound influx and efflux from these pathogens is a major bottleneck for the discovery of effective antibacterial compounds. Here we analyse the correlation between the whole-cell compound accumulation of ~200 molecules and their predicted porin permeability coefficient (influx), using a recently developed scoring function. We found a strong linear relationship (74%) between the two, confirming porins key in compound uptake in Gram-negative bacteria. The analysis of this unique dataset aids to better understand the molecular descriptors behind whole-cell accumulation and molecular uptake in Gram-negative bacteria.

Permeation of ß-Lactamase Inhibitors through the General Porins of Gram-Negative Bacteria.

Modern medicine relies upon antibiotics, but we have arrived to the point where our inability to come up with new effective molecules against resistant pathogens, together with the declining private investment, is resulting in the number of untreatable infections increasing worldwide at worrying pace. Among other pathogens, widely recognized institutions have indicated Gram-negative bacteria as particularly challenging, due to the presence of the outer membrane. The very first step in the action of every antibiotic or adjuvant is the permeation through this membrane, with small hydrophilic drugs usually crossing through protein channels. Thus, a detailed understanding of their properties at a molecular level is crucial. By making use of Molecular Dynamics simulations, we compared the two main porins of four members of the Enterobacteriaceae family, and, in this paper, we show their shared geometrical and electrostatic characteristics. Then, we used metadynamics simulations to reconstruct the free energy for permeation of selected diazobicyclooctans through OmpF. We demonstrate how porins features are coupled to those of the translocating species, modulating their passive permeation. In particular, we show that the minimal projection area of a molecule is a better descriptor than its molecular mass or the volume. Together with the magnitude and orientation of the electric dipole moment, these are the crucial parameters to gain an efficient compensation between the entropic and enthalpic contributions to the free energy barrier required for permeation. Our results confirm the possibility to predict the permeability of molecules through porins by using a few molecular parameters and bolster the general model according to which the free energy increase is mostly due to the decrease of conformational entropy, and this can be compensated by a favorable alignment of the electric dipole with respect to the channel intrinsic electric field.

A combined activation mechanism for the glucagon receptor.

We report on a combined activation mechanism for a class B G-protein-coupled receptor (GPCR), the glucagon receptor. By computing the conformational free-energy landscape associated with the activation of the receptor-agonist complex and comparing it with that obtained with the ternary complex (receptor-agonist-G protein) we show that the agonist stabilizes the receptor in a preactivated complex, which is then fully activated upon binding of the G protein. The proposed mechanism contrasts with the generally assumed GPCR activation mechanism, which proceeds through an opening of the intracellular region allosterically elicited by the binding of the agonist. The mechanism found here is consistent with electron cryo-microscopy structural data and might be general for class B GPCRs. It also helps us to understand the mode of action of the numerous allosteric antagonists of this important drug target.

Kanamycin Uptake into Escherichia coli Is Facilitated by OmpF and OmpC Porin Channels Located in the Outer Membrane.

Despite decades of therapeutic application of aminoglycosides, it is still a matter of debate if porins contribute to the translocation of the antibiotics across the bacterial outer membrane. Here, we quantified the uptake of kanamycin across the major porin channels OmpF and OmpC present in the outer membrane of Escherichia coli. Our analysis revealed that, despite its relatively large size, about 10-20 kanamycin molecules per second permeate through OmpF and OmpC under a 10 µM concentration gradient, whereas OmpN does not allow the passage. Molecular simulations elucidate the uptake mechanism of kanamycin through these porins. Whole-cell studies with a defined set of E. coli porin mutants provide evidence that translocation of kanamycin via porins is relevant for antibiotic potency. The values are discussed with respect to other antibiotics.

Porins and small-molecule translocation across the outer membrane of Gram-negative bacteria.

Gram-negative bacteria and their complex cell envelope, which comprises an outer membrane and an inner membrane, are an important and attractive system for studying the translocation of small molecules across biological membranes. In the outer membrane of Enterobacteriaceae, trimeric porins control the cellular uptake of small molecules, including nutrients and antibacterial agents. The relatively slow porin-mediated passive uptake across the outer membrane and active efflux via efflux pumps in the inner membrane creates a permeability barrier. The synergistic action of outer membrane permeability, efflux pump activities and enzymatic degradation efficiently reduces the intracellular concentrations of small molecules and contributes to the emergence of antibiotic resistance. In this Review, we discuss recent advances in our understanding of the molecular and functional roles of general porins in small-molecule translocation in Enterobacteriaceae and consider the crucial contribution of porins in antibiotic resistance.

Diffusion of large particles through small pores: From entropic to enthalpic transport.

We present a statistical model for solving and predicting the transport of large molecules through small flexible channels. The average radius of the channel and the average radius of the molecule are the only two quantities determining the steric part of the potential of mean force for the translocation, in the case of a small rigid particle and a large rigid channel: the barrier is completely entropic and is described by the Fick-Jacobs model. However, the flexibility of the channel's cross section and that of the molecule's size have a significant effect on transport, especially when a large molecule goes through a narrow channel. In this case, the steric barrier changes its statistical nature becoming enthalpic, and we predict a strong temperature enhancement of the diffusion current through the channel. The flexibility is described in terms of the equilibrium fluctuations of the channel and of the molecule. The model is compared with the all-atom MD simulations of the transport of hard spheres of various radii and of drug molecules through a biological nanochannel. For the case of Gaussian fluctuations, we derived a simple analytical expression for the steric barrier, which can be quantified using average size and fluctuations of the channel and of the molecule.

Glucose transport via the pseudomonad porin OprB: implications for the design of Trojan Horse anti-infectives.

Deciphering the transport through outer-membrane porins is crucial to understand how anti-infectives enter Gram-negative bacteria and perform their function. Here we elucidated the transport mechanism of substrates through the Pseudomonads sugar-specific porin OprB by means of multiscale modeling. We used molecular dynamics simulations to quantify the energetics of transport and thus a diffusion model to quantify the macroscopic flux of molecules through OprB. Our results show that Trp171 and several glutamate residues in the constriction region are key for the transport of glucose, the preferred natural substrate, through OprB. The unveiled transport mechanism suggests that 2-acetamido-1,2-dideoxynojirimycin (DNJ-NAc), an anti-infective structurally similar to glucose, can enter the cell via OprB. We quantified its energetics and macroscopic flux through OprB providing a comparative analysis with the natural substrate. Thus this pore can be considered as a promising gateway for exploiting the Trojan Horse strategy in pathogenic bacteria.

Getting Drugs into Gram-Negative Bacteria: Rational Rules for Permeation through General Porins.

Small, hydrophilic molecules, including most important antibiotics in clinical use, cross the Gram-negative outer membrane through the water-filled channels provided by porins. We have determined the X-ray crystal structures of the principal general porins from three species of Enterobacteriaceae, namely Enterobacter aerogenes, Enterobacter cloacae, and Klebsiella pneumoniae, and determined their antibiotic permeabilities as well as those of the orthologues from Escherichia coli. Starting from the structure of the porins and molecules, we propose a physical mechanism underlying transport and condense it in a computationally efficient scoring function. The scoring function shows good agreement with in vitro penetration data and will enable the screening of virtual databases to identify molecules with optimal permeability through porins and help to guide the optimization of antibiotics with poor permeation.

Getting Drugs through Small Pores: Exploiting the Porins Pathway in Pseudomonas aeruginosa.

Understanding molecular properties of outer membrane channels of Gram-negative bacteria is of fundamental significance as they are the entry point of polar antibiotics into bacteria. Outer membrane proteomics revealed OccK8 (OprE) to be among the five most expressed substrate specific channels of the clinically important Pseudomonas aeruginosa. The high-resolution X-ray structure and electrophysiology highlighted a very narrow pore. However, experimental in vitro methods showed the transport of natural amino acids and antibiotics, among them ceftazidime. We used molecular dynamics simulations to reveal the importance of the physicochemical properties of ceftazidime in modulating the translocation through OccK8, proposing a structure-function relationship. As in general porins, the internal electric field favors the translocation of polar molecules by gainful energy compensation in the central constriction region. Importantly, the comparatively narrow OccK8 pore can undergo a substrate-induced expansion to accommodate relatively large-sized substrates.

Unusual Constriction Zones in the Major Porins OmpU and OmpT from Vibrio cholerae.

The outer membranes (OM) of many Gram-negative bacteria contain general porins, which form nonspecific, large-diameter channels for the diffusional uptake of small molecules required for cell growth and function. While the porins of Enterobacteriaceae (e.g., E. coli OmpF and OmpC) have been extensively characterized structurally and biochemically, much less is known about their counterparts in Vibrionaceae. Vibrio cholerae, the causative agent of cholera, has two major porins, OmpU and OmpT, for which no structural information is available despite their importance for the bacterium. Here we report high-resolution X-ray crystal structures of V. cholerae OmpU and OmpT complemented with molecular dynamics simulations. While similar overall to other general porins, the channels of OmpU and OmpT have unusual constrictions that create narrower barriers for small-molecule permeation and change the internal electric fields of the channels. Together with electrophysiological and in vitro transport data, our results illuminate small-molecule uptake within the Vibrionaceae.

Bacterial Outer Membrane Porins as Electrostatic Nanosieves: Exploring Transport Rules of Small Polar Molecules.

Transport of molecules through biological membranes is a fundamental process in biology, facilitated by selective channels and general pores. The architecture of some outer membrane pores in Gram-negative bacteria, common to other eukaryotic pores, suggests them as prototypes of electrostatically regulated nanosieve devices. In this study, we sensed the internal electrostatics of the two most abundant outer membrane channels of Escherichia coli, using norfloxacin as a dipolar probe in single molecule electrophysiology. The voltage dependence of the association rate constant of norfloxacin interacting with these nanochannels follows an exponential trend, unexpected for neutral molecules. We combined electrophysiology, channel mutagenesis, and enhanced sampling molecular dynamics simulations to explain this molecular mechanism. Voltage and temperature dependent ion current measurements allowed us to quantify the transversal electric field inside the channel as well as the distance where the applied potential drops. Finally, we proposed a general model for transport of polar molecules through these electrostatic nanosieves. Our model helps to further understand the basis for permeability in Gram-negative pathogens, contributing to fill in the innovation gap that has limited the discovery of effective antibiotics in the last 20 years.

Rationalizing the permeation of polar antibiotics into Gram-negative bacteria.

The increasing level of antibiotic resistance in Gram-negative bacteria, together with the lack of new potential drug scaffolds in the pipeline, make the problem of infectious diseases a global challenge for modern medicine. The main reason that Gram-negative bacteria are particularly challenging is the presence of an outer cell-protecting membrane, which is not present in Gram-positive species. Such an asymmetric bilayer is a highly effective barrier for polar molecules. Several protein systems are expressed in the outer membrane to control the internal concentration of both nutrients and noxious species, in particular: (i) water-filled channels that modulate the permeation of polar molecules and ions according to concentration gradients, and (ii) efflux pumps to actively expel toxic compounds. Thus, besides expressing specific enzymes for drugs degradation, Gram-negative bacteria can also resist by modulating the influx and efflux of antibiotics, keeping the internal concentration low. However, there are no direct and robust experimental methods capable of measuring the permeability of small molecules, thus severely limiting our knowledge of the molecular mechanisms that ultimately control the permeation of antibiotics through the outer membrane. This is the innovation gap to be filled for Gram-negative bacteria. This review is focused on the permeation of small molecules through porins, considered the main path for the entry of polar antibiotics into Gram-negative bacteria. A fundamental understanding of how these proteins are able to filter small molecules is a prerequisite to design/optimize antibacterials with improved permeation. The level of sophistication of modern molecular modeling algorithms and the advances in new computer hardware has made the simulation of such complex processes possible at the molecular level. In this work we aim to share our experience and perspectives in the context of a multidisciplinary extended collaboration within the IMI-Translocation consortium. The synergistic combination of structural data, in vitro assays and computer simulations has proven to give new insights towards the identification and description of physico-chemical properties modulating permeation. Once similar general rules are identified, we believe that the use of virtual screening techniques will be very helpful in searching for new molecular scaffolds with enhanced permeation, and that molecular modeling will be of fundamental assistance to the optimization stage.