High-Resolution Imaging of Maltoporin LamB while Quantifying the Free-Energy Landscape and Asymmetry of Sugar Binding.

Maltoporins are a family of membrane proteins that facilitate the diffusion of hydrophilic molecules and maltosaccharides across the outer membrane of Gram-negative bacteria. Two contradicting models propose the sugar binding, uptake, and transport by maltoporins to be either symmetric or asymmetric. Here, we address this contradiction and introduce force-distance-based atomic force microscopy to image single maltoporin LamB trimers in the membrane at sub-nanometer resolution and simultaneously quantify the binding of different malto-oligosaccharides. We assay subtle differences of the binding free-energy landscape of maltotriose, maltotetraose, and maltopentaose, which quantifies how binding strength and affinity increase with the malto-oligosaccharide chain length. The ligand-binding parameters change considerably by mutating the extracellular loop 3, which folds into and constricts the transmembrane pore of LamB. By recording LamB topographs and structurally mapping binding events at sub-nanometer resolution, we observe LamB to preferentially bind maltodextrin from the periplasmic side, which shows sugar binding and uptake to be asymmetric. The study introduces atomic force microscopy as an analytical nanoscopic tool that can differentiate among the factors modulating and models describing the binding and uptake of substrates by membrane proteins.

Membrane perforation by the pore-forming toxin pneumolysin.

Pneumolysin (PLY), a major virulence factor of Streptococcus pneumoniae, perforates cholesterol-rich lipid membranes. PLY protomers oligomerize as rings on the membrane and then undergo a structural transition that triggers the formation of membrane pores. Structures of PLY rings in prepore and pore conformations define the beginning and end of this transition, but the detailed mechanism of pore formation remains unclear. With atomistic and coarse-grained molecular dynamics simulations, we resolve key steps during PLY pore formation. Our simulations confirm critical PLY membrane-binding sites identified previously by mutagenesis. The transmembrane ß-hairpins of the PLY pore conformation are stable only for oligomers, forming a curtain-like membrane-spanning ß-sheet. Its hydrophilic inner face draws water into the protein-lipid interface, forcing lipids to recede. For PLY rings, this zone of lipid clearance expands into a cylindrical membrane pore. The lipid plug caught inside the PLY ring can escape by lipid efflux via the lower leaflet. If this path is too slow or blocked, the pore opens by membrane buckling, driven by the line tension acting on the detached rim of the lipid plug. Interestingly, PLY rings are just wide enough for the plug to buckle spontaneously in mammalian membranes. In a survey of electron cryo-microscopy (cryo-EM) and atomic force microscopy images, we identify key intermediates along both the efflux and buckling pathways to pore formation, as seen in the simulations.

Imaging in Biologically-Relevant Environments with AFM Using Stiff qPlus Sensors.

High-resolution imaging of soft biological samples with atomic force microscopy (AFM) is challenging because they must be imaged with small forces to prevent deformation. Typically, AFM of those samples is performed with soft silicon cantilevers (k ≈ 0.1-10 N/m) and optical detection in a liquid environment. We set up a new microscope that uses a stiff qPlus sensor (k ≥ 1 kN/m). Several complex biologically-relevant solutions are non-transparent, and even change their optical properties over time, such as the cell culture medium we used. While this would be problematic for AFM setups with optical detection, it is no problem for our qPlus setup which uses electrical detection. The high stiffness of the qPlus sensor allows us to use small amplitudes in frequency-modulation mode and obtain high Q factors even in liquid. The samples are immersed in solution in a liquid cell and long tips are used, with only the tip apex submerged. We discuss the noise terms and compare the minimal detectable signal to that of soft cantilevers. Atomic resolution of muscovite mica was achieved in various liquids: H2O, Tris buffer and a cell culture medium. We show images of lipid membranes in which the individual head groups are resolved.

Mechanism of membrane pore formation by human gasdermin-D.

Gasdermin-D (GSDMD), a member of the gasdermin protein family, mediates pyroptosis in human and murine cells. Cleaved by inflammatory caspases, GSDMD inserts its N-terminal domain (GSDMDNterm) into cellular membranes and assembles large oligomeric complexes permeabilizing the membrane. So far, the mechanisms of GSDMDNterm insertion, oligomerization, and pore formation are poorly understood. Here, we apply high-resolution (≤ 2 nm) atomic force microscopy (AFM) to describe how GSDMDNterm inserts and assembles in membranes. We observe GSDMDNterm inserting into a variety of lipid compositions, among which phosphatidylinositide (PI(4,5)P2) increases and cholesterol reduces insertion. Once inserted, GSDMDNterm assembles arc-, slit-, and ring-shaped oligomers, each of which being able to form transmembrane pores. This assembly and pore formation process is independent on whether GSDMD has been cleaved by caspase-1, caspase-4, or caspase-5. Using time-lapse AFM, we monitor how GSDMDNterm assembles into arc-shaped oligomers that can transform into larger slit-shaped and finally into stable ring-shaped oligomers. Our observations translate into a mechanistic model of GSDMDNterm transmembrane pore assembly, which is likely shared within the gasdermin protein family.

Maltoporin LamB Unfolds ß Hairpins along Mechanical Stress-Dependent Unfolding Pathways.

Upon mechanical pulling at either terminal end, ß barrel outer membrane proteins stepwise unfold ß strands or ß hairpins until entirely extracted from the membrane. This unique unfolding pathway has been described for ß barrels comprising 8, 14, or 22 ß strands. Here we mechanically unfold the 18-stranded ß barrel outer membrane protein LamB from Escherichia coli. We find that its mechanical unfolding pathway is shaped by the stepwise unfolding of ß hairpins. However, we also observe that ß hairpins can unfold groupwise. Thereby, ß hairpins unfolding at higher pulling forces show a higher probability to unfold collectively, whereas ß hairpins unfolding at lower forces tend to unfold individually. This result suggests that the collective unfolding of ß hairpins resembles a far-from-equilibrium process, whereas the unfolding of individual ß hairpins describes a closer-to-equilibrium process. Our findings support a direct link between outer membrane protein structure and the unfolding pathway and contribute to a better understanding of their unfolding in response to mechanical stress.

Unraveling the Pore-Forming Steps of Pneumolysin from Streptococcus pneumoniae.

Pneumolysin (PLY) is the main virulence factor of Streptococcus pneumoniae that causes pneumonia, meningitis, and invasive pneumococcal infection. PLY is produced as monomers, which bind to cholesterol-containing membranes, where they oligomerize into large pores. To investigate the pore-forming mechanism, we determined the crystal structure of PLY at 2.4 Šand used it to design mutants on the surface of monomers. Electron microscopy of liposomes incubated with PLY mutants revealed that several mutations interfered with ring formation. Mutants that formed incomplete rings or linear arrays had strongly reduced hemolytic activity. By high-resolution time-lapse atomic force microscopy of wild-type PLY, we observed two different ring-shaped complexes. Most of the complexes protruded ∼8 nm above the membrane surface, while a smaller number protruded ∼11 nm or more. The lower complexes were identified as pores or prepores by the presence or absence of a lipid bilayer in their center. The taller complexes were side-by-side assemblies of monomers of soluble PLY that represent an early form of the prepore. Our observations suggest a four-step mechanism of membrane attachment and pore formation by PLY, which is discussed in the context of recent structural models. The functional separation of these steps is necessary for the understanding how cholesterol-dependent cytolysins form pores and lyse cells.

GSDMD membrane pore formation constitutes the mechanism of pyroptotic cell death.

Pyroptosis is a lytic type of cell death that is initiated by inflammatory caspases. These caspases are activated within multi-protein inflammasome complexes that assemble in response to pathogens and endogenous danger signals. Pyroptotic cell death has been proposed to proceed via the formation of a plasma membrane pore, but the underlying molecular mechanism has remained unclear. Recently, gasdermin D (GSDMD), a member of the ill-characterized gasdermin protein family, was identified as a caspase substrate and an essential mediator of pyroptosis. GSDMD is thus a candidate for pyroptotic pore formation. Here, we characterize GSDMD function in live cells and in vitro We show that the N-terminal fragment of caspase-1-cleaved GSDMD rapidly targets the membrane fraction of macrophages and that it induces the formation of a plasma membrane pore. In vitro, the N-terminal fragment of caspase-1-cleaved recombinant GSDMD tightly binds liposomes and forms large permeability pores. Visualization of liposome-inserted GSDMD at nanometer resolution by cryo-electron and atomic force microscopy shows circular pores with variable ring diameters around 20 nm. Overall, these data demonstrate that GSDMD is the direct and final executor of pyroptotic cell death.

Directly Observing the Lipid-Dependent Self-Assembly and Pore-Forming Mechanism of the Cytolytic Toxin Listeriolysin O.

Listeriolysin O (LLO) is the major virulence factor of Listeria monocytogenes and a member of the cholesterol-dependent cytolysin (CDC) family. Gram-positive pathogenic bacteria produce water-soluble CDC monomers that bind cholesterol-dependent to the lipid membrane of the attacked cell or of the phagosome, oligomerize into prepores, and insert into the membrane to form transmembrane pores. However, the mechanisms guiding LLO toward pore formation are poorly understood. Using electron microscopy and time-lapse atomic force microscopy, we show that wild-type LLO binds to membranes, depending on the presence of cholesterol and other lipids. LLO oligomerizes into arc- or slit-shaped assemblies, which merge into complete rings. All three oligomeric assemblies can form transmembrane pores, and their efficiency to form pores depends on the cholesterol and the phospholipid composition of the membrane. Furthermore, the dynamic fusion of arcs, slits, and rings into larger rings and their formation of transmembrane pores does not involve a height difference between prepore and pore. Our results reveal new insights into the pore-forming mechanism and introduce a dynamic model of pore formation by LLO and other CDC pore-forming toxins.

Toxicity of protein oligomers is rationalized by a function combining size and surface hydrophobicity.

The misfolding and aberrant assembly of peptides and proteins into fibrillar aggregates is the hallmark of many pathologies. Fibril formation is accompanied by oligomeric species thought to be the primary pathogenic agents in many of these diseases. With the aim of identifying the structural determinants responsible for the toxicity of misfolded oligomers, we created 12 oligomeric variants from the N-terminal domain of the E. coli HypF protein (HypF-N) by replacing one or more charged amino acid residues with neutral apolar residues and allowing the mutated proteins to aggregate under two sets of conditions. The resulting oligomeric species have different degrees of cytotoxicity when added to the extracellular medium of the cells, as assessed by the extent of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, apoptosis, and influx of Ca2+ into the cells. The structural properties of the oligomeric variants were characterized by evaluating their surface hydrophobicity with 8-anilinonaphthalene-1-sulfonate (ANS) binding and by measuring their size by means of turbidimetry as well as light scattering. We find that increases in the surface hydrophobicity of the oligomers following mutation can promote the formation of larger assemblies and that the overall toxicity correlates with a combination of both surface hydrophobicity and size, with the most toxic oligomers having high hydrophobicity and small size. These results have allowed the relationships between these three parameters to be studied simultaneously and quantitatively, and have enabled the generation of an equation that is able to rationalize and even predict toxicity of the oligomers resulting from their surface hydrophobicity and size.

Multiparametric high-resolution imaging of native proteins by force-distance curve-based AFM.

A current challenge in the life sciences is to understand how the properties of individual molecular machines adjust in order to meet the functional requirements of the cell. Recent developments in force-distance (FD) curve-based atomic force microscopy (FD-based AFM) enable researchers to combine sub-nanometer imaging with quantitative mapping of physical, chemical and biological properties. Here we present a protocol to apply FD-based AFM to the multiparametric imaging of native proteins under physiological conditions. We describe procedures for experimental FD-based AFM setup, high-resolution imaging of proteins in the native unperturbed state with simultaneous quantitative mapping of multiple parameters, and data interpretation and analysis. The protocol, which can be completed in 1-3 d, enables researchers to image proteins and protein complexes in the native unperturbed state and to simultaneously map their biophysical and biochemical properties at sub-nanometer resolution.

Salt anions promote the conversion of HypF-N into amyloid-like oligomers and modulate the structure of the oligomers and the monomeric precursor state.

An understanding of the solution factors contributing to the rate of aggregation of a protein into amyloid oligomers, to the modulation of the conformational state populated prior to aggregation and to the structure/morphology of the resulting oligomers is one of the goals of present research in this field. We have studied the influence of six different salts on the conversion of the N-terminal domain of Escherichiacoli HypF (HypF-N) into amyloid-like oligomers under conditions of acidic pH. Our results show that salts having different anions (NaCl, NaClO(4), NaI, Na(2)SO(4)) accelerate oligomerization with an efficacy that follows the electroselectivity series of the anions (SO(4)(2-)≥ ClO(4)(-)>I(-)>Cl(-)). By contrast, salts with different cations (NaCl, LiCl, KCl) have similar effects. We also investigated the effect of salts on the structure of the final and initial states of HypF-N aggregation. The electroselectivity series does not apply to the effect of anions on the structure of the oligomers. By contrast, it applies to their effect on the content of secondary structure and on the exposure of hydrophobic clusters of the monomeric precursor state. The results therefore indicate that the binding of anions to the positively charged residues of HypF-N at low pH is the mechanism by which salts modulate the rate of oligomerization and the structure of the monomeric precursor state but not the structure of the resulting oligomers. Overall, the data contribute to rationalize the effect of salts on amyloid-like oligomer formation and to explain the role of charged biological macromolecules in protein aggregation processes.