Stefania A. Mari

Gating of the MlotiK1 potassium channel involves large rearrangements of the cyclic nucleotide-binding domains

Cyclic nucleotide-regulated ion channels are present in bacteria, plants, vertebrates, and humans. In higher organisms, they are closely involved in signaling networks of vision and olfaction. Binding of cAMP or cGMP favors the activation of these ion channels. Despite a wealth of structural and studies, there is a lack of structural data describing the gating process in a full-length cyclic nucleotide-regulated channel. We used high-resolution atomic force microscopy (AFM) to directly observe the conformational change of the membrane embedded bacterial cyclic nucleotide-regulated channel MlotiK1. In the nucleotide-bound conformation, the cytoplasmic cyclic nucleotide-binding (CNB) domains of MlotiK1 are disposed in a fourfold symmetric arrangement forming a pore-like vestibule. Upon nucleotide-unbinding, the four CNB domains undergo a large rearrangement, stand up by ∼1.7 nm, and adopt a structurally variable grouped conformation that closes the cytoplasmic vestibule. This fully reversible conformational change provides insight into how CNB domains rearrange when regulating the potassium channel.

pH-Induced Conformational Change of the β-Barrel-Forming Protein OmpG Reconstituted into Native E. coli Lipids

A gating mechanism of the beta-barrel-forming outer membrane protein G (OmpG) from Escherichia coli was recently presented. The mechanism was based on X-ray structures revealed from crystals grown from solubilized OmpG at both neutral pH and acidic pH. To investigate whether these conformations represent the naturally occurring gating mechanism, we reconstituted OmpG in native E. coli lipids and applied high-resolution atomic force microscopy. The reconstituted OmpG molecules assembled into both monomers and dimers. Single monomeric and dimeric OmpG molecules showed open channel entrances at pH 7.5 and at room temperature. The extracellular loops connecting the beta-strands that form the transmembrane beta-barrel pore exhibited elevated structural flexibility. Upon lowering the pH to 5.0, the conformation of OmpG molecules changed to close the extracellular entrance of their channel. It appears that one or more of the extracellular loops collapsed onto the channel entrance. This conformational change was fully reversible. Our data confirm that the previously reported gating mechanism of OmpG occurs at physiological conditions in E. coli lipid membranes.

pH-Dependent Interactions Guide the Folding and Gate the Transmembrane Pore of the β-Barrel Membrane Protein OmpG

The physical interactions that switch the functional state of membrane proteins are poorly understood. Previously, the pH-gating conformations of the beta-barrel forming outer membrane protein G (OmpG) from Escherichia coli have been solved. When the pH changes from neutral to acidic the flexible extracellular loop L6 folds into and closes the OmpG pore. Here, we used single-molecule force spectroscopy to structurally localize and quantify the interactions that are associated with the pH-dependent closure. At acidic pH, we detected a pH-dependent interaction at loop L6. This interaction changed the (un)folding of loop L6 and of beta-strands 11 and 12, which connect loop L6. All other interactions detected within OmpG were unaffected by changes in pH. These results provide a quantitative and mechanistic explanation of how pH-dependent interactions change the folding of a peptide loop to gate the transmembrane pore. They further demonstrate how the stability of OmpG is optimized so that pH changes modify only those interactions necessary to gate the transmembrane pore.

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.

Substrate-induced changes in the structural properties of LacY

Significance Lactose permease of Escherichia coli (LacY), a model for the major facilitator superfamily, catalyzes galactopyranoside/H+ symport across the membrane by a mechanism in which large conformational changes expose the sugar-binding site in the middle of the molecule alternatively to either side of the membrane (an alternating access mechanism). Despite substantial progress with respect to static X-ray crystal structures of LacY, the dynamics of the transport mechanism are not fully understood. Here we use dynamic single-molecule force spectroscopy to quantify the structural properties that change upon substrate binding. The results reveal very significant changes in conformational, kinetic, energetic, and mechanical properties primarily in the N-terminal 6-helix bundle, while the C-terminal 6-helix bundle remains largely unaffected. The lactose permease (LacY) of Escherichia coli, a paradigm for the major facilitator superfamily, catalyzes the coupled stoichiometric translocation of a galactopyranoside and an H+ across the cytoplasmic membrane. To catalyze transport, LacY undergoes large conformational changes that allow alternating access of sugar- and H+-binding sites to either side of the membrane. Despite strong evidence for an alternating access mechanism, it remains unclear how H+- and sugar-binding trigger the cascade of interactions leading to alternating conformational states. Here we used dynamic single-molecule force spectroscopy to investigate how substrate binding induces this phenomenon. Galactoside binding strongly modifies kinetic, energetic, and mechanical properties of the N-terminal 6-helix bundle of LacY, whereas the C-terminal 6-helix bundle remains largely unaffected. Within the N-terminal 6-helix bundle, the properties of helix V, which contains residues critical for sugar binding, change most radically. Particularly, secondary structures forming the N-terminal domain exhibit mechanically brittle properties in the unbound state, but highly flexible conformations in the substrate-bound state with significantly increased lifetimes and energetic stability. Thus, sugar binding tunes the properties of the N-terminal domain to initiate galactoside/H+ symport. In contrast to wild-type LacY, the properties of the conformationally restricted mutant Cys154➝Gly do not change upon sugar binding. It is also observed that the single mutation of Cys154➝Gly alters intramolecular interactions so that individual transmembrane helices manifest different properties. The results support a working model of LacY in which substrate binding induces alternating conformational states and provides insight into their specific kinetic, energetic, and mechanical properties.

Structure and function of the glucose PTS transporter from Escherichia coli

The glucose transporter IICB of the Escherichia coli phosphotransferase system (PTS) consists of a polytopic membrane domain (IIC) responsible for substrate transport and a hydrophilic C-terminal domain (IIB) responsible for substrate phosphorylation. We have overexpressed and purified a triple mutant of IIC (mut-IIC), which had recently been shown to be suitable for crystallization purposes. Mut-IIC was homodimeric as determined by blue native-PAGE and gel-filtration, and had an eyeglasses-like structure as shown by negative-stain transmission electron microscopy (TEM) and single particle analysis. Glucose binding and transport by mut-IIC, mut-IICB and wildtype-IICB were compared with scintillation proximity and in vivo transport assays. Binding was reduced and transport was impaired by the triple mutation. The scintillation proximity assay allowed determination of substrate binding, affinity and specificity of wildtype-IICB by a direct method. 2D crystallization of mut-IIC yielded highly-ordered tubular crystals and made possible the calculation of a projection structure at 12Å resolution by negative-stain TEM. Immunogold labeling TEM revealed the sidedness of the tubular crystals, and high-resolution atomic force microscopy the surface structure of mut-IIC. This work presents the structure of a glucose PTS transporter at the highest resolution achieved so far and sets the basis for future structural studies.

Observing a Lipid-Dependent Alteration in Single Lactose Permeases

Lipids of the Escherichia coli membrane are mainly composed of 70%-80% phosphatidylethanolamine (PE) and 20%-25% phosphatidylglycerol (PG). Biochemical studies indicate that the depletion of PE causes inversion of the N-terminal helix bundle of the lactose permease (LacY), and helix VII becomes extramembranous. Here we study this phenomenon using single-molecule force spectroscopy, which is sensitive to the structure of membrane proteins. In PE and PG at a ratio of 3:1, ∼95% of the LacY molecules adopt a native structure. However, when PE is omitted and the membrane contains PG only, LacY almost equally populates a native and a perturbed conformation. The most drastic changes occur at helices VI and VII and the intervening loop. Since helix VII contains Asp237 and Asp240, zwitterionic PE may suppress electrostatic repulsion between LacY and PG in the PE:PG environment. Thus, PE promotes a native fold and prevents LacY from populating a functionally defective, nonnative conformation.

YidC assists the stepwise and stochastic folding of membrane proteins

How chaperones, insertases and translocases facilitate insertion and folding of complex cytoplasmic proteins into cellular membranes is not fully understood. Here, we utilize single-molecule force spectroscopy to observe YidC, a transmembrane chaperone/insertase, sculpting the folding trajectory of the polytopic α-helical membrane protein lactose permease (LacY). In the absence of YidC, unfolded LacY inserts individual structural segments into the membrane; however, misfolding dominates the process so that folding cannot be completed. YidC prevents LacY from misfolding by stabilizing the unfolded state from which LacY inserts structural segments stepwise into the membrane until folding is completed. During stepwise insertion, YidC and membrane together stabilize the transient folds. Remarkably, the order of insertion of structural segments is stochastic, thereby indicating that LacY can fold along variable pathways towards the native structure. Since YidC is essential in membrane protein biogenesis and LacY a paradigm for the major facilitator superfamily, our observations have general relevance.

Molecular plasticity of the human Voltage-Dependent Anion Channel embedded into a membrane.

The voltage-dependent anion channel (VDAC) regulates the flux of metabolites and ions across the outer mitochondrial membrane. Regulation of ion flow involves conformational transitions in VDAC, but the nature of these changes has not been resolved to date. By combining single-molecule force spectroscopy with nuclear magnetic resonance spectroscopy we show that the β barrel of human VDAC embedded into a membrane is highly flexible. Its mechanical flexibility exceeds by up to one order of magnitude that determined for β strands of other membrane proteins and is largest in the N-terminal part of the β barrel. Interaction with Ca(2+), a key regulator of metabolism and apoptosis, considerably decreases the barrels conformational variability and kinetic free energy in the membrane. The combined data suggest that physiological VDAC function depends on the molecular plasticity of its channel.

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.