The ß2-adrenergic receptor associates with CXCR4 multimers in human cancer cells.

While the existence and functional role of class C G-protein-coupled receptors (GPCR) dimers is well established, there is still a lack of consensus regarding class A and B GPCR multimerization. This lack of consensus is largely due to the inherent challenges of demonstrating the presence of multimeric receptor complexes in a physiologically relevant cellular context. The C-X-C motif chemokine receptor 4 (CXCR4) is a class A GPCR that is a promising target of anticancer therapy. Here, we investigated the potential of CXCR4 to form multimeric complexes with other GPCRs and characterized the relative size of the complexes in a live-cell environment. Using a bimolecular fluorescence complementation (BiFC) assay, we identified the ß2 adrenergic receptor (ß2AR) as an interaction partner. To investigate the molecular scale details of CXCR4-ß2AR interactions, we used a time-resolved fluorescence spectroscopy method called pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). PIE-FCCS can resolve membrane protein density, diffusion, and multimerization state in live cells at physiological expression levels. We probed CXCR4 and ß2AR homo- and heteromultimerization in model cell lines and found that CXCR4 assembles into multimeric complexes larger than dimers in MDA-MB-231 human breast cancer cells and in HCC4006 human lung cancer cells. We also found that ß2AR associates with CXCR4 multimers in MDA-MB-231 and HCC4006 cells to a higher degree than in COS-7 and CHO cells and in a ligand-dependent manner. These results suggest that CXCR4-ß2AR heteromers are present in human cancer cells and that GPCR multimerization is significantly affected by the plasma membrane environment.

Engineering transmembrane signal transduction in synthetic membranes using two-component systems.

Cells use signal transduction across their membranes to sense and respond to a wide array of chemical and physical signals. Creating synthetic systems which can harness cellular signaling modalities promises to provide a powerful platform for biosensing and therapeutic applications. As a first step toward this goal, we investigated how bacterial two-component systems (TCSs) can be leveraged to enable transmembrane-signaling with synthetic membranes. Specifically, we demonstrate that a bacterial two-component nitrate-sensing system (NarX-NarL) can be reproduced outside of a cell using synthetic membranes and cell-free protein expression systems. We find that performance and sensitivity of the TCS can be tuned by altering the biophysical properties of the membrane in which the histidine kinase (NarX) is integrated. Through protein engineering efforts, we modify the sensing domain of NarX to generate sensors capable of detecting an array of ligands. Finally, we demonstrate that these systems can sense ligands in relevant sample environments. By leveraging membrane and protein design, this work helps reveal how transmembrane sensing can be recapitulated outside of the cell, adding to the arsenal of deployable cell-free systems primed for real world biosensing.

Polybasic Patches in Both C2 Domains of Synaptotagmin-1 Are Required for Evoked Neurotransmitter Release.

Synaptotagmin-1 (Syt1) is a vesicular calcium sensor required for synchronous neurotransmitter release, composed of a single-pass transmembrane domain linked to two C2 domains (C2A and C2B) that bind calcium, acidic lipids, and SNARE proteins that drive fusion of the synaptic vesicle with the plasma membrane. Despite its essential role, how Syt1 couples calcium entry to synchronous release is poorly understood. Calcium binding to C2B is critical for synchronous release, and C2B additionally binds the SNARE complex. The C2A domain is also required for Syt1 function, but it is not clear why. Here, we asked what critical feature of C2A may be responsible for its functional role and compared this to the analogous feature in C2B. We focused on highly conserved poly-lysine patches located on the sides of C2A (K189-192) and C2B (K324-327). We tested effects of charge-neutralization mutations in either region (Syt1K189-192A and Syt1K326-327A) side by side to determine their relative contributions to Syt1 function in cultured cortical neurons from mice of either sex and in single-molecule experiments. Combining electrophysiological recordings and optical tweezers measurements to probe dynamic single C2 domain-membrane interactions, we show that both C2A and C2B polybasic patches contribute to membrane binding, and both are required for evoked release. The size of the readily releasable vesicle pool and the rate of spontaneous release were unaffected, so both patches are likely required specifically for synchronization of release. We suggest these patches contribute to cooperative membrane binding, increasing the overall affinity of Syt1 for negatively charged membranes and facilitating evoked release.SIGNIFICANCE STATEMENT Synaptotagmin-1 is a vesicular calcium sensor required for synchronous neurotransmitter release. Its tandem cytosolic C2 domains (C2A and C2B) bind calcium, acidic lipids, and SNARE proteins that drive fusion of the synaptic vesicle with the plasma membrane. How calcium binding to Synaptotagmin-1 leads to release and the relative contributions of the C2 domains are unclear. Combining electrophysiological recordings from cultured neurons and optical tweezers measurements of single C2 domain-membrane interactions, we show that conserved polybasic regions in both domains contribute to membrane binding cooperatively, and both are required for evoked release, likely by increasing the overall affinity of Synaptotagmin-1 for acidic membranes.

MEMBRANE PROTEIN STRUCTURES AND INTERACTIONS FROM COVALENT LABELING COUPLED WITH MASS SPECTROMETRY.

Membrane proteins are incredibly important biomolecules because they mediate interactions between a cell's external and internal environment. Obtaining information about membrane protein structure and interactions is thus important for understanding these essential biomolecules. Compared with the analyses of water-soluble proteins, the structural analysis of membrane proteins is more challenging owing to their unique chemical properties and the presence of lipid components that are necessary to solubilize them. The combination of covalent labeling (CL) and mass spectrometry (MS) has recently been applied with great success to study membrane protein structure and interactions. These studies have demonstrated the many advantages that CL-MS methods have over other traditional biophysical techniques. In this review, we discuss both amino acid-specific and non-specific labeling approaches and the special considerations needed to address the unique challenges associated with interrogating membrane proteins. This review highlights the aspects of this approach that require special care to be applied correctly and provides a comprehensive review of the membrane protein systems that have been studied by CL-MS. © 2020 John Wiley & Sons Ltd. Mass Spec Rev.

Cholesterol-dependent cytolysins: The outstanding questions.

The cholesterol-dependent cytolysins (CDCs) are a major family of bacterial pore-forming proteins secreted as virulence factors by Gram-positive bacterial species. CDCs are produced as soluble, monomeric proteins that bind specifically to cholesterol-rich membranes, where they oligomerize into ring-shaped pores of more than 30 monomers. Understanding the details of the steps the toxin undergoes in converting from monomer to a membrane-spanning pore is a continuing challenge. In this review we summarize what we know about CDCs and highlight the remaining outstanding questions that require answers to obtain a complete picture of how these toxins kill cells.

The Mechanics and Thermodynamics of Tubule Formation in Biological Membranes.

Membrane tubulation is a ubiquitous process that occurs both at the plasma membrane and on the membranes of intracellular organelles. These tubulation events are known to be mediated by forces applied on the membrane either due to motor proteins, by polymerization of the cytoskeleton, or due to the interactions between membrane proteins binding onto the membrane. The numerous experimental observations of tube formation have been amply supported by mathematical modeling of the associated membrane mechanics and have provided insights into the force-displacement relationships of membrane tubes. Recent advances in quantitative biophysical measurements of membrane-protein interactions and tubule formation have necessitated the need for advances in modeling that will account for the interplay of multiple aspects of physics that occur simultaneously. Here, we present a comprehensive review of experimental observations of tubule formation and provide context from the framework of continuum modeling. Finally, we explore the scope for future research in this area with an emphasis on iterative modeling and experimental measurements that will enable us to expand our mechanistic understanding of tubulation processes in cells.

Investigation of the conserved reentrant membrane helix in the monotopic phosphoglycosyl transferase superfamily supports key molecular interactions with polyprenol phosphate substrates.

Long-chain polyprenol phosphates feature in membrane-associated glycoconjugate biosynthesis pathways across domains of life. These unique amphiphilic molecules are best known as substrates of polytopic membrane proteins, including polyprenol-phosphate phosphoglycosyl and glycosyl transferases, and as components of more complex substrates. The linear polyprenols are constrained by double bond geometry and lend themselves well to interactions with polytopic membrane proteins, in which multiple transmembrane helices form a rich landscape for interactions. Recently, a new superfamily of monotopic phosphoglycosyl transferase enzymes has been identified that interacts with polyprenol phosphate substrates via a single reentrant membrane helix. Intriguingly, despite the dramatic differences in their membrane-interaction domains, both polytopic and monotopic enzymes similarly favor a unique cis/trans geometry in their polyprenol phosphate substrates. Herein, we present a multipronged biochemical and biophysical study of PglC, a monotopic phosphoglycosyl transferase that catalyzes the first membrane-committed step in N-linked glycoprotein biosynthesis in Campylobacter jejuni. We probe the significance of polyprenol phosphate geometry both in mediating substrate binding to PglC and in modulating the local membrane environment. Geometry is found to be important for binding to PglC; a conserved proline residue in the reentrant membrane helix is determined to drive polyprenol phosphate recognition and specificity. Pyrene fluorescence studies show that polyprenol phosphates at physiologically-relevant levels increase the disorder of the local lipid bilayer; however, this effect is confined to polyprenol phosphates with specific isoprene geometries. The molecular insights from this study may shed new light on the interactions of polyprenol phosphates with diverse membrane-associated proteins in glycoconjugate biosynthesis.

Quaternary structure of the yeast pheromone receptor Ste2 in living cells.

Transmembrane proteins known as G protein-coupled receptors (GPCRs) have been shown to form functional homo- or hetero-oligomeric complexes, although agreement has been slow to emerge on whether homo-oligomerization plays functional roles. Here we introduce a platform to determine the identity and abundance of differing quaternary structures formed by GPCRs in living cells following changes in environmental conditions, such as changes in concentrations. The method capitalizes on the intrinsic capability of FRET spectrometry to extract oligomer geometrical information from distributions of FRET efficiencies (or FRET spectrograms) determined from pixel-level imaging of cells, combined with the ability of the statistical ensemble approaches to FRET to probe the proportion of different quaternary structures (such as dimers, rhombus or parallelogram shaped tetramers, etc.) from averages over entire cells. Our approach revealed that the yeast pheromone receptor Ste2 forms predominantly tetramers at average expression levels of 2 to 25 molecules per pixel (2.8·10-6 to 3.5·10-5molecules/nm2), and a mixture of tetramers and octamers at expression levels of 25-100 molecules per pixel (3.5·10-5 to 1.4·10-4molecules/nm2). Ste2 is a class D GPCR found in the yeast Saccharomyces cerevisiae of the mating type a, and binds the pheromone α-factor secreted by cells of the mating type α. Such investigations may inform development of antifungal therapies targeting oligomers of pheromone receptors. The proposed FRET imaging platform may be used to determine the quaternary structure sub-states and stoichiometry of any GPCR and, indeed, any membrane protein in living cells. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova.

Modeling the mechanochemical feedback for membrane-protein interactions using a continuum mesh model.

The Helfrich free energy is widely used to model the generation of membrane curvature due to different physical and chemical components. The governing equations resulting from the energy minimization procedure are a system of coupled higher order partial differential equations. Simulations of membrane deformation for obtaining quantitative comparisons against experimental observations require computational schemes that will allow us to solve these equations without restrictions to axisymmetric coordinates. Here, we describe one such tool that we developed in our group based on discrete differential geometry to solve these equations along with examples.

Applications of phase-separating multi-bilayers in protein-membrane domain interactions.

Synthetic model membranes are important tools to elucidate lipid domain and protein interactions due to predefined lipid compositions and characterizable biophysical properties. Here, we introduce a model membrane with multiple lipid bilayers (multi-bilayers) stacked on a mica substrate that is prepared through a spin-coating technique. The spin-coated multi-bilayers are useful in the study of phase separated membranes with a high cholesterol content, mobile lipids, microscopic and reversible phase separation, and easy conjugation with proteins, which make them a good model to study interactions between proteins and membrane domains.

Tuning Chemoreceptor Signaling by Positioning Aromatic Residues at the Lipid-Aqueous Interface.

Aromatic tuning facilitates stimulus-independent modulation of receptor output. The methodology is based upon the affinity of amphipathic aromatic residues, namely Trp and Tyr, for the polar-hydrophobic interfaces found within biological membranes. Here, we describe the application of aromatic tuning within the aspartate chemoreceptor of Escherichia coli (Tar). We have also employed the method within other related proteins, such as sensor histidine kinases (SHKs), and therefore hope that other research groups find it useful to modulate signal output from their receptor of interest.

Lipopeptide daptomycin: Interactions with bacterial and phospholipid membranes, stability of membrane aggregates and micellation in solution.

Daptomycin is a cyclic lipopeptide effective against multidrug Gram-positive bacteria. Despite having a net negative charge, it is selective against negatively charged bacterial membranes. It has been established that daptomycin's antibiotic activity is based on directly targeting the bacterial membranes and that this antibacterial activity depends on calcium ions. Importantly, however, both the precise role of ions and the physical mechanisms responsible for daptomycin's action remain poorly understood. We investigate these issues using three types of molecular dynamics simulations: umbrella sampling free energy calculations for a single daptomycin, unbiased simulations for daptomycin tetramers, and unbiased simulations of micellation of daptomycin both in the absence and presence of calcium ions. The simulations are in the excess of 4 µs. As the most important finding, we establish that binding of the calcium ions on the aspartic acid residues is the key to stabilizing daptomycin tetramers inside the model material membrane. These complexes are vital for daptomycin's antibacterial activity. In the absence of binding, the tetramer is not stable and moves slowly out of the membrane. We also demonstrate that in solution, micellation of daptomycin occurs both in the presence and absence of calcium ions, and discuss the similarities between the behaviors of daptomycin and amyloid peptides in membranes.

Cholesterol-dependent cytolysins: from water-soluble state to membrane pore.

The cholesterol-dependent cytolysins (CDCs) are a family of bacterial toxins that are important virulence factors for a number of pathogenic Gram-positive bacterial species. CDCs are secreted as soluble, stable monomeric proteins that bind specifically to cholesterol-rich cell membranes, where they assemble into well-defined ring-shaped complexes of around 40 monomers. The complex then undergoes a concerted structural change, driving a large pore through the membrane, potentially lysing the target cell. Understanding the details of this process as the protein transitions from a discrete monomer to a complex, membrane-spanning protein machine is an ongoing challenge. While many of the details have been revealed, there are still questions that remain unanswered. In this review, we present an overview of some of the key features of the structure and function of the CDCs, including the structure of the secreted monomers, the process of interaction with target membranes, and the transition from bound monomers to complete pores. Future directions in CDC research and the potential of CDCs as research tools will also be discussed.

Nanotechnology in Auditory Research: Membrane Electromechanics in Hearing.

The soft, thin membranes that envelop all living cells are 2D, nanoscale, fluid assemblies of phospholipids, sterols, proteins, and other molecules. Mechanical interactions between these components facilitate membrane function, a key example of which is ion flow mediated by the mechanical opening and closing of channels. Hearing and balance are initiated by the modulation of ion flow through mechanoreceptor channels in stereocilia membranes. Cochlear amplification by the outer hair cell involves modulation of ion movement by the membrane protein prestin. Voltage-gated ion channels shape the receptor potential in hair cells and are responsible for the initiation of action potentials that are at the heart of sensory processing in the brain. All three processes require a membrane and their kinetics are modulated by the mechanical (i.e., material) properties of the membrane. This chapter reviews the methodology for measuring the mechanics of cellular membranes and introduces a method for examining membrane electromechanics. The approach allows examination of electromechanically mediated interactions between the different molecular species in the membrane that contribute to the biology of hearing and balance.

Membrane lipid therapy: Modulation of the cell membrane composition and structure as a molecular base for drug discovery and new disease treatment.

Nowadays we understand cell membranes not as a simple double lipid layer but as a collection of complex and dynamic protein-lipid structures and microdomains that serve as functional platforms for interacting signaling lipids and proteins. Membrane lipids and lipid structures participate directly as messengers or regulators of signal transduction. In addition, protein-lipid interactions participate in the localization of signaling protein partners to specific membrane microdomains. Thus, lipid alterations change cell signaling that are associated with a variety of diseases including cancer, obesity, neurodegenerative disorders, cardiovascular pathologies, etc. This article reviews the newly emerging field of membrane lipid therapy which involves the pharmacological regulation of membrane lipid composition and structure for the treatment of diseases. Membrane lipid therapy proposes the use of new molecules specifically designed to modify membrane lipid structures and microdomains as pharmaceutical disease-modifying agents by reversing the malfunction or altering the expression of disease-specific protein or lipid signal cascades. Here, we provide an in-depth analysis of this emerging field, especially its molecular bases and its relevance to the development of innovative therapeutic approaches.