In the past few years there has been a growth in the use of nanoparticles for stabilizing lipid membranes that contain embedded proteins. These bionanoparticles provide a solution to the challenging ...problem of membrane protein isolation by maintaining a lipid bilayer essential to protein integrity and activity. We have previously described the use of an amphipathic polymer （poly（styrene-co-maleic add）, SMA） to produce discoidal nanoparticles with a lipid bilayer core containing the embedded protein. However the structure of the nanoparticle itself has not yet been determined. This leaves a major gap in understanding how the SMA stabilizes the encapsulated bilayer and how the bilayer relates physically and structurally to an unencapsulated lipid bilayer. In this paper we address this issue by describing the structure of the SMA lipid particle （SMALP） using data from small angle neutron scattering （SANS）, electron microscopy （EM）, attenuated total reflection Fourier transform infrared spectroscopy （ATR-FTIR）, differential scanning calorimetry （DSC） and nuclear magnetic resonance spectroscopy （NMR）. We show that the particle is disc shaped containing a polymer ＂bracelet＂ encircling the lipid bilayer. The structure and orientation of the individual components within the bilayer and polymer are determined showing that styrene moieties within SMA intercalate between the lipid acyl chains. The dimensions of the encapsulated bilayer are also determined and match those measured for a natural membrane. Taken together, the description of the structure of the SMALP forms the foundation for future development and applications of SMALPs in membrane protein production and analysis.
ABC (ATP-binding-cassette) transporters carry out many vital functions and are involved in numerous diseases, but study of the structure and function of these proteins is often hampered by their ...large size and membrane location. Membrane protein purification usually utilizes detergents to solubilize the protein from the membrane, effectively removing it from its native lipid environment. Subsequently, lipids have to be added back and detergent removed to reconstitute the protein into a lipid bilayer. In the present study, we present the application of a new methodology for the extraction and purification of ABC transporters without the use of detergent, instead, using a copolymer, SMA (polystyrene-co-maleic acid). SMA inserts into a bilayer and assembles into discrete particles, essentially solubilizing the membrane into small discs of bilayer encircled by a polymer, termed SMALPs (SMA lipid particles). We show that this polymer can extract several eukaryotic ABC transporters, P-glycoprotein (ABCB1), MRP1 (multidrug-resistance protein 1; ABCC1), MRP4 (ABCC4), ABCG2 and CFTR (cystic fibrosis transmembrane conductance regulator; ABCC7), from a range of different expression systems. The SMALP-encapsulated ABC transporters can be purified by affinity chromatography, and are able to bind ligands comparably with those in native membranes or detergent micelles. A greater degree of purity and enhanced stability is seen compared with detergent solubilization. The present study demonstrates that eukaryotic ABC transporters can be extracted and purified without ever being removed from their lipid bilayer environment, opening up a wide range of possibilities for the future study of their structure and function.
G-protein coupled receptors (GPCRs) constitute the largest class of membrane proteins and are a major drug target. A serious obstacle to studying GPCR structure/function characteristics is the ...requirement to extract the receptors from their native environment in the plasma membrane, coupled with the inherent instability of GPCRs in the detergents required for their solubilization. In the present study, we report the first solubilization and purification of a functional GPCR human adenosine A2A receptor (A2AR), in the total absence of detergent at any stage, by exploiting spontaneous encapsulation by styrene maleic acid (SMA) co-polymer direct from the membrane into a nanoscale SMA lipid particle (SMALP). Furthermore, the A2AR-SMALP, generated from yeast (Pichia pastoris) or mammalian cells, exhibited increased thermostability (~5°C) compared with detergent DDM (n-dodecyl-β-D-maltopyranoside)-solubilized A2AR controls. The A2AR-SMALP was also stable when stored for prolonged periods at 4°C and was resistant to multiple freeze-thaw cycles, in marked contrast with the detergent-solubilized receptor. These properties establish the potential for using GPCR-SMALP in receptor-based drug discovery assays. Moreover, in contrast with nanodiscs stabilized by scaffold proteins, the non-proteinaceous nature of the SMA polymer allowed unobscured biophysical characterization of the embedded receptor. Consequently, CD spectroscopy was used to relate changes in secondary structure to loss of ligand binding ((3)HZM241385) capability. SMALP-solubilization of GPCRs, retaining the annular lipid environment, will enable a wide range of therapeutic targets to be prepared in native-like state to aid drug discovery and understanding of GPCR molecular mechanisms.
In bacteria, the translocation of proteins across the cytoplasmic membrane by the Sec machinery requires the ATPase SecA. SecA binds ribosomes and recognises nascent substrate proteins, but the ...molecular mechanism of nascent substrate recognition is unknown. We investigated the role of the C-terminal tail (CTT) of SecA in nascent polypeptide recognition. The CTT consists of a flexible linker (FLD) and a small metal-binding domain (MBD). Phylogenetic analysis and ribosome binding experiments indicated that the MBD interacts with 70S ribosomes. Disruption of the MBD only or the entire CTT had opposing effects on ribosome binding, substrate-protein binding, ATPase activity and in vivo function, suggesting that the CTT influences the conformation of SecA. Site-specific crosslinking indicated that F399 in SecA contacts ribosomal protein uL29, and binding to nascent chains disrupts this interaction. Structural studies provided insight into the CTT-mediated conformational changes in SecA. Our results suggest a mechanism for nascent substrate protein recognition.
The EvgS/EvgA two-component system of
is activated in response to low pH and alkali metals and regulates many genes, including those for the glutamate-dependent acid resistance system and a number of ...efflux pumps. EvgS, the sensor kinase, is one of five unconventional histidine kinases (HKs) in
and has a large periplasmic domain and a cytoplasmic PAS domain in addition to phospho-acceptor, HK and dimerization, internal receiver, and phosphotransfer domains. Mutations that constitutively activate the protein at pH 7 map to the PAS domain. Here, we built a homology model of the periplasmic region of EvgS, based on the structure of the equivalent region of the BvgS homologue, to guide mutagenesis of potential key residues in this region. We show that histidine 226 is required for induction and that it is structurally colocated with a proline residue (P522) at the top of the predicted transmembrane helix that is expected to play a key role in passing information to the cytoplasmic domains. We also show that the constitutive mutations in the PAS domain can be further activated by low external pH. Expression of the cytoplasmic part of the protein alone also gives constitutive activation, which is lost if the constitutive PAS mutations are present. These findings are consistent with a model in which EvgS senses both external and internal pH and is activated by a shift from a tight inactive to a weak active dimer, and we present an analysis of the purified cytoplasmic portion of EvgS that supports this.
One of the ways bacteria sense their environment is through two-component systems, which have one membrane-bound protein to do the sensing and another inside the cell to turn genes on or off in response to what the membrane-bound protein has detected. The membrane-bound protein must thus be able to detect the stress and signal this detection event to the protein inside the cell. To understand this process, we studied a protein that helps
to survive exposure to low pH, which it must do before taking up residence in the gastrointestinal tract. We describe a predicted structure for the main sensing part of the protein and identify some key residues within it that are involved in the sensing and signaling processes. We propose a mechanism for how the protein may become activated and present some evidence to support our proposal.
Bacterial proteins with MCE domains were first described as being important for Mammalian Cell Entry. More recent evidence suggests they are components of lipid ABC transporters. In Escherichia coli, ...the single-domain protein MlaD is known to be part of an inner membrane transporter that is important for maintenance of outer membrane lipid asymmetry. Here we describe two multi MCE domain-containing proteins in Escherichia coli, PqiB and YebT, the latter of which is an orthologue of MAM-7 that was previously reported to be an outer membrane protein. We show that all three MCE domain-containing proteins localise to the inner membrane. Bioinformatic analyses revealed that MCE domains are widely distributed across bacterial phyla but multi MCE domain-containing proteins evolved in Proteobacteria from single-domain proteins. Mutants defective in mlaD, pqiAB and yebST were shown to have distinct but partially overlapping phenotypes, but the primary functions of PqiB and YebT differ from MlaD. Complementing our previous findings that all three proteins bind phospholipids, results presented here indicate that multi-domain proteins evolved in Proteobacteria for specific functions in maintaining cell envelope homeostasis.
The transmembrane domain proteins of the claudin superfamily are the major structural components of cellular tight junctions. One family member, claudin-1, also associates with tetraspanin CD81 as ...part of a receptor complex that is essential for hepatitis C virus (HCV) infection of the liver. To understand the molecular basis of claudin-1/CD81 association we previously produced and purified milligram quantities of functional, full-length CD81, which binds a soluble form of HCV E2 glycoprotein (sE2). Here we report the production, purification and characterization of claudin-1. Both yeast membrane-bound and detergent-extracted, purified claudin-1 were antigenic and recognized by specific antibodies. Analytical ultracentrifugation demonstrated that extraction with n-octyl-β-d-glucopyranoside yielded monodispersed, dimeric pools of claudin-1 while extraction with profoldin-8 or n-decylphosphocholine yielded a dynamic mixture of claudin-1 oligomers. Neither form bound sE2 in line with literature expectations, while further functional analysis was hampered by the finding that incorporation of claudin-1 into proteoliposomes rendered them intractable to study. Dynamic light scattering demonstrated that claudin-1 oligomers associate with CD81 in vitro in a defined molar ratio of 1∶2 and that complex formation was enhanced by the presence of cholesteryl hemisuccinate. Attempts to assay the complex biologically were limited by our finding that claudin-1 affects the properties of proteoliposomes. We conclude that recombinant, correctly-folded, full-length claudin-1 can be produced in yeast membranes, that it can be extracted in different oligomeric forms that do not bind sE2 and that a dynamic preparation can form a specific complex with CD81 in vitro in the absence of any other cellular components. These findings pave the way for the structural characterization of claudin-1 alone and in complex with CD81.
Tetraspanins are small transmembrane proteins, found in all higher eukaryotes, that compartmentalize cellular membranes through interactions with partner proteins. CD81 is a prototypical tetraspanin ...and contributes to numerous physiological and pathological processes, including acting as a critical entry receptor for hepatitis C virus (HCV). Antibody engagement of tetraspanins can induce a variety of effects, including actin cytoskeletal rearrangements, activation of MAPK-ERK signaling and cell migration. However, the epitope specificity of most anti-tetraspanin antibodies is not known, limiting mechanistic interpretation of these studies.
We generated a panel of monoclonal antibodies (mAbs) specific for CD81 second extracellular domain (EC2) and performed detailed epitope mapping with a panel of CD81 mutants. All mAbs were screened for their ability to inhibit HCV infection and E2-CD81 association. Nanoscale distribution of cell surface CD81 was investigated by scanning electron microscopy.
The antibodies were classified in two epitope groups targeting opposing sides of EC2. We observed a wide range of anti-HCV potencies that were independent of their epitope grouping, but associated with their relative affinity for cell-surface expressed CD81. Scanning electron microscopy identified at least two populations of CD81; monodisperse and higher-order assemblies, consistent with tetraspanin-enriched microdomains.
These novel antibodies provide well-characterised tools to investigate CD81 function, including HCV entry, and have the potential to provide insights into tetraspanin biology in general.
Despite the great importance of membrane proteins, structural and functional studies of these proteins present major challenges. A significant hurdle is the extraction of the functional protein from ...its natural lipid membrane. Traditionally achieved with detergents, purification procedures can be costly and time consuming. A critical flaw with detergent approaches is the removal of the protein from the native lipid environment required to maintain functionally stable protein. This protocol describes the preparation of styrene maleic acid (SMA) co-polymer to extract membrane proteins from prokaryotic and eukaryotic expression systems. Successful isolation of membrane proteins into SMA lipid particles (SMALPs) allows the proteins to remain with native lipid, surrounded by SMA. We detail procedures for obtaining 25 g of SMA (4 d); explain the preparation of protein-containing SMALPs using membranes isolated from Escherichia coli (2 d) and control protein-free SMALPS using E. coli polar lipid extract (1-2 h); investigate SMALP protein purity by SDS-PAGE analysis and estimate protein concentration (4 h); and detail biophysical methods such as circular dichroism (CD) spectroscopy and sedimentation velocity analytical ultracentrifugation (svAUC) to undertake initial structural studies to characterize SMALPs (∼2 d). Together, these methods provide a practical tool kit for those wanting to use SMALPs to study membrane proteins.
SecA is an essential component of the Sec machinery in bacteria, which is responsible for transporting proteins across the cytoplasmic membrane. Recent work from our laboratory indicates that SecA ...binds to ribosomes. Here, we used two different approaches to demonstrate that SecA also interacts with nascent polypeptides in vivo and that these polypeptides are Sec substrates. First, we photo-cross-linked SecA to ribosomes in vivo and identified mRNAs that copurify with SecA. Microarray analysis of the copurifying mRNAs indicated a strong enrichment for proteins containing Sec-targeting sequences. Second, we used a 2-dimensional (2-D) gel approach to analyze radioactively labeled nascent polypeptides that copurify with SecA, including maltose binding protein, a well-characterized SecA substrate. The interaction of SecA with nascent chains was not strongly affected in cells lacking SecB or trigger factor, both of which also interact with nascent Sec substrates. Indeed, the ability of SecB to interact with nascent chains was disrupted in strains in which the interaction between SecA and the ribosome was defective. Analysis of the interaction of SecA with purified ribosomes containing arrested nascent chains in vitro indicates that SecA can begin to interact with a variety of nascent chains when they reach a length of ∼110 amino acids, which is considerably shorter than the length required for interaction with SecB. Our results suggest that SecA cotranslationally recognizes nascent Sec substrates and that this recognition could be required for the efficient delivery of these proteins to the membrane-embedded Sec machinery.
SecA is an ATPase that provides the energy for the translocation of proteins across the cytoplasmic membrane by the Sec machinery in bacteria. The translocation of most of these proteins is uncoupled from protein synthesis and is frequently described as "posttranslational." Here, we show that SecA interacts with nascent Sec substrates. This interaction is not dependent on SecB or trigger factor, which also interact with nascent Sec substrates. Moreover, the interaction of SecB with nascent polypeptides is dependent on the interaction of SecA with the ribosome, suggesting that interaction of the nascent chain with SecA precedes interaction with SecB. Our results suggest that SecA could recognize substrate proteins cotranslationally in order to efficiently target them for uncoupled protein translocation.
PDF
