Microbial expression systems for membrane proteins.

Despite many high-profile successes, recombinant membrane protein production remains a technical challenge; it is still the case that many fewer membrane protein structures have been published than those of soluble proteins. However, progress is being made because empirical methods have been developed to produce the required quantity and quality of these challenging targets. This review focuses on the microbial expression systems that are a key source of recombinant prokaryotic and eukaryotic membrane proteins for structural studies. We provide an overview of the host strains, tags and promoters that, in our experience, are most likely to yield protein suitable for structural and functional characterization. We also catalogue the detergents used for solubilization and crystallization studies of these proteins. Here, we emphasize a combination of practical methods, not necessarily high-throughput, which can be implemented in any laboratory equipped for recombinant DNA technology and microbial cell culture.

Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo.

This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a so-called fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting. Y-FAST is as bright as common fluorescent proteins, exhibits good photostability, and allows the efficient labeling of proteins in various organelles and hosts. Upon fluorogen binding, fluorescence appears instantaneously, allowing monitoring of rapid processes in near real time. Y-FAST distinguishes itself from other tagging systems because the fluorogen binding is highly dynamic and fully reversible, which enables rapid labeling and unlabeling of proteins by addition and withdrawal of the fluorogen, opening new exciting prospects for the development of multiplexing imaging protocols based on sequential labeling.

The Stable Interaction Between Signal Peptidase LepB of Escherichia coli and Nuclease Bacteriocins Promotes Toxin Entry into the Cytoplasm.

LepB is a key membrane component of the cellular secretion machinery, which releases secreted proteins into the periplasm by cleaving the inner membrane-bound leader. We showed that LepB is also an essential component of the machinery hijacked by the tRNase colicin D for its import. Here we demonstrate that this non-catalytic activity of LepB is to promote the association of the central domain of colicin D with the inner membrane before the FtsH-dependent proteolytic processing and translocation of the toxic tRNase domain into the cytoplasm. The novel structural role of LepB results in a stable interaction with colicin D, with a stoichiometry of 1:1 and a nanomolar Kd determined in vitro. LepB provides a chaperone-like function for the penetration of several nuclease-type bacteriocins into target cells. The colicin-LepB interaction is shown to require only a short peptide sequence within the central domain of these bacteriocins and to involve residues present in the short C-terminal Box E of LepB. Genomic screening identified the conserved LepB binding motif in colicin-like ORFs from 13 additional bacterial species. These findings establish a new paradigm for the functional adaptability of an essential inner-membrane enzyme.

Functional Modulation of a G Protein-Coupled Receptor Conformational Landscape in a Lipid Bilayer.

Mapping the conformational landscape of G protein-coupled receptors (GPCRs), and in particular how this landscape is modulated by the membrane environment, is required to gain a clear picture of how signaling proceeds. To this end, we have developed an original strategy based on solution-state nuclear magnetic resonance combined with an efficient isotope labeling scheme. This strategy was applied to a typical GPCR, the leukotriene B4 receptor BLT2, reconstituted in a lipid bilayer. Because of this, we are able to provide direct evidence that BLT2 explores a complex landscape that includes four different conformational states for the unliganded receptor. The relative distribution of the different states is modulated by ligands and the sterol content of the membrane, in parallel with the changes in the ability of the receptor to activate its cognate G protein. This demonstrates a conformational coupling between the agonist and the membrane environment that is likely to be fundamental for GPCR signaling.

Structure of the prolyl-acyl carrier protein oxidase involved in the biosynthesis of the cyanotoxin anatoxin-a.

Anatoxin-a and homoanatoxin-a are two potent cyanobacterial neurotoxins biosynthesized from L-proline by a short pathway involving polyketide synthases. Proline is first loaded onto AnaD, an acyl carrier protein, and prolyl-AnaD is then oxidized to 1-pyrroline-5-carboxyl-AnaD by a flavoprotein, AnaB. Three polyketide synthases then transform this imine into anatoxin-a or homoanatoxin-a. AnaB was crystallized in its holo form and its three-dimensional structure was determined by X-ray diffraction at 2.8 Šresolution. AnaB is a homotetramer and its fold is very similar to that of the acyl-CoA dehydrogenases (ACADs). The active-site base of AnaB, Glu244, superimposed very well with that of human isovaleryl-CoA dehydrogenase, confirming previous site-directed mutagenesis experiments and mechanistic proposals. The substrate-binding site of AnaB is small and is likely to be fitted for the pyrrolidine ring of proline. However, in contrast to ACADs, which use an electron-transport protein, AnaB uses molecular oxygen as the electron acceptor, as in acyl-CoA oxidases. Calculation of the solvent-accessible surface area around the FAD in AnaB and in several homologues showed that it is significantly larger in AnaB than in its homologues. A protonated histidine near the FAD in AnaB is likely to participate in oxygen activation. Furthermore, an array of water molecules detected in the AnaB structure suggests a possible path for molecular oxygen towards FAD. This is consistent with AnaB being an oxidase rather than a dehydrogenase. The structure of AnaB is the first to be described for a prolyl-ACP oxidase and it will contribute to defining the structural basis responsible for oxygen reactivity in flavoenzymes.

Membrane Protein Production in Escherichia coli: Protocols and Rules.

Despite recent progresses in the use of eukaryotic expression system, production of membrane proteins for structural studies still relies on microbial expression systems. In this review, we provide protocols to achieve high level expression of membrane proteins in Escherichia coli, especially using the T7 RNA polymerase based expression system. From the design of the construct, the choice of the appropriate vector-host combination, the assessment of the bacterial fitness, to the selection of bacterial mutant adapted to the production of the target membrane protein, the chapter covers all necessary methods for a rapid optimization of a specific target membrane protein. In addition, we provide a protocol for membrane protein solubilization based on our recent analysis of the Protein Data Bank.

Exploration of the dynamic interplay between lipids and membrane proteins by hydrostatic pressure.

Cell membranes represent a complex and variable medium in time and space of lipids and proteins. Their physico-chemical properties are determined by lipid components which can in turn influence the biological function of membranes. Here, we used hydrostatic pressure to study the close dynamic relationships between lipids and membrane proteins. Experiments on the ß-barrel OmpX and the α-helical BLT2 G Protein-Coupled Receptor in nanodiscs of different lipid compositions reveal conformational landscapes intimately linked to pressure and lipids. Pressure can modify the conformational landscape of the membrane protein per se, but also increases the gelation of lipids, both being monitored simultaneously at high atomic resolution by NMR. Our study also clearly shows that a membrane protein can modulate, at least locally, the fluidity of the bilayer. The strategy proposed herein opens new perspectives to scrutinize the dynamic interplay between membrane proteins and their surrounding lipids.

Structural models of mitochondrial uncoupling proteins obtained in DPC micelles are not functionally relevant.

Uncoupling protein 1 (UCP1) is found in the inner mitochondrial membrane of brown adipocytes. In the presence of long-chain fatty acids (LCFAs), UCP1 increases the proton conductance, which, in turn, increases fatty acid oxidation and energy release as heat. Atomic models of UCP1 and UCP2 have been generated based on the NMR backbone structure of UCP2 in dodecylphosphocholine (DPC), a detergent known to inactivate UCP1. Based on NMR titration experiments on UCP1 with LCFA, it has been proposed that K56 and K269 are crucial for LCFA binding and UCP1 activation. Given the numerous controversies on the use of DPC for structure-function analyses of membrane proteins, we revisited those UCP1 mutants in a more physiological context by expressing them in the mitochondria of Saccharomyces cerevisiae. Mitochondrial respiration, assayed on permeabilized spheroplasts, enables the determination of UCP1 activation and inhibition. The K56S, K269S, and K56S/K269S mutants did not display any default in activation, which shows that the NMR titration experiments in DPC detergent are not relevant to UCP1 function.

Study of membrane deformations induced by Hepatitis C protein NS4B and its terminal amphipathic peptides.

Many viruses destabilize cellular membranous compartments to form their replication complexes, but the mechanism(s) underlying membrane perturbation remains unknown. Expression in eukaryotic cells of NS4B, a protein of the hepatitis C virus (HCV), alters membranous complexes and induces structures similar to the so-called membranous web that appears crucial to the formation of the HCV replication complex. As over-expression of the protein is lethal to both prokaryotic and eukaryotic cells, NS4B was produced in large quantities in a "cell-free" system in the presence of detergent, after which it was inserted into lipid membranes. X-ray diffraction revealed that NS4B modifies the phase diagram of synthetic lipid aqueous phases considerably, perturbing the transition temperature and cooperativity. Cryo-electron microscopy demonstrated that NS4B introduces significant disorder in the synthetic membrane as well as discontinuities that could be interpreted as due to the formation of pores and membrane merging events. C- and N-terminal fragments of NS4B are both able to destabilize liposomes. While most NS4B amphipathic peptides perforate membranes, one NS4B peptide induces membrane fusion. Cryo-electron microscopy reveals a particular structure that can be interpreted as arising from hemi-fusion-like events. Amphipathic domains are present in many proteins, and if exposed to the aqueous cytoplasmic medium are sufficient to destabilize membranes in order to form viral replication complexes. These domains have important functions in the viral replication cycle, and thus represent potential targets for the development of anti-viral molecules.

Structure of the agonist 12-HHT in its BLT2 receptor-bound state.

G Protein-Coupled receptors represent the main communicating pathway for signals from the outside to the inside of most of eukaryotic cells. They define the largest family of integral membrane receptors at the surface of the cells and constitute the main target of the current drugs on the market. The low affinity leukotriene receptor BLT2 is a receptor involved in pro- and anti-inflammatory pathways and can be activated by various unsaturated fatty acid compounds. We present here the NMR structure of the agonist 12-HHT in its BLT2-bound state and a model of interaction of the ligand with the receptor based on a conformational homology modeling associated with docking simulations. Put into perspective with the data obtained with leukotriene B4, our results illuminate the ligand selectivity of BLT2 and may help define new molecules to modulate the activity of this receptor.

NMR analysis of GPCR conformational landscapes and dynamics.

Understanding the signal transduction mechanism mediated by the G Protein-Coupled Receptors (GPCRs) in eukaryote cells represents one of the main issues in modern biology. At the molecular level, various biophysical approaches have provided important insights on the functional plasticity of these complex allosteric machines. In this context, X-ray crystal structures published during the last decade represent a major breakthrough in GPCR structural biology, delivering important information on the activation process of these receptors through the description of the three-dimensional organization of their active and inactive states. In complement to crystals and cryo-electronic microscopy structures, information on the probability of existence of different GPCR conformations and the dynamic barriers separating those structural sub-states is required to better understand GPCR function. Among the panel of techniques available, nuclear magnetic resonance (NMR) spectroscopy represents a powerful tool to characterize both conformational landscapes and dynamics. Here, we will outline the potential of NMR to address such biological questions, and we will illustrate the functional insights that NMR has brought in the field of GPCRs in the recent years.

Escherichia coli as host for membrane protein structure determination: a global analysis.

The structural biology of membrane proteins (MP) is hampered by the difficulty in producing and purifying them. A comprehensive analysis of protein databases revealed that 213 unique membrane protein structures have been obtained after production of the target protein in E. coli. The primary expression system used was the one based on the T7 RNA polymerase, followed by the arabinose and T5 promoter based expression systems. The C41λ(DE3) and C43λ(DE3) bacterial mutant hosts have contributed to 28% of non E. coli membrane protein structures. A large scale analysis of expression protocols demonstrated a preference for a combination of bacterial host-vector together with a bimodal distribution of induction temperature and of inducer concentration. Altogether our analysis provides a set of rules for the optimal use of bacterial expression systems in membrane protein production.

The PIR domain of Grb14 is an intrinsically unstructured protein: implication in insulin signaling.

Grb14 belongs to the Grb7 family of adapter proteins and was identified as a negative regulator of insulin signal transduction. Its inhibitory effect on the insulin receptor kinase activity is controlled by a newly discovered domain called PIR. To investigate the biochemical and biophysical characteristics of this new domain, we cloned and purified recombinant PIR-SH2, PIR, and SH2 domains. The isolated PIR and PIR-SH2 domains were physiologically active and inhibited insulin-induced reinitiation of meiosis in the Xenopus oocytes system. However, NMR experiments on (15)N-labelled PIR revealed that it did not present secondary structure. These results suggest that the PIR domain belongs to the growing family of intrinsically unstructured proteins.

Regulation and functional roles of Grb14.

Grb14 is the last described member of the Grb7 family of adaptors, containing Grb7, Grb10 and Grb14. These proteins share a series of conserved domains involved in protein-protein and protein-lipid interactions: an amino terminal proline-rich region, a C-terminal SH2 domain, and a central GM region containing a RA, a PH domain, and a newly described PIR (BPS) region. As shown for the other members of the Grb7/10/14 family, Grb14 binds to various receptor tyrosine kinases (RTKs) under ligand induction. This interaction involves the SH2 and PIR domains, and the respective participation of these domains is likely to be a determinant in the specificity of action of Grb14. At the present time, a role for this Grb14-RTK interaction was established only for insulin (IR) and FGF receptors (FGFR). Grb14, through its PIR, is an inhibitor of IR tyrosine kinase activity and thus of insulin effects. Grb14 also decreases FGF signaling, but more probably by interfering with cellular effectors downstream from the receptor. Only a few cytosolic partners of Grb14 are identified. One of them, the adaptor ZIP, allows phosphorylation of Grb14, and regulation of its inhibitory action on IR signaling. The identification of further proteins interacting with Grb14 is required to elucidate the biological role of this protein.

Cyclopiazonic acid is complexed to a divalent metal ion when bound to the sarcoplasmic reticulum Ca2+-ATPase.

We have determined the structure of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) in an E2.P(i)-like form stabilized as a complex with MgF(4)(2-), an ATP analog, adenosine 5'-(beta,gamma-methylene)triphosphate (AMPPCP), and cyclopiazonic acid (CPA). The structure determined at 2.5A resolution leads to a significantly revised model of CPA binding when compared with earlier reports. It shows that a divalent metal ion is required for CPA binding through coordination of the tetramic acid moiety at a characteristic kink of the M1 helix found in all P-type ATPase structures, which is expected to be part of the cytoplasmic cation access pathway. Our model is consistent with the biochemical data on CPA function and provides new measures in structure-based drug design targeting Ca(2+)-ATPases, e.g. from pathogens. We also present an extended structural basis of ATP modulation pinpointing key residues at or near the ATP binding site. A structural comparison to the Na(+),K(+)-ATPase reveals that the Phe(93) side chain occupies the equivalent binding pocket of the CPA site in SERCA, suggesting an important role of this residue in stabilization of the potassium-occluded E2 state of Na(+),K(+)-ATPase.

Dimeric structure of human Na+/H+ exchanger isoform 1 overproduced in Saccharomyces cerevisiae.

The Na(+)/H(+) exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH by extruding an intracellular H(+) in exchange for one extracellular Na(+). The human NHE1 isoform is involved in heart disease and cell growth and proliferation. Although details of NHE1 regulation and transport are being revealed, there is little information available on the structure of the intact protein. In this report, we demonstrate overexpression, purification, and characterization of the human NHE1 (hNHE1) protein in Saccharomyces cerevisiae. Overproduction of the His-tagged protein followed by purification via nickel-nitrilotriacetic acid-agarose chromatography yielded 0.2 mg of pure protein/liter of cell culture. Reconstitution of hNHE1 in proteoliposomes demonstrated that the protein was active and responsive to an NHE1-specific inhibitor. Circular dichroism spectroscopy of purified hNHE1 revealed that the protein contains 41% alpha-helix, 23% beta-sheet, and 36% random coil. Size exclusion chromatography indicated that the protein-detergent micelle was in excess of 200 kDa, consistent with an hNHE1 dimer. Electron microscopy and single particle reconstruction of negatively stained hNHE1 confirmed that the protein was a dimer, with a compact globular domain assigned to the transmembrane region and an apical ridge assigned to the cytoplasmic domain. The transmembrane domain of the hNHE1 reconstruction was clearly dimeric, where each monomer had a size and shape consistent with the predicted 12 membrane-spanning segments for hNHE1.

The molecular basis for cyclopiazonic acid inhibition of the sarcoplasmic reticulum calcium pump.

The sarcoplasmic reticulum Ca(2+)-ATPase is essential for calcium reuptake in the muscle contraction-relaxation cycle. Here we present structures of a calcium-free state with bound cyclopiazonic acid (CPA) and magnesium fluoride at 2.65 A resolution and a calcium-free state with bound CPA and ADP at 3.4A resolution. In both structures, CPA occupies the calcium access channel delimited by transmembrane segments M1-M4. Inhibition of Ca(2+)-ATPase is stabilized by a polar pocket that surrounds the tetramic acid of CPA and a hydrophobic platform that cradles the inhibitor. The calcium pump residues involved include Gln(56), Leu(61), Val(62), and Asn(101). We conclude that CPA inhibits the calcium pump by blocking the calcium access channel and immobilizing a subset of transmembrane helices. In the E2(CPA) structure, ADP is bound in a distinct orientation within the nucleotide binding pocket. The adenine ring is sandwiched between Arg(489) of the nucleotide-binding domain and Arg(678) of the phosphorylation domain. This mode of binding conforms to an adenine recognition motif commonly found in ATP-dependent proteins.