Insights into outer membrane protein crystallization

Outer membrane proteins are structurally distinct from those that reside in the inner membrane and play important roles in bacterial pathogenicity and human metabolism. X-ray crystallography studies on >40 different outer membrane proteins have revealed that the transmembrane portion of these proteins can be constructed from either β-sheets or less commonly from α-helices. The most common architecture is the β-barrel, which can be formed from either a single anti-parallel sheet, fused at both ends to form a barrel or from multiple peptide chains. Outer membrane proteins exhibit considerable rigidity and stability, making their study through x-ray crystallography particularly tractable. As the number of structures of outer membrane proteins increases a more rational approach to their crystallization can be made. Herein we analyse the crystallization data from 53 outer membrane proteins and compare the results to those obtained for inner membrane proteins. A targeted sparse matrix screen for outer membrane protein crystallization is presented based on the present analysis.     

Heat shock induction by a misassembled cytoplasmic membrane protein complex in Escherichia coli

We analysed the effects of the overproduction of parts or all of a multisubunit ATP-binding cassette (ABC) transporter, the MalFGK2 complex, involved in the uptake of maltose and maltodextrins in Escherichia coli . We found that production of the MalF protein alone was inducing the phtrA promoter, which is under the control of a recently discovered sigma factor, σ²⁴, involved in the response to extracytoplasmic stresses. The production level, stability and localization of MalF were not altered when produced without its partners, suggesting that the protein was correctly inserted in the membrane. Our results indicate that a large periplasmic loop located between the third and fourth transmembrane segment of MalF, the L3 loop, is responsible for phtrA induction: (i) deleted MalF proteins with no L3 loop or with a L3 loop lacking 120 amino acids do not induce the phtrA promoter; (ii) the export to the periplasm of the L3 loop alone or fused to MalE induces the phtrA promoter. Moreover, the proteolytic sensitivity of MalF is different when it is produced alone and when MalF and MalG are produced together, suggesting a change in the conformation and/or accessibility of MalF. These results suggest that some inner membrane proteins can be sensed outside the cytoplasm by a quality control apparatus or by the export machinery. Moreover, the observation of the phtrA induction by MalF could be a useful new tool for studying the insertion and assembly of the MalFGK2 complex.     

Insights into the association of FcgammaRII and TCR with detergent-resistant membrane domains: isolation of the domains in detergent-free density gradients facilitates membrane fragment reconstitution

Plasma membrane rafts are routinely isolated as detergent-resistant membranes (DRMs) floating in detergent-free density gradients. Here we show that both the presence and exclusion of TX-100 during the density gradient fractionation have profound effects on the location of FcgammaRII and TCR in DRM fractions. The presence of TX-100 during fractionation promoted solubilization of non-cross-linked FcgammaRII when the receptor was insufficiently dissolved upon cell lysis. In the detergent-supplemented gradients, TX-100 micelles floated, further enhancing dissociation of FcgammaRII and TCR from DRMs and promoting a shift of the receptors toward higher-density fractions. Hence, fractionation of cell lysates over the detergent-containing gradients enables isolation of DRMs devoid of weakly associated proteins, like nonactivated FcgammaRII and TCR. On the other hand, in a detergent-free gradient, non-cross-linked FcgammaRII, fully soluble in 0.2% TX-100, was recovered in DRM fractions. Moreover, employment of the TX-100-free gradient for refractionation of intermediate-density fractions, derived from detergent-supplemented gradients and containing FcgammaRII and TCR, resulted in flotation of the receptors to buoyant fractions. An analysis of the TX-100 concentration revealed that after fractionation of 0.2% TX-100 cell lysates in the absence of detergent, the level of TX-100 in DRM fractions was reduced to 0.01%, below the critical micelle concentration. Therefore, fractionation of detergent cell lysates over detergent-free gradients can mimic conditions for a membrane reconstitution, evoking association of a distinct subset of membrane proteins, including FcgammaRII and TCR, with DRMs.     

Clustering induces redistribution of syndecan-4 core protein into raft membrane domains

Syndecan-4 is a heparan sulfate-carrying core protein that has been directly implicated in fibroblast growth factor 2 (FGF2) signaling. Recent studies have suggested that many signaling proteins localize to the raft compartment of the plasma cell membrane. To establish whether syndecan-4 is present in the raft compartment, we have studied the distribution of the core protein and an Fc receptor (FcR)-syndecan-4 chimera prior to and following clustering with FGF2 or antibodies. Whereas unclustered syndecan-4 was present predominantly in the non-raft membrane compartment, clustering induced extensive syndecan-4 redistribution to the rafts as demonstrated by the sucrose gradient centrifugation and life confocal microscopy. Although syndecan-4 and caveolin-1 moved in tandem, syndecan-4 was not present in caveolae, a major subset of raft compartments. We conclude that syndecan-4 clustering induces its redistribution to the non-caveolae raft compartment. This process may play an important role in syndecan-4-mediation of FGF2 signaling.     

Insights into complex processes of animal development

Life is very complicated. Recently, a group of review papers on the complexity of various biological behaviors were published by Cell to celebrate its 40th anniversary. The developmental process must be one of the most complex events in multi-cellular organisms. JMCB has published several research papers in the previous Collection ＇Understanding and Mani-pulating Developmental Complexity＇ （Wu, 2013）. This issue publishes more articles focusing on the molecular mechanisms that regulate animal developmental processes, which should provide evidences for a better understanding of the developmental complexity.     

Rearrangements of sea urchin egg cytoplasmic membrane domains at fertilization

Fertilization in the sea urchin is accompanied by rapid reorganization of the egg endoplasmic reticulum (ER). ER-derived vesicles contribute to one of three classes of membranes used in assembling the male pronuclear envelope in vitro. We provide here biochemical evidence for the rearrangement of sea urchin egg cytoplasmic membrane domains at fertilization up to the first mitosis, with respect to two nuclear envelope markers, lamin B and lamin B receptor (LBR), using purified vesicles prepared from homogenates fractionated by floatation on sucrose gradients. In unfertilized eggs, immunoprecipitation data indicate that most of lamin B and LBR are localized in the same vesicles but do not interact. By 3 min post-fertilization, both proteins are more widely distributed across the gradients and by 12 min most of lamin B and LBR are localized in vesicles of different densities. This partitioning is maintained throughout S phase. At mitosis, most lamin B and LBR remain in distinct vesicles, while a small proportion of lamin B and LBR, likely derived from the disassembled nuclear envelope, associate in a minor subset of vesicles. The results illustrate a dynamic reorganization of egg cytoplasmic membranes at fertilization, and the establishment of distinct membrane domains enriched in specific nuclear envelope markers during the first cell cycle of sea urchin development. Additionally, we demonstrate that male pro-nuclear membrane assembly occurs only when both cytosol and membranes originate from fertilized but not unfertilized eggs, suggesting that fertilization-induced membrane rearrangements contribute to the ability of the egg to assemble the male pronuclear envelope.     

The Rieske protein from Paracoccus denitrificans is inserted into the cytoplasmic membrane by the twin-arginine translocase

The Rieske [2Fe-2S] protein (ISP) is an essential subunit of cytochrome bc(1) complexes in mitochondrial and bacterial respiratory chains. Based on the presence of two consecutive arginines, it was argued that the ISP of Paracoccus denitrificans, a Gram-negative soil bacterium, is inserted into the cytoplasmic membrane via the twin-arginine translocation (Tat) pathway. Here, we provide experimental evidence that membrane integration of the bacterial ISP indeed relies on the Tat translocon. We show that targeting of the ISP depends on the twin-arginine motif. A strict requirement is established particularly for the second arginine residue (R16); conservative replacement of the first arginine (R15K) still permits substantial ISP transport. Comparative sequence analysis reveals characteristics common to Tat signal peptides in several bacterial ISPs; however, there are distinctive features relating to the fact that the presumed ISP Tat signal simultaneously serves as a membrane anchor. These differences include an elevated hydrophobicity of the h-region compared with generic Tat signals and the absence of an otherwise well-conserved '+5'-consensus motif lysine residue. Substitution of the +5 lysine (Y20K) compromises ISP export and/or cytochrome bc(1) stability to some extent and points to a specific role for this deviation from the canonical Tat motif. EPR spectroscopy confirms cytosolic insertion of the [2Fe-2S] cofactor. Mutation of an essential cofactor binding residue (C152S) decreases the ISP membrane levels, possibly indicating that cofactor insertion is a prerequisite for efficient translocation along the Tat pathway.     

Pullulanase secretion in Escherichia coli K-12 requires a cytoplasmic protein and a putative polytopic cytoplasmic membrane protein

The previously uncharacterized third and fourth genes (pulE and pulF) of the pullulanase secretion gene operon of Klebsiella oxytoca strain UNF5023 are, respectively, predicted to encode a 55 kDa polypeptide with a putative nucleotide-binding site, and a highly hydrophobic 44 kDa polypeptide that probably spans the cytoplasmic membrane several times. Expression of pulE in minicells or under the control of a strong bacteriophage T7 promoter resulted in the production of a c. 58 kDa cytoplasmic protein. A representative PulE-beta-galactosidase hybrid protein created by Tnlac mutagenesis was also found mainly in the cytoplasm. These results are in line with the predicted absence from PulE of a region of sufficient hydrophobicity to function as a signal sequence. The PulF polypeptide could not be detected either in minicells or when the gene was transcribed from the T7 promoter, but the acquirement of three pulF-lacZ gene fusions that encoded hybrid proteins with relatively high levels of beta-galactosidase activity indicates that this gene can be transcribed and translated. Gene disruption experiments indicated that both pulE and pulF are required for pullulanase secretion in Escherichia coli K-12. Both proteins exhibit considerable homology throughout their entire lengths with other proteins involved in protein secretion, pilin assembly, conjugation and transformation competence in a variety of bacteria. In addition, PulE protein has consensus sequences found in a wide variety of nucleotide-binding proteins. This study completes the initial characterization of the pullulanase secretion gene operon, which comprises 13 genes that are all essential for the transport of pullulanase across the outer membrane.     

Insights into the mechanism of HIV-1 envelope induced membrane fusion as revealed by its inhibitory peptides

HIV-1 fusion with its target cells is mediated by the glycoprotein 41 (gp41) transmembrane subunit of the viral envelope glycoprotein (ENV). The current models propose that gp41 undergoes several conformational changes between the apposing viral and cell membranes to facilitate fusion. In this review we focus on the progress that has been made in revealing the dynamic role of the N-terminal heptad repeat (NHR) and the C-terminal heptad repeat (CHR) regions within gp41 to the fusion process. The involvement of these regions in the formation of the gp41 pre-hairpin and hairpin conformations during an ongoing fusion event was mainly discovered by their derived inhibitory peptides. For example, the core structure within the hairpin conformation in a dynamic fusion event is suggested to be larger than its high resolution structure and its minimal boundaries were determined in situ. Also, inhibitory peptides helped reveal the dual contribution of the NHR to the fusion process. Finally, we will also discuss several developments in peptide design that has led to a deeper understanding of the mechanism of viral membrane fusion.     

Investigating the conformational coupling between the transmembrane and cytoplasmic domains of a single-spanning membrane protein. A 1H-NMR study

PMP1 is a 38-residue single-spanning membrane protein whose C-terminal cytoplasmic domain, Y25-F38, is highly positively charged. The conformational coupling between the transmembrane span and the cytoplasmic domain of PMP1 was investigated from 1H-nuclear magnetic resonance data of two synthetic fragments: F9-F38, i.e. 80% of the whole sequence, and Y25-F38, the isolated cytoplasmic domain. Highly disordered in aqueous solution, the Y25-F38 peptide adopts a well-defined conformation in the presence of dodecylphosphocholine micelles. Compared with the long PMP1 fragment, this structure exhibits both native and non-native elements. Our results make it possible to assess the influence of a hydrophobic anchor on the intrinsic conformational propensity of a cytoplasmic domain.     

Insights into substrate recognition by the Escherichia coli Orf135 protein through its solution structure

Escherichia coli Orf135 hydrolyzes oxidatively damaged nucleotides such as 2-hydroxy-dATP, 8-oxo-dGTP and 5-hydroxy-CTP, in addition to 5-methyl-dCTP, dCTP and CTP. Nucleotide pool sanitization by Orf135 is important since nucleotides are continually subjected to potential damage by reactive oxygen species produced during respiration. Orf135 is a member of the Nudix family of proteins which hydrolyze nucleoside diphosphate derivatives. Nudix hydrolases are characterized by the presence of a conserved motif, even though they recognize various substrates and possess a variety of substrate binding pockets. We investigated the tertiary structure of Orf135 and its interaction with a 2-hydroxy-dATP analog using NMR. We report on the solution structure of Orf135, which should contribute towards a structural understanding of Orf135 and its interaction with substrates.     

Oligomerization of a membrane protein correlates with its retention in the Golgi complex

The first membrane-spanning domain (m1) of the M glycoprotein of avian coronavirus (formerly called E1) is sufficient to retain this protein in the cis-Golgi. When the membrane-spanning domain of a protein which is efficiently delivered to the plasma membrane (VSV G protein) is replaced with m1, the resulting chimera (Gm1) is retained in the Golgi (Swift, A. M., and C. E. Machamer. 1991. J. Cell Biol. 115:19-30). When assayed in sucrose gradients, we observed that Gm1 formed a large oligomer, and that much of this oligomer was SDS resistant and stayed near the top of the stacking gel of an SDS-polyacrylamide gel. The unusual stability of the oligomer allowed it to be detected easily. Gm1 mutants with single amino acid substitutions in the m1 domain that were retained in the Golgi complex formed SDS-resistant oligomers, whereas mutants that were rapidly released to the plasma membrane did not. Oligomerization was not detected immediately after synthesis of Gm1, but occurred gradually with a lag of approximately 10 min, suggesting that it is not merely aggregation of misfolded proteins. Furthermore, oligomerization did not occur under several conditions that block ER to Golgi transport. The lumenal domain was not required for oligomerization since another chimera (alpha m1G), where the lumenal domain of Gm1 was replaced by the alpha subunit of human chorionic gonadotropin, also formed an SDS-resistant oligomer, and was able to form hetero-oligomers with Gm1 as revealed by coprecipitation experiments. SDS resistance was conferred by the cytoplasmic tail of VSV G, because proteolytic digestion of the tail in microsomes containing Gm1 oligomers resulted in loss of SDS resistance, although the protease-treated material continued to migrate as a large oligomer on sucrose gradients. Interestingly, treatment of cells with cytochalasin D blocked formation of SDS-resistant (but not SDS- sensitive) oligomers. Our data suggest that SDS-resistant oligomers form as newly synthesized molecules of Gm1 arrive at the Golgi complex and may interact (directly or indirectly) with an actin-based cytoskeletal matrix. The oligomerization of Gm1 and other resident proteins could serve as a mechanism for their retention in the Golgi complex.     

Insights into the regulation of eukaryotic elongation factor 2 kinase and the interplay between its domains

eEF2K (eukaryotic elongation factor 2 kinase) is a Ca2+/CaM (calmodulin)-dependent protein kinase which regulates the translation elongation machinery. eEF2K belongs to the small group of so-called 'α-kinases' which are distinct from the main eukaryotic protein kinase superfamily. In addition to the α-kinase catalytic domain, other domains have been identified in eEF2K: a CaM-binding region, N-terminal to the kinase domain; a C-terminal region containing several predicted α-helices (resembling SEL1 domains); and a probably rather unstructured 'linker' region connecting them. In the present paper, we demonstrate: (i) that several highly conserved residues, implicated in binding ATP or metal ions, are critical for eEF2K activity; (ii) that Ca2+/CaM enhance the ability of eEF2K to bind to ATP, providing the first insight into the allosteric control of eEF2K; (iii) that the CaM-binding/α-kinase domain of eEF2K itself possesses autokinase activity, but is unable to phosphorylate substrates in trans; (iv) that phosphorylation of these substrates requires the SEL1-like domains of eEF2K; and (v) that highly conserved residues in the C-terminal tip of eEF2K are essential for the phosphorylation of eEF2, but not a peptide substrate. On the basis of these findings, we propose a model for the functional organization and control of eEF2K.     

Structural characterization of ordered domains in a hydrophobic membrane protein

A delipidized proteolipid protein fraction was purified from organic solvent extracts of bovine cerebral cortex and studied by means of diffraction, electron microscopic, and ir techniques. Special use was made of an electron diffraction procedure which minimized the electron damage to the biological specimens. The ir spectroscopy of the apoprotein fraction indicated the presence of polypeptides in extended β-conformation, possibly in the antiparallel mode of packing. Electron microscopy of the fraction, negatively stained in organic media, made apparent the presence of both ordered and amorphous material. Only the former, characterized by repeating units of about 40–45 Å in diameter and varying length, produced diffraction patterns in the selected area mode exhibiting a highly undistorted lattice. The two-dimensional cell parameters of the protein fraction were a = 4.79 Å, b = 7.20 Å, and γ = 90°. The plane group symmetry, corresponding to the systematic absences, was p 2gg, consistent with the β-pleated sheet structure of simple polypeptides.     

Synergistic interactions between aqueous and membrane domains of a designed protein determine its fold and stability

Membrane-spanning proteins contain both aqueous and membrane-spanning regions, both of which contribute to folding and stability. To explore the interplay between these two domains we have designed and studied the assembly of coiled-coil peptides that span from the membrane into the aqueous phase. The membrane-spanning segment is based on MS1, a transmembrane coiled coil that contains a single Asn at a buried a position of a central heptad in its sequence. This Asn has been shown to drive assembly of the monomeric peptide in a membrane environment to a mixture of dimers and trimers. The coiled coil has now been extended into the aqueous phase by addition of water-soluble helical extensions. Although too short to fold in isolation, these helical extensions were expected to interact synergistically with the transmembrane domain and modulate its stability as well as its conformational specificity for forming dimers versus trimers. One design contains Asn at a position of the aqueous helical extension, which was expected to specify a dimeric state; a second peptide, which contains Val at this position, was expected to form trimers. The thermodynamics of assembly of the hybrid peptides were studied in micelles by sedimentation equilibrium ultracentrifugation. The aqueous helical extensions indeed conferred additional stability and conformational specificity to MS1 in the expected manner. These studies highlight the delicate interplay between membrane-spanning and water-soluble regions of proteins, and demonstrate how these different environments define the thermodynamics of a given specific interaction. In this case, an Asn in the transmembrane domain provided a strong driving force for folding but failed to specify a unique oligomerization state, while an Asn in the water-soluble domain was able to define specificity for a specific aggregation state as well as modulate stability.     

Visualization of membrane protein domains by cryo-electron microscopy of dengue virus

Improved technology for reconstructing cryo-electron microscopy (cryo-EM) images has now made it possible to determine secondary structural features of membrane proteins in enveloped viruses. The structure of mature dengue virus particles was determined to a resolution of 9.5 A by cryo-EM and image reconstruction techniques, establishing the secondary structural disposition of the 180 envelope (E) and 180 membrane (M) proteins in the lipid envelope. The alpha-helical 'stem' regions of the E molecules, as well as part of the N-terminal section of the M proteins, are buried in the outer leaflet of the viral membrane. The 'anchor' regions of E and the M proteins each form antiparallel E-E and M-M transmembrane alpha-helices, leaving their C termini on the exterior of the viral membrane, consistent with the predicted topology of the unprocessed polyprotein. This is one of only a few determinations of the disposition of transmembrane proteins in situ and shows that the nucleocapsid core and envelope proteins do not have a direct interaction in the mature virus.     

Translational diffusion of class II major histocompatibility complex molecules is constrained by their cytoplasmic domains

Site-directed mutagenesis in vitro was used to introduce stop codons in the genomic DNA of the alpha and beta chains of the murine class II major histocompatibility complex antigen, I-Ak. Mutated DNA was transfected into B lymphoma cells that were then selected by neomycin resistance and for their ability to express I-Ak molecules on their plasma membrane. The translational diffusion coefficient (Dlat) of I-Ak molecules composed of a wild-type beta chain paired with an alpha chain missing either 6 or 12 amino acids from the cytoplasmic domain is on the average threefold higher than the Dlat of wild-type I-Ak molecules as measured by fluorescence photobleaching and recovery. The removal of 12 amino acids from the cytoplasmic domain of the beta chain did not change the Dlat value from that of wild-type I-Ak if the truncated beta chain was paired with a wild-type alpha chain. Removing all amino acids of the cytoplasmic domains of both the alpha and beta chains resulted in a 10-fold increase in the Dlat, the highest value for any of the truncated I-Ak molecules tested. These data indicate that the carboxy- terminal six amino acids of the cytoplasmic domain of the alpha chain and the six plasma membrane-proximal amino acids of the beta chain are important in constraining the translational diffusion of I-Ak molecules in the plasma membrane.