The protein–protein interactions required for assembly of the Tn3 resolution synapse

The site-specific recombinase Tn3 resolvase initiates DNA strand exchange when two res recombination sites and six resolvase dimers interact to form a synapse. The detailed architecture of this intricate recombination machine remains unclear. We have clarified which of the potential dimer–dimer interactions are required for synapsis and recombination, using a novel complementation strategy that exploits a previously uncharacterized resolvase from Bartonella bacilliformis (“Bart”). Tn3 and Bart resolvases recognize different DNA motifs, via diverged C-terminal domains (CTDs). They also differ substantially at N-terminal domain (NTD) surfaces involved in dimerization and synapse assembly. We designed NTD-CTD hybrid proteins, and hybrid res sites containing both Tn3 and Bart dimer binding sites. Using these components in in vivo assays, we demonstrate that productive synapsis requires a specific “R” interface involving resolvase NTDs at all three dimer-binding sites in res. Synapses containing mixtures of wild-type Tn3 and Bart resolvase NTD dimers are recombination-defective, but activity can be restored by replacing patches of Tn3 resolvase R interface residues with Bart residues, or vice versa. We conclude that the Tn3/Bart family synapse is assembled exclusively by R interactions between resolvase dimers, except for the one special dimer–dimer interaction required for catalysis.     

GPCR: G protein complexes—the fundamental signaling assembly

G protein coupled receptors (GPCR) constitute the largest group of cell surface receptors that transmit various signals across biological membranes through the binding and activation of heterotrimeric G proteins, which amplify the signal and activate downstream effectors leading to the biological responses. Thus, the first critical step in this signaling cascade is the interaction between receptor and its cognate G protein. Understanding this critical event at the molecular level is of high importance because abnormal function of GPCRs is associated with many diseases. Thus, these receptors are targets for drug development.     

The SufBCD protein complex is the scaffold for iron–sulfur cluster assembly in Thermus thermophiles HB8

Iron–sulfur (Fe–S) clusters are the oldest and most versatile inorganic cofactors that are required to sustain fundamental life processes. Bacteria have three systems of [Fe–S] cluster biogenesis, designated ISC, NIF, and SUF. In contrast, the Thermus thermophiles HB8 has only one system, formed mostly by SUF homologs that contain six proteins: SufA, SufB, SufC, SufD, SufS and SufE. The kinetics of SufC ATPase was studied using a linked enzyme assay method. In the presence of SufB, SufD or SufBD complexes, the activity of SufC was enhanced. The cysteine desulfurase activity of SufS was also stimulated by the presence of the SufBCD complex. The results obtained through enzymology revealed that aconitase activity was activated by [Fe–S] clusters reconstituted on the SufBCD complex. Consolidated results from spectral and enzymatic analysis suggest that the SufBCD complex is a novel type of Fe–S scaffold system that can assemble Fe/S clusters de novo.     

The β-barrel assembly machinery (BAM) is required for the assembly of a primitive S-layer protein in the ancient outer membrane of Thermus thermophilus

The ancient bacterial lineage Thermus spp has a primitive form of outer membrane attached to the cell wall through SlpA, a protein that shows intermediate properties between S-layer proteins and outer membrane (OM) porins. In E. coli and related Proteobacteria, porins are secreted through the BAM (β-barrel assembly machinery) pathway, whose main component is BamA. A homologue to this protein is encoded in all the Thermus spp so far sequenced, so we wondered if this pathway could be responsible for SlpA secretion in this ancient bacterial model. To analyse this hypothesis, we attempted to get mutants on this BamA^th of T. thermophilus HB27. Knockout and deletion mutants lacking the last 10 amino acids were not viable, whereas its depletion by means of a BamA antisense RNA lead defective attachment to the cell wall of its OM-like envelope. Such defects were related to defective folding of the SlpA protein that was more sensitive to proteases than in a wild-type strain. A similar phenotype was found in mutants lacking the terminal Phe of SlpA. Further protein–protein interaction assays confirmed the existence of specific binding between SlpA and BamA^th. Taking together, these data suggest that SlpA is secreted through a BAM-like pathway in this ancestral bacterial lineage, supporting an ancient origin of this pathway before the evolution of the Proteobacteria.     

Mapping iron binding sites on human frataxin: implications for cluster assembly on the ISU Fe–S cluster scaffold protein

Frataxin is an iron binding mitochondrial matrix protein that has been shown to mediate iron delivery during iron-sulfur cluster and heme biosynthesis. There is a high degree of structural homology for frataxin proteins from diverse sources, and all possess an anionic surface defined by acidic residues. In the human protein these residues principally lie on a surface defined by the alpha1 helix and beta1 sheet and the impact of multiple substitutions of these carboxylate residues on iron binding is described. Full-length human frataxin has previously been shown to undergo self-cleavage to produce a truncated form both in vitro and in vivo. This truncated protein has been shown to bind approximately seven iron centers that are presumably associated with the acidic patch. Relative to this native protein, the stoichiometry decreases according to the number and sites of mutations. Nevertheless, the iron-dependent binding affinity of each frataxin derivative to the iron-sulfur cluster scaffold protein ISU is found to be similar to that of native frataxin, as defined by isothermal titration calorimetry experiments, requiring only one iron center to promote nanomolar binding. While frataxins from various cell types appear to bind differing numbers of iron centers, the physiologically relevant number of bound irons appears to be small, with significantly higher binding affinity following complex formation with partner proteins (micromolar compared with nanomolar binding). By contrast, in reconstitution assays for frataxin-promoted [2Fe-2S](2+) cluster assembly on ISU, one derivative does display a modestly lower reconstitution rate. The overall consensus from these data is to consider a pool of potential sites that can stably bind an iron center when bridged to a variety of physiological targets.     

The protein–protein interaction network of the human Sirtuin family

Protein–protein interaction networks are useful for studying human diseases and to look for possible health care through a holistic approach. Networks are playing an increasing and important role in the understanding of physiological processes such as homeostasis, signaling, spatial and temporal organizations, and pathological conditions. In this article we show the complex system of interactions determined by human Sirtuins (Sirt) largely involved in many metabolic processes as well as in different diseases. The Sirtuin family consists of seven homologous Sirt-s having structurally similar cores but different terminal segments, being rather variable in length and/or intrinsically disordered. Many studies have determined their cellular location as well as biological functions although molecular mechanisms through which they act are actually little known therefore, the aim of this work was to define, explore and understand the Sirtuin-related human interactome. As a first step, we have integrated the experimentally determined protein–protein interactions of the Sirtuin-family as well as their first and second neighbors to a Sirtuin-related sub-interactome. Our data showed that the second-neighbor network of Sirtuins encompasses 25% of the entire human interactome, and exhibits a scale-free degree distribution and interconnectedness among top degree nodes. Moreover, the Sirtuin sub interactome showed a modular structure around the core comprising mixed functions. Finally, we extracted from the Sirtuin sub-interactome subnets related to cancer, aging and post-translational modifications for information on key nodes and topological space of the subnets in the Sirt family network.     

The clathrin assembly protein AP180 regulates the generation of amyloid-β peptide

The overproduction and extracellular buildup of amyloid-β peptide (Aβ) is a critical step in the etiology of Alzheimer’s disease. Recent data suggest that intracellular trafficking is of central importance in the production of Aβ. Here we use a neuronal cell line to examine two structurally similar clathrin assembly proteins, AP180 and CALM. We show that RNA interference-mediated knockdown of AP180 reduces the generation of Aβ1-40 and Aβ1-42, whereas CALM knockdown has no effect on Aβ generation. Thus AP180 is among the traffic controllers that oversee and regulate amyloid precursor protein processing pathways. Our results also suggest that AP180 and CALM, while similar in their domain structures and biochemical properties, are in fact dedicated to separate trafficking pathways in neurons.     

Surface-tethered planar membranes containing the β-barrel assembly machinery: a platform for investigating bacterial outer membrane protein folding

The outer membrane of Gram-negative bacteria presents a robust physicochemical barrier protecting the cell from both the natural environment and acting as the first line of defense against antimicrobial materials. The proteins situated within the outer membrane are responsible for a range of biological functions including controlling influx and efflux. These outer membrane proteins (OMPs) are ultimately inserted and folded within the membrane by the β-barrel assembly machine (Bam) complex. The precise mechanism by which the Bam complex folds and inserts OMPs remains unclear. Here, we have developed a platform for investigating Bam-mediated OMP insertion. By derivatizing a gold surface with a copper-chelating self-assembled monolayer, we were able to assemble a planar system containing the complete Bam complex reconstituted within a phospholipid bilayer. Structural characterization of this interfacial protein-tethered bilayer by polarized neutron reflectometry revealed distinct regions consistent with known high-resolution models of the Bam complex. Additionally, by monitoring changes of mass associated with OMP insertion by quartz crystal microbalance with dissipation monitoring, we were able to demonstrate the functionality of this system by inserting two diverse OMPs within the membrane, pertactin, and OmpT. This platform has promising application in investigating the mechanism of Bam-mediated OMP insertion, in addition to OMP function and activity within a phospholipid bilayer environment.     

The ability to convert the 4 S glucocorticoid receptor to the 7–8 S form is dependent on both RNA and protein factors

The DEAE-cellulose-purified 4 S form of the rat liver glucocorticoid receptor can associate with cytosolic factors, as evidenced by an alteration of the sedimentation value of the 7–8 S form. On the basis of sedimentation profile, this form is indistinguishable from the activated, low-salt 7–8 S form isolated from rat liver cytosol. In addition, both the endogenous and reconstituted 7–8 S receptor can bind DNA as the 7–8 S form. In keeping with our reports that the endogenous form of the 7–8 S receptor is sensitive to RNAase digestion, treatment of the cytosol with RNAase prior to mixing with the 4 S receptor prevents the formation of the 7–8 S material. Moreover, warming the cytosol to 50°C prior to mixing with the 4 S receptor also eliminates the ability to form the heavier material. Since RNA is heat-stable, this suggests that other factors may be involved. Treatment of the cytosol with N-ethylmaleimide, a reagent reported to be specific for sulfhydryl groups, also eliminates 7–8 S generating ability. These observations suggest that a protein may be a component of the 7–8 S generating material. This is substantiated by the observation that trypsin or chymotrypsin treatment of the cytosol mitigates the ability of the cytosol to form the 7–8 S material and results in the appearance of a form of the receptor that sediments at approximately 6 S. Protease treatment of partially purified material eliminates the 7–8 S generating activity entirely. We conclude that the 7–8 S form of the receptor can be reconstituted from the 4 S receptor via association with at least two other cytosolic factors, a protein and an RNA.     

Identification of a protein–protein interaction between KCNE1 and the activation gate machinery of KCNQ1

KCNQ1 channels assemble with KCNE1 transmembrane (TM) peptides to form voltage-gated K⁺ channel complexes with slow activation gate opening. The cytoplasmic C-terminal domain that abuts the KCNE1 TM segment has been implicated in regulating KCNQ1 gating, yet its interaction with KCNQ1 has not been described. Here, we identified a protein–protein interaction between the KCNE1 C-terminal domain and the KCNQ1 S6 activation gate and S4–S5 linker. Using cysteine cross-linking, we biochemically screened over 300 cysteine pairs in the KCNQ1–KCNE1 complex and identified three residues in KCNQ1 (H363C, P369C, and I257C) that formed disulfide bonds with cysteine residues in the KCNE1 C-terminal domain. Statistical analysis of cross-link efficiency showed that H363C preferentially reacted with KCNE1 residues H73C, S74C, and D76C, whereas P369C showed preference for only D76C. Electrophysiological investigation of the mutant K⁺ channel complexes revealed that the KCNQ1 residue, H363C, formed cross-links not only with KCNE1 subunits, but also with neighboring KCNQ1 subunits in the complex. Cross-link formation involving the H363C residue was state dependent, primarily occurring when the KCNQ1–KCNE1 complex was closed. Based on these biochemical and electrophysiological data, we generated a closed-state model of the KCNQ1–KCNE1 cytoplasmic region where these protein–protein interactions are poised to slow activation gate opening.     

The translocation of transportin–cargo complexes through nuclear pores is independent of both Ran and energy

Active transport between nucleus and cytoplasm proceeds through nuclear pore complexes (NPCs) and is mediated largely by shuttling transport receptors that use direct RanGTP binding to coordinate loading and unloading of cargo [1], [2], [3], [4]. Import receptors such as importin β or transportin bind their substrates at low RanGTP levels in the cytoplasm and release them upon encountering RanGTP in the nucleus, where a high RanGTP concentration is predicted. This substrate release is, in the case of import by the importin α/β heterodimer, coupled directly to importin β release from the NPCs. If the importin β –RanGTP interaction is prevented, import intermediates arrest at the nuclear side of the NPCs [5], [6]. This arrest makes it difficult to probe directly the Ran and energy requirements of the actual translocation from the cytoplasmic to the nuclear side of the NPC, which immediately precedes substrate release. Here, we have shown that in the case of transportin, dissociation of transportin–substrate complexes is uncoupled from transportin release from NPCs. This allowed us to dissect the requirements of translocation through the NPC, substrate release and transportin recycling. Surprisingly, translocation of transportin–substrate complexes into the nucleus requires neither Ran nor nucleoside triphosphates (NTPs). It is only nuclear RanGTP, not GTP hydrolysis, that is needed for dissociation of transportin–substrate complexes and for re-export of transportin to the cytoplasm. GTP hydrolysis is apparently required only to restore the import competence of the re-exported transportin and, thus, for multiple rounds of transportin-dependent import. In addition, we provide evidence that at least one type of substrate can also complete NPC passage mediated by importin β independently of Ran and energy.     

The conformational transition during G protein–coupled receptor (GPCR) and G protein interaction

The precise structural mechanism of G protein–coupled receptor (GPCR)–G protein coupling has been of significant research interest because it provides fundamental knowledge on cellular signaling and valuable information for GPCR-targeted drug development. Over the last decade, several GPCR–G protein complex structures have been identified. However, these structures are mere snapshots of guanosine diphosphate (GDP)-released stable GPCR–G protein complexes, which have limited the understanding of the allosteric conformational transition during receptor binding to GDP release and the GPCR–G protein coupling selectivity. Recently, deeper insights into the mechanism underlying stepwise conformational changes during GPCR–G protein coupling were obtained using hydrogen/deuterium exchange mass spectrometry, hydroxyl radical footprinting-mass spectrometry, X-ray crystallography, cryoelectron microscopy, and molecular dynamics simulation techniques. This review summarizes these recent developments.     

Stimulation of ribonucleic acid polymerase activity in vitro by prostatic steroid–protein receptor complexes

A system has been developed which allows the stimulation in vitro of prostatic RNA polymerase by prostatic 5alpha-dihydrotestosterone-protein receptor complexes prepared from the tissues of castrated rats. The reconstitution in vitro of such a system necessitates the purification of several subcellular components. Two 5alpha-dihydrotestosterone-receptor complexes are located in the prostatic soluble supernatant fraction, separable by selective ammonium sulphate fractionation, and one complex can be isolated from the nuclear fraction. In the presence of all these complexes, stimulation of RNA polymerase in intact nuclei and nucleoli was observed. The complexes also increased the activity of the enzyme solubilized from whole nuclei. Greater stimulation of this system was noted in the presence of prostatic chromatin as template, as compared with that observed with calf thymus DNA or liver chromatin as template. The effects of the complexes on subnuclear forms of RNA polymerase, of nucleolar and extranucleolar origin, are also described. RNA polymerase solubilized from nucleoli is more susceptible to stimulation by the 5alpha-dihydrotestosterone-receptor complexes than is the ;nucleoplasmic' enzyme. Stimulation occurs less readily in the presence of Mn(2+) and at high ionic strength than in the presence of Mg(2+) and at low ionic strength. Preliminary experiments show that prostatic nucleolar RNA polymerase transcribes prostatic chromatin poorly as compared with the nucleoplasmic enzyme. The observations reported indicate an involvement of non-histone proteins associated with DNA in the process by which stimulation of enzyme activity by the 5alpha-dihydrotestosterone-receptor complexes is achieved. The implications of these findings in the mechanism of steroid hormone action is considered.     

p62 links the autophagy pathway and the ubiqutin–proteasome system upon ubiquitinated protein degradation

The ubiquitin–proteasome system (UPS) and autophagy are two distinct and interacting proteolytic systems. They play critical roles in cell survival under normal conditions and during stress. An increasing body of evidence indicates that ubiquitinated cargoes are important markers of degradation. p62, a classical receptor of autophagy, is a multifunctional protein located throughout the cell and involved in many signal transduction pathways, including the Keap1–Nrf2 pathway. It is involved in the proteasomal degradation of ubiquitinated proteins. When the cellular p62 level is manipulated, the quantity and location pattern of ubiquitinated proteins change with a considerable impact on cell survival. Altered p62 levels can even lead to some diseases. The proteotoxic stress imposed by proteasome inhibition can activate autophagy through p62 phosphorylation. A deficiency in autophagy may compromise the ubiquitin–proteasome system, since overabundant p62 delays delivery of the proteasomal substrate to the proteasome despite proteasomal catalytic activity being unchanged. In addition, p62 and the proteasome can modulate the activity of HDAC6 deacetylase, thus influencing the autophagic degradation.     

Integrating the puzzle pieces: The current atomistic picture of phospholipid–G protein coupled receptor interactions

A compelling question of how phospholipids interact with their target receptors has been of interest since the first receptor-mediated effects were reported. The recent report of a crystal structure for the S1P₁ receptor in complex with an antagonist phospholipid provides interesting perspective on the insights that had previously been gained through structure–activity studies of the phospholipids, as well as modeling and mutagenesis studies of the receptors. This review integrates these varied lines of investigation in the context of their various contributions to our current understanding of phospholipid–receptor interactions. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.     

Structurally flexible macro-organization of the pigment–protein complexes of the diatom Phaeodactylum tricornutum

By means of circular dichroism (CD) spectroscopy, we have characterized the organization of the photosynthetic complexes of the diatom Phaeodactylum tricornutum at different levels of structural complexity: in intact cells, isolated thylakoid membranes and purified fucoxanthin chlorophyll protein (FCP) complexes. We found that the CD spectrum of whole cells was dominated by a large band at (+)698 nm, accompanied by a long tail from differential scattering, features typical for psi-type (polymerization or salt-induced) CD. The CD spectrum additionally contained intense (−)679 nm, (+)445 nm and (−)470 nm bands, which were also present in isolated thylakoid membranes and FCPs. While the latter two bands were evidently produced by excitonic interactions, the nature of the (−)679 nm band remained unclear. Electrochromic absorbance changes also revealed the existence of a CD-silent long-wavelength (∼545 nm) absorbing fucoxanthin molecule with very high sensitivity to the transmembrane electrical field. In intact cells the main CD band at (+)698 nm appeared to be associated with the multilamellar organization of the thylakoid membranes. It was sensitive to the osmotic pressure and was selectively diminished at elevated temperatures and was capable of undergoing light-induced reversible changes. In isolated thylakoid membranes, the psi-type CD band, which was lost during the isolation procedure, could be partially restored by addition of Mg-ions, along with the maximum quantum yield and the non-photochemical quenching of singlet excited chlorophyll a, measured by fluorescence transients.