Pentameric Complex Research Articles

The Importance of TM3-4 Loop Subdomains for Functional Reconstitution of Glycine Receptors by Independent Domains

Truncated glycine receptors that have been found in human patients suffering from the neuromotor disorder hyperekplexia or in spontaneous mouse models resulted in non-functional ion channels. Rescue of function experiments with the lacking protein portion expressed as a separate independent domain demonstrated restoration of glycine receptor functionality in vitro. This construct harbored most of the TM3-4 loop, TM4, and the C terminus and was required for concomitant transport of the truncated α1 and the complementation domain from the endoplasmic reticulum toward the cell surface, thereby enabling complex formation of functional glycine receptors. Here, the complementation domain was stepwise truncated from its N terminus in the TM3-4 loop. Truncation of more than 49 amino acids led again to loss of functionality in the receptor complex expressed from two independent domain constructs. We identified residues 357-418 in the intracellular TM3-4 loop as being required for reconstitution of functional glycine-gated channels. All complementation constructs showed cell surface protein expression and correct orientation according to glycine receptor topology. Moreover, we demonstrated that the truncations did not result in a decreased protein-protein interaction between both glycine receptor domains. Rather, deletions of more than 49 amino acids abolished conformational changes necessary for ion channel opening. When the TM3-4 loop subdomain harboring residues 357-418 was expressed as a third independent construct together with the truncated N-terminal and C-terminal glycine receptor domains, functionality of the glycine receptor was again restored. Thus, residues 357-418 represent an important determinant in the process of conformational rearrangements following ligand binding resulting in channel opening.

Antibodies Against Neutralization Epitopes of Human Cytomegalovirus gH/gL/pUL128-130-131 Complex and Virus Spreading May Correlate with Virus Control In Vivo

Recently, human cytomegalovirus (HCMV) UL128-131 locus gene products have been found to be associated with glycoprotein H (gH) and glycoprotein L (gL) to form a pentameric glycoprotein complex gH/gL/pUL128-130-131, which is present in the virus envelope and elicits production of neutralizing antibodies. Purpose of this study was to verify whether in vitro activities of these antibodies may correlate with protection in vivo. By using potently neutralizing human monoclonal antibodies (mAbs) targeting 10 different epitopes of the pentameric complex, a competitive ELISA assay was developed, in which the pentamer bound to the solid-phase was reacted competitively with human sera and murinized human mAbs. In addition, inhibition of virus spreading (plaque formation and leukocyte transfer) by neutralizing human mAbs and sera was investigated. In the absence of any reactivity of sera from HCMV-seronegative subjects, antibodies to all 10 epitopes were detected in HCMV-seropositive individuals. During primary HCMV infection in pregnancy antibodies to some epitopes showed a trend towards an earlier appearance in mothers not transmitting the virus to the fetus as compared to transmitting mothers. In addition, the activity of neutralizing human mAbs and sera in blocking virus cell-to-cell spreading and virus transfer to leukocytes from infected endothelial cells was shown to develop during the convalescent phase of primary infection. Dissection of the neutralizing/inhibiting activities of human sera may be helpful in the study of their protective role in vivo. In particular, neutralizing antibodies to the pentamer may be a surrogate marker of protection in vivo.

Solution structure of the natively assembled yeast ribosomal stalk determined by small-angle X-ray scattering

The ribosomal stalk of the 60S subunit has been shown to play a crucial role in all steps of protein synthesis, but its structure and exact molecular function remain an unanswered question. In the present study, we show the low-resolution models of the solution structure of the yeast ribosomal stalk, composed of five proteins, P0-(P1-P2)(2). The model of the pentameric stalk complex determined by small-angle X-ray scattering reveals an elongated shape with a maximum length of 13 nm. The model displays three distinct lobes, which may correspond to the individual P1-P2 heterodimers anchored to the C-terminal domain of the P0 protein.

Conformational Dynamics of Activation for the Pentameric Complex of Dimeric G Protein-Coupled Receptor and Heterotrimeric G Protein

Photoactivation of rhodopsin (Rho), a G protein-coupled receptor, causes conformational changes that provide a specific binding site for the rod G protein, G(t). In this work we employed structural mass spectrometry techniques to elucidate the structural changes accompanying transition of ground state Rho to photoactivated Rho (Rho(∗)) and in the pentameric complex between dimeric Rho(∗) and heterotrimeric G(t). Observed differences in hydroxyl radical labeling and deuterium uptake between Rho(∗) and the (Rho(∗))(2)-G(t) complex suggest that photoactivation causes structural relaxation of Rho following its initial tightening upon G(t) coupling. In contrast, nucleotide-free G(t) in the complex is significantly more accessible to deuterium uptake allowing it to accept GTP and mediating complex dissociation. Thus, we provide direct evidence that in the critical step of signal amplification, Rho(∗) and G(t) exhibit dissimilar conformational changes when they are coupled in the (Rho(∗))(2)-G(t) complex.

Analysis of chimeric ribosomal stalk complexes from eukaryotic and bacterial sources: structural features responsible for specificity of translation factors

Ribosomal protein P0 forms a pentameric complex with two heterodimers of the flexible stalk proteins P1•P2 and plays a role in the functional interaction of eukaryotic ribosomes with translational factors. To investigate the functionality of domains of P0 characteristic to eukaryotes, we constructed various chimeras between silkworm P0 and Escherichia coli counterpart L10. Replacement of the C-terminal region of L10 with that of P0 allowed the binding of two P1•P2 heterodimers, which supported ribosomal activity dependent on eukaryotic elongation factors eEF-2/eEF-1α, but not activity dependent on bacterial factors EF-G/EF-Tu. Conversely, replacement of the C-terminal region of P0 with that of L10 allowed binding of two bacterial L12 homodimers, which resulted in a low level of activity dependent on bacterial factors. Insertion of the extended region of P0 that is absent in the bacterial counterpart into L10 did not affect L12 binding or bacterial factor-dependent activity, but deletion of this region from P0 resulted in a 40% reduction in eukaryotic factor-dependent activity. The results indicate that the C-terminal regions of P0 and L10 are responsible for binding of the cognate stalk dimers and ribosomal specificity for translation factors and suggest that the extended region participates in accessibility only for eukaryotic factors.

Pseudopod Growth and Evolution during Cell Movement Is Controlled through SCAR/WAVE Dephosphorylation

SCAR/WAVE is a principal regulator of pseudopod growth in crawling cells. It exists in a stable pentameric complex, which is regulated at multiple levels that are only beginning to be understood. SCAR/WAVE is phosphorylated at multiple sites, but how this affects its biological activity is unclear. Here we show that dephosphorylation of Dictyostelium SCAR controls normal pseudopod dynamics. We demonstrate that the C-terminal acidic domain of most Dictyostelium SCAR is basally phosphorylated at four serine residues. A small amount of singly phosphorylated SCAR is also found. SCAR phosphorylation site mutants cannot replace SCAR's role in the pseudopod cycle, though they rescue cell size and growth. Unphosphorylatable SCAR is hyperactive-excessive recruitment to the front results in large pseudopods that fail to bifurcate because they continually grow forward. Conversely, phosphomimetic SCAR is weakly active, causing frequent small, disorganized pseudopods. Even in its regulatory complex, SCAR is normally held inactive by an interaction between the phosphorylated acidic and basic domains. Loss of basic residues complementary to the acidic phosphosites yields a hyperactive protein similar to unphosphorylatable SCAR. Regulated dephosphorylation of a fraction of the cellular SCAR pool is a key step in SCAR activation during pseudopod growth. Phosphorylation increases autoinhibition of the intact complex. Dephosphorylation weakens this interaction and facilitates SCAR activation but also destabilizes the protein. We show that SCAR is specifically dephosphorylated in pseudopods, increasing activation by Rac and lipids and supporting positive feedback of pseudopod growth.

Identification of a Preassembled TRH Receptor-Gq/11 Protein Complex in HEK293 Cells

Protein-protein interactions define specificity in signal transduction and these interactions are central to transmembrane signaling by G-protein-coupled receptors (GPCRs). It is not quite clear, however, whether GPCRs and the regulatory trimeric G-proteins behave as freely and independently diffusible molecules in the plasma membrane or whether they form some preassociated complexes. Here we used clear-native polyacrylamide gel electrophoresis (CN-PAGE) to investigate the presumed coupling between thyrotropin-releasing hormone (TRH) receptor and its cognate G(q/11) protein in HEK293 cells expressing high levels of these proteins. Under different solubilization conditions, the TRH receptor (TRH-R) was identified to form a putative pentameric complex composed of TRH-R homodimer and G(q/11) protein. The presumed association of TRH-R with G(q/11)α or Gβ proteins in plasma membranes was verified by RNAi experiments. After 10- or 30-min hormone treatment, TRH-R signaling complexes gradually dissociated with a concomitant release of receptor homodimers. These observations support the model in which GPCRs can be coupled to trimeric G-proteins in preassembled signaling complexes, which might be dynamically regulated upon receptor activation. The precoupling of receptors with their cognate G-proteins can contribute to faster G-protein activation and subsequent signal transfer into the cell interior.

Monitoring of Cytomegalovirus-Specific CD8 + T-Cell Response With Major Histocompatibility Complex Pentamers in Kidney Transplant Recipients

Introduction Cytomegalovirus (CMV) can reactivate causing serious clinical problems during immunosuppression. CMV-specific CD8 + T cells play an important role in the control of CMV reactivation. Using pentameric major histocompatibility complex (MHC) peptide complexes, we investigated cellular immune responses to CMV among healthy individuals and kidney transplantation recipients in Korea, which is an endemic area of CMV infection. Materials and methods Analysis of CMV-specific T cells was performed on 28 healthy individuals and 40 recipients who bore human leukocyte antigen (HLA)-A2 or -A24. CMV pp65 pentamer-binding cells incubated with various monoclonal antibodies were measured by four-color flow cytometry. Results Detectable levels of pentamer + CD8 + T cells were present in 109/139 samples (78.4%) that stained with the A*02NLV-pentamer, while 15/67 samples (22.4%) stained with the A*24QYD-pentamer ( P < .01). Among patients with HLA-A2, 22/24 (91.7%) samples showing positive CMV antigenemia revealed detectable pentamer + CD8 + T cells, while 87/115 (75.7%) displaying negative CMV antigenemia had detectable pentamer + CD8 + T cells ( P = .04). There was no significant difference in percentages of pentamer + CD8 + T cells between patients who did versus who did not experience episodes of CMV infection. The subpopulation of CMV-specific CD8 + T cells in transplantation recipients was evaluated using phenotypic markers; memory cells comprised the majority of the CMV-specific CD8 + T-cell population. Conclusion The A*02NLV-pentamer complex was useful to monitor CMV-specific T cells. However, MHC pentamer-based techniques did not provide a clear distinction between patients who are or are not at risk for CMV infection.

Serum antibody response to the gH/gL/pUL128–131 five-protein complex of human cytomegalovirus (HCMV) in primary and reactivated HCMV infections

Background Recently, a new human cytomegalovirus (HCMV) glycoprotein complex has been identified and potentially proposed as a vaccine. Objective The aim of this study was to determine whether the HCMV gH/gL/pUL128–pUL130–pUL131 (gH/gL/pUL128–131) 5-protein (pentameric) complex (which has been recently found to be indispensable for the infection of endothelial and epithelial cells) is able to elicit a consistent antibody response in both primary and reactivated HCMV infections. Study design The antibody response was determined by both indirect immunofluorescence (IFA) and ELISA, using fixed (IFA) or lysed (ELISA) epithelial (ARPE-19) cells infected with one or more adenoviral vectors, each carrying one HCMV gene and, in parallel, with a control adenovirus vector. Results The specificity of results was determined by the reactivity of human neutralizing mAbs recognizing two, three, or four proteins of the complex. In 14 cases of primary infection, an IgG antibody seroconversion to the UL128–131 gene products was consistently detected within 2–4 weeks after onset of infection, while antibodies persisted for at least 12 months. The IgG antibody response to UL128–131 gene products was generally superior to the response to gH and appeared to follow the neutralizing antibody response (as determined in epithelial cells). In reactivated infections, the antibody response showed a trend reminiscent of a booster response. IgG antibodies were detected in HCMV-seropositive healthy adult controls, but not in HCMV-seronegative individuals. Conclusions The IgG antibody response to the pentameric complex could be a major target for the evaluation of the antibody response to a pentamer-based vaccine.

Dynamic changes of cytotoxic T lymphocytes (CTLs), natural killer (NK) cells, and natural killer T (NKT) cells in patients with acute hepatitis B infection

BackgroundThe goal of this study is to observe changes in HBcAg-specific cytotoxic T lymphocytes (CTLs), natural killer (NK) and natural killer T (NKT) cells from peripheral blood and to relate such changes on viral clearance and liver injury in patients with acute hepatitis B (AHB).MethodsDynamic profiles on the frequency of HLA-A0201-restricted HBcAg18-27 pentamer complex (MHC-Pentamer)-specific CTLs and lymphocyte subsets in AHB patients were analyzed in addition to liver function tests, HBV serological markers, and HBV DNA levels. ELISPOT was used to detect interferon-gamma (INF-γ) secretion in specific CTLs stimulated with known T cell epitope peptides associated with HBV surface protein, polymerase, and core protein.ResultsHBV-specific CTL frequencies in AHB patients were much higher than in patients with chronic hepatitis B (CHB) (p < 0.05). HBeAg and HBV DNA disappeared earlier in AHB patients with a high frequency of HBV-specific CTLs compared with those with a low frequency of HBV-specific CTLs (p = 0.001 and 0.024, respectively). INF-γ spots of effector cells stimulated by Pol575-583, Env348-357, or Core18-27 epitope peptides were significantly greater in AHB patients than in CHB patients (p < 0.01). CD3+CD8+ T cell numbers in AHB patients was more than observed in the healthy control group from the first to the fourth week after admission (p = 0.008 and 0.01, respectively); the number of CD3+CD8+ T cells and frequency of HBcAg18-27-specific CTLs in AHB patients reached peak levels at the second week after admission. NK and NKT cell numbers were negatively correlated with the frequency of HBcAg-specific CTLs (r = -0.266, p = 0.05).ConclusionsPatients with AHB possess a higher frequency of HBcAg-specific CTLs than CHB patients. The frequency of specific CTLs in AHB patients is correlated with HBeAg clearance indicating that HBV-specific CTLs play an important role in viral clearance and the self-limited process of the disease. Furthermore, NK and NKT cells are likely involved in the early, non-specific immune response to clear the virus.

Stoichiometry of HLA Class II-Invariant Chain Oligomers

BackgroundThe HLA gene complex encodes three class II isotypes, DR, DQ, and DP. HLA class II molecules are peptide receptors that present antigens for recognition by T lymphocytes. In antigen presenting cells, the assembly of matched α and β subunits to heterodimers is chaperoned by invariant chain (Ii). Ii forms a homotrimer with three binding sites for class II heterodimers. The current model of class II and Ii structure states that three αβ heterodimers bind to an Ii trimer.Methology/Principal FindingsWe have now analyzed the composition and size of the complexes of class II and Ii using epitope tagged class II subunits and density gradient experiments. We show here that class II-Ii oligomers consist of one class II heterodimer associated with one Ii trimer, such that the DR, DQ and DP isotypes are contained within separate complexes with Ii.Conclusion/SignificanceWe propose a structural model of the class II-Ii oligomer and speculate that the pentameric class II-Ii complex is bent towards the cell membrane, inhibiting the binding of additional class II heterodimers to Ii.

Interaction map of the Trypanosoma cruzi ribosomal P protein complex (stalk) and the elongation factor 2

The large subunit of the eukaryotic ribosome possesses a long and protruding stalk formed by the ribosomal P proteins. This structure is involved in the translation step of protein synthesis through interaction with the elongation factor 2 (EF-2). The Trypanosoma cruzi stalk complex is composed of four proteins of about 11 kDa, TcP1α, TcP1β, TcP2α, TcP2β and a fifth TcP0 of about 34 kDa. In a previous work, a yeast two-hybrid (Y2H) protein-protein interaction map of T. cruzi ribosomal P proteins was generated. In order to gain new insight into the assembly of the stalk, a complete interaction map was generated by surface plasmon resonance (SPR) and the kinetics of each interaction was calculated. All previously detected interactions were confirmed and new interacting pairs were found, such as TcP1β-TcP2α and TcP1β-TcP2β. Moreover P2 but not P1 proteins were able to homo-oligomerize. In addition, the region comprising amino acids 210-270 on TcP0 was identified as the region interacting with P1/P2 proteins, using Y2H and SPR. The interaction domains on TcP2β were also mapped by SPR identifying two distinct regions. The assembly order of the pentameric complex was assessed by SPR showing the existence of a hierarchy in the association of the different P proteins forming the stalk. Finally, the TcEF-2 gene was identified, cloned, expressed and refolded. Using SPR analysis we showed that TcEF-2 bound with similar affinity to the four P1/P2 ribosomal P proteins of T. cruzi but with reduced affinity to TcP0.

Pentameric Organization of the Ribosomal Stalk Accelerates Recruitment of Ricin A Chain to the Ribosome for Depurination

Ribosome inactivating proteins (RIPs) depurinate a universally conserved adenine in the α-sarcin/ricin loop (SRL) and inhibit protein synthesis at the translation elongation step. We previously showed that ribosomal stalk is required for depurination of the SRL by ricin toxin A chain (RTA). The interaction between RTA and ribosomes was characterized by a two-step binding model, where the stalk structure could be considered as an important interacting element. Here, using purified yeast ribosomal stalk complexes assembled in vivo, we show a direct interaction between RTA and the isolated stalk complex. Detailed kinetic analysis of these interactions in real time using surface plasmon resonance (SPR) indicated that there is only one type of interaction between RTA and the ribosomal stalk, which represents one of the two binding steps of the interaction with ribosomes. Interactions of RTA with the isolated stalk were relatively insensitive to salt, indicating that nonelectrostatic interactions were dominant. We compared the interaction of RTA with the full pentameric stalk complex containing two pairs of P1/P2 proteins with its interaction with the trimeric stalk complexes containing only one pair of P1/P2 and found that the rate of association of RTA with the pentamer was higher than with either trimer. These results demonstrate that the stalk is the main landing platform for RTA on the ribosome and that pentameric organization of the stalk accelerates recruitment of RTA to the ribosome for depurination. Our results suggest that multiple copies of the stalk proteins might also increase the scavenging ability of the ribosome for the translational GTPases.

A Role for ETO-2 In Megakaryocytic Inhibition by Cyclic AMP Agonists

AbstractAbstract 289The regulation of megakaryopoiesis by cyclic AMP (cAMP) signaling forms the basis for treatment approaches to thrombocytosis in patients with myeloproliferative neoplasms. Anagrelide, the longstanding treatment of choice, is an inhibitor of the cAMP catabolizing enzyme phosphodiesterase III, and acts to lower platelet counts by inhibiting early stages of megakaryocytic differentiation. Essential for programming megakaryocytic differentiation is the transcriptionally activating pentameric complex consisting of GATA-1, SCL/TAL-1, an E-protein (either E2A or the similar HEB), and the bridging factors Lmo2 and Ldb1. This complex becomes repressive upon the recruitment of the co-repressor ETO-2 (also called MTG16 and CBFA2T3) by either SCL or E-protein components. ETO-2 has also been described as an A-kinase floating protein (AKAP). AKAPs bind the regulatory subunits of Protein kinase A (PKA) and tether the kinase within a specific compartment of the cell, ensuring proximity to appropriate substrates following kinase activation. The current study addresses the hypothesis that elevations in cAMP, such as those induced by anagrelide, inhibit megakaryocytic differentiation through the activation of a novel PKA signaling pathway involving ETO-2 and other components of the pentameric complex. Using peripheral blood mobilized human CD34+ progenitors cultured under conditions promoting megakaryopoiesis, we have confirmed that 500 nM anagrelide or 10 μ M forskolin, a direct adenylyl cyclase agonist, suppress upregulation of CD41 by two to three-fold as well as polyploidization, as determined by FACS on day 6 cultures. Pre-treatment of cultures with 10 μ M H89, a specific inhibitor of PKA, prevented the inhibitory effect of forskolin on megakaryopoiesis, as reflected by CD41 upregulation at day 6. Analysis of the kinetics of PKA activation, by immunoblotting for phospho-CREB and for phospho-PKA substrates (Cell Signaling), surprisingly showed only transient PKA induction by forskolin, with signals returning to baseline level after 4 hours. Additionally, washout experiments in which cells were treated with forskolin for only the first 4 hours of megakaryocytic culture showed severely blunted CD41 upregulation on day 6. These findings argue that elevations in cAMP inhibit megakaryocytic differentiation via activation of PKA and that a brief window of PKA activation during early megakaryopoiesis suffices for a stable blockade of differentiation. To determine a potential role for ETO-2, expression levels were analyzed at various times during megakaryocytic culture. In contrast to the rapid downregulation described in fetal murine megakaryocyte culture (Hamlett I. et al. Blood 112:2738-2749, 2008), human CD34+ progenitors showed no downregulation of ETO-2 mRNA or protein during megakaryocytic differentiation, nor were levels significantly affected by forskolin treatment. To study the functional role of ETO-2, we utilized shRNA knockdown in CD34+ hematopoietic progenitor cells undergoing megakaryocytic culture. In contrast to cells transduced with control vector, cells expressing shRNA targeting ETO-2 showed resistance to the inhibitory effects of elevated cAMP (n = 3). In examining potential targets within the pentameric complex, we found that elevations in cAMP reproducibly lowered the protein and mRNA levels of E2A by 50% while not affecting mRNA levels of another E protein, HEB. Altogether, these data suggest that cAMP mediates inhibition of megakaryopoiesis via activation of PKA, which utilizes ETO-2, possibly as an AKAP, to modulate the composition of an essential activating complex by downregulating E2A. Interestingly, E2A+/− mice are impaired in their ability to generate megakaryocytic progenitors from bipotent megakaryocyte-erythroid precursors (Semerad CL et al. PNAS 106:1930-1935, 2009). This novel signaling pathway contributes to an understanding of the mechanism of action of anagrelide and also further elucidates the basic regulatory circuitry governing determination of the megakaryocytic lineage. Disclosures:No relevant conflicts of interest to declare.

Dimeric Arrangement of the Parathyroid Hormone Receptor and a Structural Mechanism for Ligand-induced Dissociation

The parathyroid hormone receptor (PTH1R) is a class B G protein-coupled receptor that is activated by parathyroid hormone (PTH) and PTH-related protein (PTHrP). Little is known about the oligomeric state of the receptor and its regulation by hormone. The crystal structure of the ligand-free PTH1R extracellular domain (ECD) reveals an unexpected dimer in which the C-terminal segment of both ECD protomers forms an alpha-helix that mimics PTH/PTHrP by occupying the peptide binding groove of the opposing protomer. ECD-mediated oligomerization of intact PTH1R was confirmed in living cells by bioluminescence and fluorescence resonance energy transfer experiments. As predicted by the structure, PTH binding disrupted receptor oligomerization. A receptor rendered monomeric by mutations in the ECD retained wild-type PTH binding and cAMP signaling ability. Our results are consistent with the hypothesis that PTH1R forms constitutive dimers that are dissociated by ligand binding and that monomeric PTH1R is capable of activating G protein.

Ginkgolide X Is a Potent Antagonist of Anionic Cys-loop Receptors with a Unique Selectivity Profile at Glycine Receptors

The novel ginkgolide analog ginkgolide X was characterized functionally at human glycine and gamma-aminobutyric acid type A receptors (GlyRs and GABA(A)Rs, respectively) in the fluorescence-based FLIPR(TM) Membrane Potential assay. The compound inhibited the signaling of all GABA(A)R subtypes included in the study with high nanomolar/low micromolar IC(50) values, except the rho 1 receptor at which it was a significantly weaker antagonist. Ginkgolide X also displayed high nanomolar/low micromolar IC(50) values at the homomeric alpha1 and alpha2 GlyRs, whereas it was inactive at the heteromeric alpha 1 beta and alpha 2 beta subtypes at concentrations up to 300 microm. Thus, the functional properties of the compound were significantly different from those of the naturally occurring ginkgolides A, B, C, J, and M but similar to those of picrotoxin. In a mutagenesis study the 6' M2 residues in the GlyR ion channel were identified as the primary molecular determinant of the selectivity profile of ginkgolide X, and a 6' M2 ring consisting of five Thr residues was found to be of key importance for its activity at the GABA(A)R. Conformational analysis and docking of low-energy conformations of the native ginkgolide A and ginkgolide X into a alpha1 GlyR homology model revealed two distinct putative binding sites formed by the 6' M2 residues together with the 2' residues and the 10' and 13' residues, respectively. Thus, we propose that the distinct functionalities of ginkgolide X compared with the other ginkgolides could arise from different flexibility and thus different binding modes to the ion channel of the anionic Cys-loop receptor.

Structural Basis for Translation Factor Recruitment to the Eukaryotic/Archaeal Ribosomes

The archaeal ribosomal stalk complex has been shown to have an apparently conserved functional structure with eukaryotic pentameric stalk complex; it provides access to eukaryotic elongation factors at levels comparable to that of the eukaryotic stalk. The crystal structure of the archaeal heptameric (P0(P1)(2)(P1)(2)(P1)(2)) stalk complex shows that the rRNA anchor protein P0 consists of an N-terminal rRNA-anchoring domain followed by three separated spine helices on which three P1 dimers bind. Based on the structure, we have generated P0 mutants depleted of any binding site(s) for P1 dimer(s). Factor-dependent GTPase assay of such mutants suggested that the first P1 dimer has higher activity than the others. Furthermore, we constructed a model of the archaeal 50 S with stalk complex by superposing the rRNA-anchoring domain of P0 on the archaeal 50 S. This model indicates that the C termini of P1 dimers where translation factors bind are all localized to the region between the stalk base of the 50 S and P0 spine helices. Together with the mutational experiments we infer that the functional significance of multiple copies of P1 is in creating a factor pool within a limited space near the stalk base of the ribosome.

Generation of branched actin networks: assembly and regulation of the N‐WASP and WAVE molecular machines

The Arp2/3 complex is a molecular machine that generates branched actin networks responsible for membrane remodeling during cell migration, endocytosis, and other morphogenetic events. This machine requires activators, which themselves are multiprotein complexes. This review focuses on recent advances concerning the assembly of stable complexes containing the most-studied activators, N-WASP and WAVE proteins, and the level of regulation that is provided by these complexes. N-WASP is the paradigmatic auto-inhibited protein, which is activated by a conformational opening. Even though this regulation has been successfully reconstituted in vitro with isolated N-WASP, the native dimeric complex with a WIP family protein has unique additional properties. WAVE proteins are part of a pentameric complex, whose basal state and activated state when bound to the Rac GTPase were recently clarified. Moreover, this review attempts to put together diverse observations concerning the WAVE complex in the conceptual frame of an in vivo assembly pathway that has gained support from the recent identification of a precursor.

Inactivation of the Bacterial Mechanosensitive Channel MscL Involves Flexible Transmembrane Helices and a ‘Dry’ Gate

MscL, a mechanosensitive channel of large conductance, is an emergency release valve residing in the cytoplasmic membrane of E. coli. Under osmotic shock, when membrane tension approaches the lytic limit of ∼10 mN/m, MscL opens a 3 nm pore relieving osmotic stress. The conformational transition in the pentameric complex was previously envisioned as a tilting iris-like motion of tightly coupled pairs of the centrally located TM1 and peripheral TM2 helices. Wetting the hydrophobic constriction formed by the rings of L19 and V23 was identified as is the rate-limiting step over a barrier of more than 50 kT. While adaptation of MscL was reported, the channel was generally considered non-inactivating. Special pressure protocols involving prolonged conditioning steps and short saturating test pulses revealed that after a 30 s exposure to half-saturating pressure (p0.5) in spheroplast patches, about 20% of MscL population reversibly inactivates. The channels return to the resting state within 1 s upon pressure release. Introduction of a flexible double glycine motif (A91G/I92G) in TM2 dramatically increased the rate of inactivation resulting in a 90% silent channel population after a 10 s step to p0.5. Single-channel traces revealed a split of concerted 70 pA opening transitions into a staircase of irregular ∼7 pA substates in the double glycine mutant. The additional hydrophilic substitution in the constriction (V23T/A91G/I92G) pre-hydrates the pore, reduces p0.5 by ∼55% and, while generating multiple substates, completely abolishes inactivation. In extrapolated-motion simulations TM2 kinks at A91G/I92G and its C-terminal end separates from TM1. We propose that in WT MscL TM1s and TM2s are tightly coupled, whereas in the double glycine mutant the unsupported TM1s may reform the tight hydrophobic seal independent of the positions of TM2s thus creating a tension-insensitive non-conductive state.

Accelerated induction of mycobacterial antigen‐specific CD8+ T cells in the Mycobacterium tuberculosis‐infected lung by subcutaneous vaccination with Mycobacterium bovis bacille Calmette–Guérin

Both CD4(+) and CD8(+) T cells are important in protection against Mycobacterium tuberculosis infection. To evaluate the effect of vaccination with Mycobacterium bovis bacille Calmette-Guérin (BCG) on the CD8(+) T-cell response to pulmonary M. tuberculosis infection, we analyzed the kinetics of CD8(+) T cells specific to the mycobacterial Mtb32a(309-318) epitope, which is shared by M. tuberculosis and M. bovis BCG, in the lung of mice infected with M. tuberculosis. The CD8(+) T cells were detected by staining lymphocytes with pentameric major histocompatibility complex (MHC) class I H-2D(b-)Mtb32a(209-318) peptide complex and were analysed by flow cytometry. Mtb32a-specific CD8(+) T cells became detectable on day 14, and reached a plateau on day 21, in the lung of M. tuberculosis-infected unvaccinated mice. Subcutaneous vaccination with M. bovis BCG in the footpads induced Mtb32a-specific CD8(+) T cells in the draining lymph nodes (LNs) on day 7 and their numbers further increased on day 14. When M. bovis BCG-vaccinated mice were exposed to pulmonaryinfection with M. tuberculosis 4 weeks after vaccination, the Mtb32a-specific CD8(+) T cells in the infected lung became detectable on day 7 and reached a plateau on day 14, which was 1 week earlier than in the unvaccinated mice. The pulmonary CD8(+) T cells from the BCG-vaccinated M. tuberculosis-infected mice produced interferon-gamma in response to Mtb32a(209-318) peptide on day 7 of the infection, whereas those of unvaccinated mice did not. The results demonstrate that induction of mycobacterial antigen-specific protective CD8(+) T cells in the M. tuberculosis-infected lung is accelerated by subcutaneous vaccination with M. bovis BCG.