Glycine Zipper Research Articles

Thermodynamic Details of Pinholin S2168 Activation Revealed Using Alchemical Free Energy Simulations.

Pinholin S2168 is a viral integral membrane protein whose function is to form nanoscopic "pinholes" in bacterial cell membranes to induce cell lysis as part of the viral replication cycle. Pinholin can transition from an inactive to an active conformation by exposing a transmembrane domain (TMD1) to the extracellular fluid. Upon activation, several copies of the protein assemble via interactions among a second transmembrane domain (TMD2) to form a single pore, thus hastening cell lysis and viral escape. The following experiments provide conformational descriptors of pinholin in active and inactive states and elucidate the molecular driving forces that control pinholin activity. In the present study, molecular dynamics (MD) simulations have been used to refine experimentally derived conformational descriptors into an atomistically detailed model of irsS2168, an antiholin mutant. To provide additional details about the thermodynamics of pinholin activation and to overcome large intrinsic kinetic barriers to activation, alchemical free energy simulations have been conducted. Alchemical mutations reveal the change in folding free energy upon mutation. The results suggest that alchemical mutations are an effective tool to rationalize experimental observations and predict the effects of site mutations on conformational states for proteins integrated into lipid bilayers. S16F, A17Q, A17Q+G21Q, and A17Q+G21Q+G14Q mutants reveal how changes in hydrophilicity and disruption of the glycine zipper motif influence pinholin's thermodynamic equilibrium, favoring the active conformation. These findings align with experimental observations from DEER spectroscopy, demonstrating that mutations increasing the hydrophilicity of TMD1 promote activation by making TMD1 more likely to exit the membrane and enter the extracellular fluid.

SlyB encapsulates outer membrane proteins in stress-induced lipid nanodomains.

The outer membrane in Gram-negative bacteria consists of an asymmetric phospholipid-lipopolysaccharide bilayer that is densely packed with outer-membrane β-barrel proteins (OMPs) and lipoproteins1. The architecture and composition of this bilayer is closely monitored and is essential to cell integrity and survival2-4. Here we find that SlyB, a lipoprotein in the PhoPQ stress regulon, forms stable stress-induced complexes with the outer-membrane proteome. SlyB comprises a 10 kDa periplasmic β-sandwich domain and a glycine zipper domain that forms a transmembrane α-helical hairpin with discrete phospholipid- and lipopolysaccharide-binding sites. After loss in lipid asymmetry, SlyB oligomerizes into ring-shaped transmembrane complexes that encapsulate β-barrel proteins into lipid nanodomains of variable size. We find that the formation of SlyB nanodomains is essential during lipopolysaccharide destabilization by antimicrobial peptides or acute cation shortage, conditions that result in a loss of OMPs and compromised outer-membrane barrier function in the absence of a functional SlyB. Our data reveal that SlyB is a compartmentalizing transmembrane guard protein that is involved in cell-envelope proteostasis and integrity, and suggest that SlyB represents a larger family of broadly conserved lipoproteins with 2TM glycine zipper domains with the ability to form lipid nanodomains.

Characterization of TelE, a T7SS LXG Effector Exhibiting a Conserved C-Terminal Glycine Zipper Motif Required for Toxicity.

Streptococcus gallolyticus subsp. gallolyticus (SGG) is an opportunistic bacterial pathogen strongly associated with colorectal cancer. Here, through comparative genomics analysis, we demonstrated that the genetic locus encoding the type VIIb secretion system (T7SSb) machinery is uniquely present in SGG in two different arrangements. SGG UCN34 carrying the most prevalent T7SSb genetic arrangement was chosen as the reference strain. To identify the effectors secreted by this secretion system, we inactivated the essC gene encoding the motor of this machinery. A comparison of the proteins secreted by UCN34 wild type and its isogenic ΔessC mutant revealed six T7SSb effector proteins, including the expected WXG effector EsxA and three LXG-containing proteins. In this work, we characterized an LXG-family toxin named herein TelE promoting the loss of membrane integrity. Seven homologs of TelE harboring a conserved glycine zipper motif at the C terminus were identified in different SGG isolates. Scanning mutagenesis of this motif showed that the glycine residue at position 470 was crucial for TelE membrane destabilization activity. TelE activity was antagonized by a small protein TipE belonging to the DUF5085 family. Overall, we report herein a unique SGG T7SSb effector exhibiting a toxic activity against nonimmune bacteria. IMPORTANCE In this study, 38 clinical isolates of Streptococcus gallolyticus subsp. gallolyticus (SGG) were sequenced and a genetic locus encoding the type VIIb secretion system (T7SSb) was found conserved and absent from 16 genomes of the closely related S. gallolyticus subsp. pasteurianus (SGP). The T7SSb is a bona fide pathogenicity island. Here, we report that the model organism SGG strain UCN34 secretes six T7SSb effectors. One of the six effectors named TelE displayed a strong toxicity when overexpressed in Escherichia coli. Our results indicate that TelE is probably a pore-forming toxin whose activity can be antagonized by a specific immunity protein named TipE. Overall, we report a unique toxin-immunity protein pair and our data expand the range of effectors secreted through T7SSb.

A glycine zipper motif is required for the translocation of a T6SS toxic effector into target cells.

Type VI secretion systems (T6SSs) can deliver diverse toxic effectors into eukaryotic and bacterial cells. Although much is known about the regulation and assembly of T6SS, the translocation mechanism of effectors into the periplasm and/or cytoplasm of target cells remains elusive. Here, we use the Agrobacterium tumefaciens DNase effector Tde1 to unravel the mechanism of translocation from attacker to prey. We demonstrate that Tde1 binds to its adaptor Tap1 through the N-terminus, which harbors continuous copies of GxxxG motifs resembling the glycine zipper structure found in proteins involved in the membrane channel formation. Amino acid substitutions on G39 xxxG43 motif do not affect Tde1-Tap1 interaction and secretion but abolish its membrane permeability and translocation of its fluorescent fusion protein into prey cells. The data suggest that G39 xxxG43 governs the delivery of Tde1 into target cells by permeabilizing the cytoplasmic membrane. Considering the widespread presence of GxxxG motifs in bacterial effectors and pore-forming toxins, we propose that glycine zipper-mediated permeabilization is a conserved mechanism used by bacterial effectors for translocation across target cell membranes.

Modulation of the Human Thrombopoietin Receptor Conformation Uncouples JAK2 V617F-Driven from Cytokine-Induced Activation

Myeloproliferative Neoplasms are clonal diseases where mutations in hematopoietic stem cells (HSCs) result in cytokine-independent activation of the Thrombopoietin Receptor (TpoR) and aberrant proliferation and differentiation of HSCs and myeloid progenitors1. The most prevalent mutation, JAK2 V617F, is the basis of >95% of Polycythemia Vera (PV) and 60% of Essential Thrombocythemia (ET) and Myelofibrosis cases2. Current treatment strategies have focused on direct inhibition of JAK2 with little or no success and with poor specificity for the V617F mutant. Unlike JAK2, the TpoR is present at the cell surface and is more easily targetable. Using a system of controlled dimerization, we previously reported that different dimeric orientations of murine TpoR result in distinct signaling pathways and variable activation intensity3. Recently, others have demonstrated that extracellular ligands (diabodies) are able to modulate human TpoR conformation to influence downstream signaling4. Here, we investigated the possibility to modulate human TpoR conformation to specifically target JAK2 V617F driven activation while preserving natural cytokine-induced signaling. Using our system of controlled dimerization of TpoR transmembrane (TM) domain, we characterized the active and inactive conformation of human TpoR in presence of JAK2 WT or JAK2 V617F in hematopoietic cells. While murine TpoR is active in almost all (6 out of 7) dimeric conformations, only 3 conformations allowed activation of human TpoR. The discrepancy between murine and human TpoR is due to sequence differences in the TM domain that modulates dimerization. Remarkably, we discovered that different dimeric conformations mediate activation of JAK2 WT and JAK2 V617F in human but not in murine TpoR. Using a cysteine cross-linking assay on live cells, we validated that TpoR dimers exhibit a different conformation in presence of JAK2 V617F than this induced by the thrombopoietin (Tpo). Given the different dimeric conformations of TpoR induced by Tpo and JAK2 V617F, we hypothesize that modulation of TpoR conformation could allow specific inhibition of JAK2 V617F-driven activation while preserving Tpo-induced activation. We probed this by introducing a glycine zipper GxxxG motif to favor dimerization in a conformation that renders JAK2 V617F inactive but allows Tpo induced activation. Conversely, we mutated residues of the TM domain to specifically disrupt the conformation required for JAK2 V617F-mediated activation. Both strategies resulted in complete inactivation of JAK2-V617F driven activation of TpoR but preserved its full activation by Tpo and cell surface localization. Together, these results provide a first demonstration that modulation of TpoR dimeric conformation is a suitable strategy for specific inhibition of the JAK2 V617F-driven activation and opens the road for novel therapeutic avenues. 1. Vainchenker W, Kralovics R. Genetic basis and molecular pathophysiology of classical myeloproliferative neoplasms. Blood. 2017;129(6):667-679. 2. Constantinescu SN, Vainchenker W, Levy G, Papadopoulos N. Functional Consequences of Mutations in Myeloproliferative Neoplasms. Hemasphere. 2021;5(6):e578. 3. Staerk J, Defour JP, Pecquet C, et al. Orientation-specific signalling by thrombopoietin receptor dimers. Embo j. 2011;30(21):4398-4413. 4. Cui L, Moraga I, Lerbs T, et al. Tuning MPL signaling to influence hematopoietic stem cell differentiation and inhibit essential thrombocythemia progenitors. Proceedings of the National Academy of Sciences. 2021;118(2):e2017849118. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Analysis of virulence proteins in pathogenic Acinetobacter baumannii to provide early warning of zoonotic risk

Acinetobacter baumannii is a ubiquitous opportunistic pathogen usually with low virulence. In recent years, reports of increased pathogenicity of A. baumannii in livestock due to the migratory behaviour of wildlife have attracted public health attention. Our previous study reported that an A. baumannii strain isolated from dead chicks, CCGGD201101, showed enhanced pathogenicity, but the mechanism for increased virulence is not understood. Here, to screen potential virulence factors, the proteomes of the isolated strain CCGGD201101 and the standard strain ATCC19606 of A. baumannii were compared, and the possible virulence-enhancing mechanisms were further analysed. The 50 % lethal dose (LD50) values of CCGGD201101 and standard strain ATCC19606 in ICR mice were determined to verify their bacterial toxicity. 2D fluorescence difference gel electrophoresis (2D-DIGE) combined with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/TOF-MS) and quantitative real-time PCR (RTqPCR) were applied to screen and identify differentially expressed proteins or genes that may be related to virulence enhancement. Bioinformatics analyses based on proteinprotein interaction (PPI) networks were used to explore the function of potential virulence proteins. The pathogenicity of potential virulence factors was assessed by phylogenetic analyses and an animal infection model. The results showed that the LD50 of CCGGD201101 for mice was 1.186 × 106 CFU/mL, and the virulence was increased by 180.5-fold compared to ATCC19606. Forty-seven protein spots were significantly upregulated for the A. baumannii CCGGD201101 strain (fold change ≥1.5, p < 0.05). In total, 14 upregulated proteins were identified using proteomic analysis, and the mRNA expression levels of these proteins were nearly identical, with few exceptions. According to the PPI network and phylogenetic analyses, the I78 family peptidase inhibitor, 3-oxoacyl-ACP reductase FabG, and glycine zipper were screened as being closely related to the pathogenicity of bacteria. Furthermore, the I78 overexpression strains exhibited higher lethality in mouse infection models, which indicated that the I78 family peptidase inhibitor was a potential new virulence factor to enhance the pathogenicity of the A. baumannii CCGGD201101 strain. The present study helped us to better understand the mechanisms of virulence enhancement and provided a scientific basis for establishing an early warning system for enhanced virulence of A. baumannii from animals.

Assembly of higher-order SMN oligomers is essential for metazoan viability and requires an exposed structural motif present in the YG zipper dimer.

Protein oligomerization is one mechanism by which homogenous solutions can separate into distinct liquid phases, enabling assembly of membraneless organelles. Survival Motor Neuron (SMN) is the eponymous component of a large macromolecular complex that chaperones biogenesis of eukaryotic ribonucleoproteins and localizes to distinct membraneless organelles in both the nucleus and cytoplasm. SMN forms the oligomeric core of this complex, and missense mutations within its YG box domain are known to cause Spinal Muscular Atrophy (SMA). The SMN YG box utilizes a unique variant of the glycine zipper motif to form dimers, but the mechanism of higher-order oligomerization remains unknown. Here, we use a combination of molecular genetic, phylogenetic, biophysical, biochemical and computational approaches to show that formation of higher-order SMN oligomers depends on a set of YG box residues that are not involved in dimerization. Mutation of key residues within this new structural motif restricts assembly of SMN to dimers and causes locomotor dysfunction and viability defects in animal models.

The ATP synthase glycine zipper of the c subunits: From the structural to the functional role in mitochondrial biology of cardiovascular diseases

The ATP synthase glycine zipper of the c subunits: From the structural to the functional role in mitochondrial biology of cardiovascular diseases

Topology-Switching Membrane Peptides for Determination of the Active Transmembrane Dimer Interface of the Epha2 Receptor

The EphA2 receptor tyrosine kinase is an interesting target for cancer therapeutics, as its rogue activation promotes tumor growth and metastasis. EphA2 activation is sensitive to dimerization induced by its single transmembrane (TM) domain. We thus previously designed the pH-sensitive TYPE7 peptide, which selectively activates EphA2 by binding its TM domain (Alves et al. eLife, 2018). TYPE7 comprises the TM sequence of EphA2, but with Glutamate residues added onto one helical face. To study the mechanism of TYPE7-induced EphA2 activation, we systematically rotated the Glu-containing helical interface. We performed biophysical and cell biology techniques on the six resulting peptides and molecular dynamics (MD) simulations on two, to study their effect on TM binding and activation of EphA2. First, we studied the interaction of the peptides with the membrane region of EphA2 reconstituted in lipid vesicles. We observed that the peptides are helical in the membrane, and most of them interact with EphA2. However, several peptides failed to activate EphA2 in cells, as assessed by EphA2-mediated cell migration. Next, we performed MD simulations to understand why similar peptides affect EphA2 activation differently. We observed that an activating peptide stabilizes the EphA2 TM dimer in a glycine zipper conformation. In contrast, a non-activating peptide stabilizes a complex in which the EphA2 helices are separated in a non-native, not-activation-competent conformation. The MD results suggest a mechanism for TYPE7-induced EphA2 activation, where peptide binding stabilizes the more active TM conformation. This provides support for the ligand-induced rotation hypothesis for RTK activation, where different helical interfaces correspond to active and inactive states. Additionally, these studies provide a basis for the specificity of TYPE7, since changing the available TM interface results in an EphA2 TM conformation that is less activation-competent.

Functional Common and Rare ERBB2 Germline Variants Cooperate in Familial and Sporadic Cancer Susceptibility.

We investigated a Spanish and Catalan family in which multiple cancer types tracked across three generations, but for which no genetic etiology had been identified. Whole-exome sequencing of germline DNA from multiple affected family members was performed to identify candidate variants to explain this occurrence of familial cancer. We discovered in all cancer-affected family members a single rare heterozygous germline variant (I654V, rs1801201) in ERBB2/HER2, which is located in a transmembrane glycine zipper motif critical for ERBB2-mediated signaling and in complete linkage disequilibrium (D' = 1) with a common polymorphism (I655V, rs1136201) previously reported in some populations as associated with cancer risk. Because multiple cancer types occurred in this family, we tested both the I654V and the I655V variants for association with cancer across multiple tumor types in 6,371 cases of Northern European ancestry drawn from The Cancer Genome Atlas and 6,647 controls, and found that the rare variant (I654V) was significantly associated with an increased risk for cancer (OR = 1.40; P = 0.021; 95% confidence interval (CI), 1.05-1.89). Functional assays performed in HEK 293T cells revealed that both the I655V single mutant (SM) and the I654V;I655V double mutant (DM) stabilized ERBB2 protein and activated ERBB2 signaling, with the DM activating ERBB2 significantly more than the SM alone. Thus, our results suggest a model whereby heritable genetic variation in the transmembrane domain activating ERBB2 signaling is associated with both sporadic and familial cancer risk, with increased ERBB2 stabilization and activation associated with increased cancer risk. PREVENTION RELEVANCE: By performing whole-exome sequencing on germline DNA from multiple cancer-affected individuals belonging to a family in which multiple cancer types track across three generations, we identified and then characterized functional common and rare variation in ERBB2 associated with both sporadic and familial cancer. Our results suggest that heritable variation activating ERBB2 signaling is associated with risk for multiple cancer types, with increases in signaling correlated with increases in risk, and modified by ancestry or family history.

Structural and functional characterization of the pore-forming domain of pinholin S2168

Pinholin S2168 triggers the lytic cycle of bacteriophage φ21 in infected Escherichia coli Activated transmembrane dimers oligomerize into small holes and uncouple the proton gradient. Transmembrane domain 1 (TMD1) regulates this activity, while TMD2 is postulated to form the actual "pinholes." Focusing on the TMD2 fragment, we used synchrotron radiation-based circular dichroism to confirm its α-helical conformation and transmembrane alignment. Solid-state 15N-NMR in oriented DMPC bilayers yielded a helix tilt angle of τ = 14°, a high order parameter (Smol = 0.9), and revealed the azimuthal angle. The resulting rotational orientation places an extended glycine zipper motif (G40xxxS44xxxG48) together with a patch of H-bonding residues (T51, T54, N55) sideways along TMD2, available for helix-helix interactions. Using fluorescence vesicle leakage assays, we demonstrate that TMD2 forms stable holes with an estimated diameter of 2 nm, as long as the glycine zipper motif remains intact. Based on our experimental data, we suggest structural models for the oligomeric pinhole (right-handed heptameric TMD2 bundle), for the active dimer (right-handed Gly-zipped TMD2/TMD2 dimer), and for the full-length pinholin protein before being triggered (Gly-zipped TMD2/TMD1-TMD1/TMD2 dimer in a line).

Aminoacid substitutions in the glycine zipper affect the conformational stability of amyloid beta fibrils

The aggregation of amyloid-beta peptides is associated with the pathogenesis of Alzheimer’s disease. The hydrophobic core of the amyloid beta sequence contains a GxxxG repeated motif, called glycine zipper, which involves crucial residues for assuring stability and promoting the process of fibril formation. Mutations in this motif lead to a completely different oligomerization pathway and rate of fibril formation. In this work, we have tested G33L and G37L residue substitutions by molecular dynamics simulations. We found that both protein mutations may lead to remarkable changes in the fibril conformational stability. Results suggest the disruption of the glycine zipper as a possible strategy to reduce the aggregation propensity of amyloid beta peptides. On the basis of our data, further investigations may consider this key region as a binding site to design/discover novel effective inhibitors.Communicated by Ramaswamy H. Sarma

Biogenic nanoparticles: Synthesis, stability and biocompatibility mediated by proteins of Pseudomonas aeruginosa

The development of environmental friendly new procedures for the synthesis of metallic nanoparticles is one of the main objectives of nanotechnology. Plants, algae, fungi and bacteria for the production of nanomaterials are viable alternatives due to their low cost, the absence of toxic waste production and their highly energy efficiency. It is also known that biosynthesized silver nanoparticles (AgNPs) show higher biocompatibility compared to the chemically-synthesized ones. In previous results, biosynthesized AgNPs were obtained from the supernatant of Pseudomonas aeruginosa, and they showed a bigger antimicrobial activity against different bacterial species compared to the chemically-synthesized ones. The aim of this work was to analyze the capping of biosynthesized AgNPs using techniques such as transmission electron microscopy (TEM), infrared spectroscopy (IR), and protein identification through mass spectrometry (MS) in order to identify the compounds responsible for their formation, stability and biocompatibility. The TEM images showed that AgNPs were surrounded by an irregular coverage. The IR spectrum showed that this coverage was composed of carbohydrates and/or proteins. Different proteins were identified in the capping associated to biosynthesized AgNPs. Some proteins seem to be important for their formation (Alkyl hydroperoxide reductase and Azurin) and stabilization (Outer membrane protein OprG and Glycine zipper 2 T M domain-containing protein). The proteins identified with the capability to interact with some biomolecules can be responsible for the biocompatibility and may be responsible for the bigger antimicrobial activity than AgNPs have previously shown. These results are pioneers in the identification of proteins in the capping of biosynthesized AgNPs.

G37V mutation of Aβ42 induces a nontoxic ellipse-like aggregate: An in vitro and in silico study

The glycine zipper motif at the C-terminus of the β-amyloid (Aβ) peptide have been shown to strongly influence the formation of neurotoxic aggregates. A previous study showed that the G37L mutation dramatically reduces the Aβ toxicity in vivo and in vitro. However, the primary cause and mechanism of the glycine zipper motif on Aβ properties remain unknown. To gain molecular insights into the impact of glycine zipper on Aβ properties, we substituted the residue 37 of Glycine by Valine and studied the structural and biochemical properties of G37V mutation, Aβ42(37V), by using in vitro and in silico approaches. Unlike G37L mutation, the G37V mutation reduced toxicity substantially but did not significantly accelerate the aggregation rate or change the content of secondary structures. Further TEM analyses showed that the G37V mutation formed an ellipse-like aggregate rather than a network-like fibril as wild type or G37L mutation of Aβ42 form. This different aggregation morphology may be highly linked with the reduction of toxicity. To gain the insight for the different properties of Aβ42(37V), we studied the structure of Aβ42 and G37V mutation using the replica exchange molecular dynamics simulation. Our results demonstrate that although the overall secondary structure population is similar with Aβ42 and Aβ42(G37V), Aβ42(G37V) shows an increase in the β-turn and β-hairpin at residues 36–37 and the flexibility of the Asp23-Lys28 salt bridge. These unique structural features may be the possible reason to account for the ellipse-like morphology.

Transmembrane regions of bovine herpesvirus 1-encoded UL49.5 and glycoprotein M regulate complex maturation and ER-Golgi trafficking.

Bovine herpesvirus 1 (BoHV-1)-encoded UL49.5 (a homologue of herpesvirus glycoprotein N) can combine different functions, regulated by complex formation with viral glycoprotein M (gM). We aimed to identify the mechanisms governing the immunomodulatory activity of BoHV-1 UL49.5. In this study, we addressed the impact of gM/UL49.5-specific regions on heterodimer formation, folding and trafficking from the endoplasmic reticulum (ER) to the trans-Golgi network (TGN) - events previously found to be responsible for abrogation of the UL49.5-mediated inhibition of the transporter associated with antigen processing (TAP). We first established, using viral mutants, that no other viral protein could efficiently compensate for the chaperone function of UL49.5 within the complex. The cytoplasmic tail of gM, containing putative trafficking signals, was dispensable either for ER retention of gM or for the release of the complex. We constructed cell lines with stable co-expression of BoHV-1 gM with chimeric UL49.5 variants, composed of the BoHV-1 N-terminal domain fused to the transmembrane region (TM) from UL49.5 of varicella-zoster virus or TM and the cytoplasmic tail of influenza virus haemagglutinin. Those membrane-anchored N-terminal domains of UL49.5 were sufficient to form a complex, yet gM/UL49.5 folding and ER-TGN trafficking could be affected by the UL49.5 TM sequence. Finally, we found that leucine substitutions in putative glycine zipper motifs within TM helices of gM resulted in strong reduction of complex formation and decreased ability of gM to interfere with UL49.5-mediated major histocompatibility class I downregulation. These findings highlight the importance of gM/UL49.5 transmembrane domains for the biology of this conserved herpesvirus protein complex.

Coherent Feedforward Regulation of Gene Expression by Caulobacter σT and GsrN during Hyperosmotic Stress.

GsrN is a conserved small RNA that is under transcriptional control of the general stress sigma factor, σT, and that functions as a posttranscriptional regulator of Caulobacter crescentus survival under multiple stress conditions. We have defined features of GsrN structure that determine survival under hyperosmotic stress, and we have applied transcriptomic and proteomic methods to identify regulatory targets of GsrN under hyperosmotic conditions. The 5' end of GsrN, which includes a conserved cytosine-rich stem-loop structure, is necessary for cell survival after osmotic upshock. GsrN both activates and represses gene expression in this stress condition. Expression of an uncharacterized open reading frame predicted to encode a glycine zipper protein, osrP, is strongly activated by GsrN. Our data support a model in which GsrN physically interacts with osrP mRNA through its 5' C-rich stem-loop to enhance OsrP protein expression. We conclude that sigT, gsrN, and osrP form a coherent feedforward loop in which σT activates gsrN and osrP transcription during stress and GsrN activates OsrP protein expression at the posttranscriptional level. This study delineates transcriptional and posttranscriptional layers of Caulobacter gene expression control during hyperosmotic stress, uncovers a new regulatory target of GsrN, and defines a coherent feedforward motif in the Caulobacter general stress response (GSR) regulatory network.IMPORTANCE Bacteria inhabit diverse niches and must adapt their physiology to constant environmental fluctuations. A major response to environmental perturbation is to change gene expression. Caulobacter and other alphaproteobacteria initiate a complex gene expression program known as the general stress response (GSR) under conditions including oxidative stress, osmotic stress, and nutrient limitation. The GSR enables cell survival in these environments. Understanding how bacteria survive stress requires that we dissect gene expression responses, such as the GSR, at the molecular level. This study is significant, as it defines transcriptional and posttranscriptional layers of gene expression regulation in response to hyperosmotic stress. We further provide evidence that a coherent feedforward motif influences the system properties of the Caulobacter GSR pathway.

Glycine Zipper Motifs in Hepatitis C Virus Nonstructural Protein 4B Are Required for the Establishment of Viral Replication Organelles.

Hepatitis C virus (HCV) RNA replication occurs in tight association with remodeled host cell membranes, presenting as cytoplasmic accumulations of single-, double-, and multimembrane vesicles in infected cells. Formation of these so-called replication organelles is mediated by a complex interplay of host cell factors and viral replicase proteins. Of these, nonstructural protein 4B (NS4B), an integral transmembrane protein, appears to play a key role, but little is known about the molecular mechanisms of how this protein contributes to organelle biogenesis. Using forward and reverse genetics, we identified glycine zipper motifs within transmembrane helices 2 and 3 of NS4B that are critically involved in viral RNA replication. Foerster resonance energy transfer analysis revealed the importance of the glycine zippers in NS4B homo- and heterotypic self-interactions. Additionally, ultrastructural analysis using electron microscopy unraveled a prominent role of glycine zipper residues for the subcellular distribution and the morphology of HCV-induced double-membrane vesicles. Notably, loss-of-function NS4B glycine zipper mutants prominently induced single-membrane vesicles with secondary invaginations that might represent an arrested intermediate state in double-membrane vesicle formation. These findings highlight a so-far-unknown role of glycine residues within the membrane integral core domain for NS4B self-interaction and functional as well as structural integrity of HCV replication organelles.IMPORTANCE Remodeling of the cellular endomembrane system leading to the establishment of replication organelles is a hallmark of positive-strand RNA viruses. In the case of HCV, expression of the nonstructural proteins induces the accumulation of double-membrane vesicles that likely arise from a concerted action of viral and coopted cellular factors. However, the underlying molecular mechanisms are incompletely understood. Here, we identify glycine zipper motifs within HCV NS4B transmembrane segments 2 and 3 that are crucial for the protein's self-interaction. Moreover, glycine residues within NS4B transmembrane helices critically contribute to the biogenesis of functional replication organelles and, thus, efficient viral RNA replication. These results reveal how glycine zipper motifs in NS4B contribute to structural and functional integrity of the HCV replication organelles and, thus, viral RNA replication.

Contact-dependent killing by Caulobacter crescentus via cell surface-associated, glycine zipper proteins.

Most bacteria are in fierce competition with other species for limited nutrients. Some bacteria can kill nearby cells by secreting bacteriocins, a diverse group of proteinaceous antimicrobials. However, bacteriocins are typically freely diffusible, and so of little value to planktonic cells in aqueous environments. Here, we identify an atypical two-protein bacteriocin in the α-proteobacterium Caulobacter crescentus that is retained on the surface of producer cells where it mediates cell contact-dependent killing. The bacteriocin-like proteins CdzC and CdzD harbor glycine-zipper motifs, often found in amyloids, and CdzC forms large, insoluble aggregates on the surface of producer cells. These aggregates can drive contact-dependent killing of other organisms, or Caulobacter cells not producing the CdzI immunity protein. The Cdz system uses a type I secretion system and is unrelated to previously described contact-dependent inhibition systems. However, Cdz-like systems are found in many bacteria, suggesting that this form of contact-dependent inhibition is common.

Retraction notice: the SMN structure reveals its crucial role in snRNP assembly.

The spliceosome plays a fundamental role in RNA metabolism by facilitating pre-RNA splicing. To understand how this essential complex is formed, we have used protein crystallography to determine the first complete structures of the key assembler protein, SMN, and the truncated isoform, SMNΔ7, which is found in patients with the disease spinal muscular atrophy (SMA). Comparison of the structures of SMN and SMNΔ7 shows many similar features, including the presence of two Tudor domains, but significant differences are observed in the C-terminal domain, including 12 additional amino acid residues encoded by exon 7 in SMN compared with SMNΔ7. Mapping of missense point mutations found in some SMA patients reveals clustering around three spatial locations, with the largest cluster found in the C-terminal domain. We propose a structural model of SMN binding with the Gemin2 protein and a heptameric Sm ring, revealing a critical assembly role of the residues 260-294, with the differences at the C-terminus of SMNΔ7 compared with SMN likely leading to loss of small nuclear ribonucleoprotein (snRNP) assembly. The SMN complex is proposed to form a dimer driven by formation of a glycine zipper involving α helix formed by amino acid residues 263-294. These results explain how structural changes of SMN give rise to loss of SMN-mediated snRNP assembly and support the hypothesis that this loss results in atrophy of neurons in SMA.

Probing Structure and Dynamics of Transmembrane Alpha Helices of the S21 Pinholin Protein using Electron Paramagnetic Resonance Spectroscopy

The final step of the bacteriophage infection cycle is cell death through membrane lysis. The mechanism of lysis uses a small membrane protein called holin and a muralytic enzyme called endolysin. The holin protein of this study is the S21 pinholin, a small hole forming membrane protein comprised of two transmembrane α-helical domains, TMD1 and TMD2. Only TMD2 is required for membrane hole formation, whereas TMD1 acts as the active inhibitory domain of TMD2. Therefore, in the mechanism of hole formation TMD1 must externalize from the cell membrane. Although the function of TMD2 is well characterized, a lack of information exists regarding TMD1. It is currently believed that TMD1 serves no functionality once externalized from the membrane. However, there is literary evidence suggesting otherwise. It is hypothesized that externalized TMD1s interact with each other using the glycine zipper packing motif to inhibit any TMD1s from looping over to close off the holin hole once formed. These stabilizing interactions could aid in hole formation by stabilizing conformational changes of internal TMD2.This study will be the first time solid phase peptide synthesis (SPPS) is used to create the S21 pinholin protein. The pinholin will be site-specifically spin labeled with MTSL and purified using RP-HPLC. Next will be to determine the degree to which TMD1 is externalized through lipid system incorporation and CW-EPR techniques, followed by confirmation of TMD1 α-helical structure using Circular Dichroism. The TMD1 interactions will be probed using Pulsed EPR DEER technique.Additionally, the local secondary structure of the pinholin's α-helices will be probed using a newly developed deuterated amino acid substituted Pulsed EPR - ESEEM technique. This will be the first application of the technique to a real protein system.