GxxxG Motif Research Articles

Distinct phenotypes of salivaricin-producing Ligilactobacillus salivarius isolated from the gastrointestinal tract of broiler chickens and laying hens: SALIVARICIN-PRODUCING LIGILACTOBACILLUS SALIVARIUS

Ligilactobacillus salivarius harbors bacteriocin genes in its repA-type megaplasmid, specifically salivaricin P (salP), a class IIb bacteriocin. This study aimed to differentiate 25 salP-positive Lig. salivarius strains isolated from the gastrointestinal tract (GIT) of broilers and laying hens. Results showed that 12 isolates were classified as Type A, with active bacteriocins, while the rest were Type B, with no active bacteriocins. In vitro and in silico characterization of salP bacteriocins revealed narrow-spectrum antibacterial activity against Listeria monocytogenes and Enterococcus faecalis. SalP bacteriocins were predicted as positively-charged, hydrophobic, small molecular weight (α, 4.097 kDa; ß, 4.285 kDa) bacteriocins with characteristic GXXXG motif. Investigation of the salP gene cluster based on genomic data revealed that Type B strains lacked the lanT and hlyD genes that encode export proteins dedicated to the modification and extracellular transport of mature salP peptides. However, two Type B strains (B4311 and B5258) showed inhibitory activity against L. monocytogenes ATCC19114. Multiplex PCR analysis and synteny mapping analysis revealed that B4311 and B5258 strains harbored the lanT gene, highlighting the importance of LanT protein in the cleavage of leader peptide and excretion of mature peptides. Further analysis revealed that the resistance of Type B strains to salP was attributable to the presence of a dedicated immunity protein, blurring the evolutionary significance of producing active bacteriocins for competitive advantage. Additionally, the loss of export proteins occurred in a polyphyletic manner, consistent with the genetic plasticity of the repA-type megaplasmid. This suggests that the loss of lanT and hlyD is likely in the presence of limited nutritional competitors. In conclusion, the observed differences in salivaricin production of Lig. salivarius exists independent of isolation host and that Type A and Type B strains can co-exist in the same environment. Finally, the functional characterization of active salP allows for a better understanding of its potential to control specific bacteria in human food and animal production.

On the abundance and importance of AXXXA sequence motifs in globular proteins and their involvement in Cβ[sbnd]Cβ interaction

The AXXXA and GXXXG motifs are frequently observed in helices, especially in membrane proteins. The motif GXXXG is known to stabilize helix-helix association in membrane proteins via CαHO bonding. AXXXA sequence motif additionally stabilizes the folded state of proteins. We found 27,000 and 18,000 occurrences of AXXXA and GXXXG motifs in a non-redundant set of 6000 obligate homodimeric (OD) complexes. Interestingly, this is less pronounced in transient homodimers (TD) and heterodimers (HetD). On average each obligate homodimer contains four AXXXA motifs, it is 2 and 3.5 for HetD and TD, respectively. Focusing on the binding surface it is seen that 27 % of the ODs contain at least one AXXXA motif at the interface, whereas it is 17 % and 15 % for HetD and TD respectively. AXXXA predominantly stabilizes the OD quaternary structure via the side chain CβCβ interactions. This interaction is energetically favorable and is found to be a major driving force for OD quaternary structure stability. Cβ-Cβ interactions are observed ∼6 times higher than the known CαHO interaction for helix-helix stabilization. Two additional new interactions of CβO and OO are observed at the AXXXA containing interface regions. The occurrence of the motif gets drastically reduced if any of the terminal Ala residues are replaced by Gly. Our findings show the importance of AXXXA in providing stability to the quaternary structure through specific hydrophobic interactions and the specificity of the Ala residue at motif termini. The knowledge gained can be used for designing synthetic proteins of improved stability and for designing peptide-based therapeutics.

Delineating the Role of GxxxG Motif in Amyloidogenesis: A New Perspective in Targeting Amyloid-Beta Mediated AD Pathogenesis.

The pursuit of a novel structural motif that can shed light on the key functional attributes is a primary focus in the study of protein folding disorders. Decades of research on Alzheimer's disease (AD) have centered on the Amyloid β (Aβ) pathway, highlighting its significance in understanding the disorder. The diversity in the Aβ pathway and the possible silent tracks which are yet to discover, makes it exceedingly intimidating to the interdisciplinary scientific community. Over the course of AD research, Aβ has consistently been at the forefront of scientific inquiry and discussion. In this review, we epitomize the role of a potential structural motif (GxxxG motif) that may provide a new horizon to the Aβ conflict. We emphasize on how comprehensive understanding of this motif from a structure-function perspective may pave the way for designing novel therapeutics intervention in AD and related diseases.

Differential pairing of transmembrane domain GxxxG dimerization motifs defines two HLA-DR MHC class II conformers

MHC class II molecules function to present exogenous antigen-derived peptides to CD4 T cells to both drive T cell activation and to provide signals back into the class II antigen-presenting cell. Previous work established the presence of multiple GxxxG dimerization motifs within the transmembrane domains of MHC class II α and β chains across a wide range of species and revealed a role for differential GxxxG motif pairing in the formation of two discrete mouse class II conformers with distinct functional properties (i.e., M1-and M2-paired I-Ak class II). Biochemical and mutagenesis studies detailed herein extend this model to human class II by identifying an anti-HLA-DR mAb (Tü36) that selectively binds M1-paired HLA-DR molecules. Analysis of the HLA-DR allele reactivity of the Tü36 mAb helped define other HLA-DR residues involved in mAb binding. In silico modeling of both TM domain interactions and whole protein structure is consistent with the outcome of biochemical/mutagenesis studies and provides insight into the possible structural differences between the two HLA-DR conformers. Cholesterol depletion studies indicate a role for cholesterol-rich membrane domains in the formation/maintenance of Tü36 mAb reactive DR molecules. Finally, phylogenetic analysis of the amino acid sequences of Tü36-reactive HLA-DR β chains reveals a unique pattern of both Tü36 mAb reactivity and key amino acid polymorphisms. In total, these studies bring the paradigm M1/M2-paired MHC class II molecules to the human HLA-DR molecule and suggest that the functional differences between these conformers defined in mouse class II extend to the human immune system.

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 G39xxxG43 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 G39xxxG43 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.

Computer modeling of the hexameric channel of VacA from H. pylori.

Helicobacter pylori (H. pylori) infects over half of the human population in the world and is a main cause of stomach ulcers. As one of the major virulence factors secreted by H. pylori, Vacuolating toxin (VacA) molecules can insert into lipid bilayers and oligomerize to form a hexameric membrane channel. It has been proposed that cell vacuolation results primarily from the formation of such channels. However, structural and functional studies of VacA transmembrane (TM) channel have been hindered by the challenge of resolving the structure of this TM domain experimentally. Here, we report a computational protocol for modeling symmetric TM helical bundles and propose a structural model of the hexameric VacA channel. In our model, van der Waals packing and Cα-H···O hydrogen bonding of small residues Gly14 and Gly18 stabilize the association of adjacent helical pairs, consistent with the frequent occurrence of GxxxG motif observed in TM helix-helix interface. Through performing molecular dynamics (MD) simulations iteratively, we refine and validate our model, demonstrating that the resulting channel is stable and supports continuous pore water flow. Additionally, we simulate the single VacA TM helix, capturing the spontaneous partial insertion of the VacA N-terminal hydrophobic region into a lipid bilayer. Our results provide molecular details of how VacA participates in vacuolating the cell and inducing cell apoptosis. The computational protocol thus established may be employed to model other pore-forming toxins without a known structure.

GxxxG Motif Stabilize Ion-Channel like Pores through Cα-H···O Interaction in Aβ (1-40).

Aβ (1-40) can transfer from the aqueous phase to the bilayer and thus form stable ion-channel-like pores where the protein has alpha-helical conformation. The stability of the pores is due to the presence of the GXXXG motif. It has been reported that these ion-channel-like pores are stabilized by a Cα-H···O hydrogen bond that is established between a glycine of the GXXXG sequence of an alpha-helix and another amino acid of a vicinal alpha-helix. However, conflicting data are reported in the literature. Some authors have suggested that hydrogen bonding does not have a stabilizing function. Here we synthesized pentapeptides having a GXXXG motif to explore its role in pore stability. We used molecular dynamics simulations, quantum mechanics, and experimental biophysical techniques to determine whether hydrogen bonding was formed and had a stabilizing function in ion-channel-like structures. Starting from our previous molecular dynamics data, molecular quantum mechanics simulations, and ATR data showed that a stable ion-channel-like pore formed and a band centered at 2910 cm-1 was attributed to the interaction between Gly 7 of an alpha-helix and Asp 23 of a vicinal alpha-helix.

Helical stability of the GnTV transmembrane domain impacts on SPPL3 dependent cleavage

Signal-Peptide Peptidase Like-3 (SPPL3) is an intramembrane cleaving aspartyl protease that causes secretion of extracellular domains from type-II transmembrane proteins. Numerous Golgi-localized glycosidases and glucosyltransferases have been identified as physiological SPPL3 substrates. By SPPL3 dependent processing, glycan-transferring enzymes are deactivated inside the cell, as their active site-containing domain is cleaved and secreted. Thus, SPPL3 impacts on glycan patterns of many cellular and secreted proteins and can regulate protein glycosylation. However, the characteristics that make a substrate a favourable candidate for SPPL3-dependent cleavage remain unknown. To gain insights into substrate requirements, we investigated the function of a GxxxG motif located in the transmembrane domain of N-acetylglucosaminyltransferase V (GnTV), a well-known SPPL3 substrate. SPPL3-dependent secretion of the substrate’s ectodomain was affected by mutations disrupting the GxxxG motif. Using deuterium/hydrogen exchange and NMR spectroscopy, we studied the effect of these mutations on the helix flexibility of the GnTV transmembrane domain and observed that increased flexibility facilitates SPPL3-dependent shedding and vice versa. This study provides first insights into the characteristics of SPPL3 substrates, combining molecular biology, biochemistry, and biophysical techniques and its results will provide the basis for better understanding the characteristics of SPPL3 substrates with implications for the substrates of other intramembrane proteases.

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

A CD36 transmembrane domain peptide interrupts CD36 interactions with membrane partners on macrophages and inhibits atherogenic functions

CD36 is a transmembrane glycoprotein receptor for oxidized low density lipoprotein (LDL) and other endogenous danger signals and promotes athero-thrombotic processes. CD36 has been shown to associate physically with other transmembrane proteins, including integrins, tetraspanins, and toll-like receptors, which modulate CD36-mediated cell signaling. The CD36 N-terminal transmembrane domain (nTMD) contains a GXXXG sequence motif that mediates protein-protein interactions in many membrane proteins. We thus hypothesized that the nTMD is involved in CD36 interactions with other membrane proteins. CD36 interactions with partner cell surface proteins on murine peritoneal macrophages were detected with an immunofluorescence-based proximity ligation cross linking assay (PLA) and confirmed by immunoprecipitation/immunoblot. Prior to performing these assays, cells were incubated with a synthetic 29 amino acid peptide containing the 22 amino acid of CD36 nTMD or a control peptide in which the glycine residues in GXXXG motif were replaced by valines. In functional experiments, macrophages were preincubated with peptides and then treated with oxLDL to assess LDL uptake, foam cell formation, ROS formation and cell migration. CD36 nTMD peptide treated cells compared to untreated or control peptide treated cells showed decreased CD36 surface associations with tetraspanin CD9 and ameliorated pathologically important CD36 mediated responses to oxLDL, including uptake of DiI-labeled oxLDL, foam cell formation, ROS generation, and inhibition of migration.

Importance of transmembrane helix 4 of l-alanine exporter AlaE in oligomer formation and substrate export activity in Escherichia coli.

AlaE is the smallest amino acid exporter identified in Escherichia coli. It exports l-alanine using the proton motive force and plays a pivotal role in maintaining intracellular l-alanine homeostasis by acting as a safety valve. However, our understanding of the molecular mechanisms of substrate export by AlaE is still limited because structural information is lacking. Due to its small size (149 amino acid residues), it has been speculated that AlaE functions by forming an oligomer. In this study, we performed chemical cross-linking and pull-down assays and showed that AlaE indeed generates homo-oligomers as a functional unit. Previous random mutagenesis experiments identified three loss-of-function AlaE point mutations in the predicted transmembrane helix 4 (TM4) region, two of which are present in the GxxxG motif. When alanine-scanning mutagenesis was applied to the TM4 region, the AlaE derivatives that had amino acid substitutions around the GxxxG motif showed low l-alanine export activities, indicating that the GxxxG motif in TM4 plays an important role in substrate export. However, these AlaE variants with low activity could still form oligomers. We therefore concluded that AlaE forms homo-oligomers and that the GxxxG motif in the TM4 region plays an essential role in AlaE activity but is not involved in AlaE oligomer formation.

Whole-Genome Sequencing, Phylogenetic and Genomic Analysis of Lactiplantibacillus pentosus L33, a Potential Probiotic Strain Isolated From Fermented Sausages

Lactobacillus is a diverse genus that includes species of industrial and biomedical interest. Lactiplantibacillus pentosus, formerly known as Lactobacillus pentosus, is a recently reclassified species, that contains strains isolated from diverse environmental niches, ranging from fermented products to mammalian gut microbiota. Importantly, several L. pentosus strains present health-promoting properties, such as immunomodulatory and antiproliferative activities, and are regarded as potential probiotic strains. In this study, we present the draft genome sequence of the potential probiotic strain L. pentosus L33, originally isolated from fermented sausages. Comprehensive bioinformatic analysis and whole-genome annotation were performed to highlight the genetic loci involved in host-microbe interactions and the probiotic phenotype. Consequently, we found that this strain codes for bile salt hydrolases, adhesins and moonlighting proteins, and for Class IIb bacteriocin peptides lacking the GxxxG and GxxxG-like motifs, crucial for their inhibitory activity. Its adhesion ability was also validated in vitro, on human cancer cells. Furthermore, L. pentosus L33 contains an exopolysaccharide (EPS) biosynthesis cluster, and it does not carry transferable antibiotic resistance genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and CAZymes analyses showed that L. pentosus L33 possesses biosynthetic pathways for seven amino acids, while it can degrade a wide array of carbohydrates. In parallel, Clusters of Orthologous Groups (COGs) and KEGG profiles of L. pentosus L33 are similar to those of 26 L. pentosus strains, as well as of two well documented L. plantarum probiotic strains. Conclusively, L. pentosus L33 exhibits good probiotic potential, although further studies are needed to elucidate the extent of its biological properties.

Autophagy-competent mitochondrial translation elongation factor TUFM inhibits caspase-8-mediated apoptosis

Mitochondria support multiple cell functions, but an accumulation of dysfunctional or excessive mitochondria is detrimental to cells. We previously demonstrated that a defect in the autophagic removal of mitochondria, termed mitophagy, leads to the acceleration of apoptosis induced by herpesvirus productive infection. However, the exact molecular mechanisms underlying activation of mitophagy and regulation of apoptosis remain poorly understood despite the identification of various mitophagy-associated proteins. Here, we report that the mitochondrial translation elongation factor Tu, a mitophagy-associated protein encoded by the TUFM gene, locates in part on the outer membrane of mitochondria (OMM) where it acts as an inhibitor of altered mitochondria-induced apoptosis through its autophagic function. Inducible depletion of TUFM potentiated caspase-8-mediated apoptosis in virus-infected cells with accumulation of altered mitochondria. In addition, TUFM depletion promoted caspase-8 activation induced by treatment with TNF-related apoptosis-inducing ligand in cancer cells, potentially via dysregulation of mitochondrial dynamics and mitophagy. Importantly, we revealed the existence of and structural requirements for autophagy-competent TUFM on the OMM; the GxxxG motif within the N-terminal mitochondrial targeting sequences of TUFM was required for self-dimerization and mitophagy. Furthermore, we found that autophagy-competent TUFM was subject to ubiquitin-proteasome-mediated degradation but stabilized upon mitophagy or autophagy activation. Moreover, overexpression of autophagy-competent TUFM could inhibit caspase-8 activation. These studies extend our knowledge of mitophagy regulation of apoptosis and could provide a novel strategic basis for targeted therapy of cancer and viral diseases.

Conserved GxxxG and WN motifs of MIC13 are essential for bridging two MICOS subcomplexes

Mitochondrial ultrastructure is highly adaptable and undergoes dynamic changes upon physiological and energetic cues. MICOS (mitochondrial contact site and cristae organizing system), a large oligomeric protein complex, maintains mitochondrial ultrastructure as it is required for formation of crista junctions (CJs) and contact sites. MIC13 acts as a critical bridge between two MICOS subcomplexes. Deletion of MIC13 causes loss of CJs resulting in cristae accumulating as concentric rings and specific destabilization of the MIC10-subcomplex. Mutations in MIC13 are associated with infantile lethal mitochondrial hepato-encephalopathy, yet functional regions within MIC13 were not known. To identify and characterize such regions, we systemically generated 20 amino-acids deletion variants across the length of MIC13. While deletion of many of these regions of MIC13 is dispensable for its stability, the N-terminal region and a stretch between amino acid residues 84 and 103 are necessary for the stability and functionality of MIC13. We could further locate conserved motifs within these regions and found that a GxxxG motif in the N-terminal transmembrane segment and an internal WN motif are essential for stability of MIC13, formation of the MIC10-subcomplex, interaction with MIC10- and MIC60-subcomplexes and maintenance of cristae morphology. The GxxxG motif is required for membrane insertion of MIC13. Overall, we systematically found important conserved residues of MIC13 that are required to perform the bridging between the two MICOS subcomplexes. The study improves our understanding of the basic molecular function of MIC13 and has implications for its role in the pathogenesis of a severe mitochondrial disease.

All-Atom Molecular Dynamics Elucidating Molecular Mechanisms of Single-Transmembrane Model Peptide Dimerization in a Lipid Bilayer.

Protein–protein interactions between transmembrane helices are essential elements for membrane protein structures and functions. To understand the effects of peptide sequences and lipid compositions on these interactions, single-molecule experiments using model systems comprising artificial peptides and membranes have been extensively performed. However, their dynamic behavior at the atomic level remains largely unclear. In this study, we applied the all-atom molecular dynamics (MD) method to simulate the interactions of single-transmembrane helical peptide dimers in membrane environments, which has previously been analyzed by single-molecule experiments. The simulations were performed with two peptides (Ala- and Leu-based artificially designed peptides, termed “host peptide”, and the host peptide added with the GXXXG motif, termed “GXXXG peptide”), two membranes (pure-POPC and POPC mixed with 30% cholesterols), and two dimer directions (parallel and antiparallel), consistent with those in the previous experiment. As a result, the MD simulations with parallel dimers reproduced the experimentally observed tendency that introducing cholesterols weakened the interactions in the GXXXG dimer and facilitated those in the host dimer. Our simulation suggested that the host dimer formed hydrogen bonds but the GXXXG dimer did not. However, some discrepancies were also observed between the experiments and simulations. Limitations in the space and time scales of simulations restrict the large-scale undulation and peristaltic motions of the membranes, resulting in differences in lateral pressure profiles. This effect could cause a discrepancy in the rotation angles of helices against the membrane normal.

Leucine heptad motifs within transmembrane domains affect function and oligomerization of human organic anion transporting polypeptide 1B1

Organic anion transporting polypeptides (OATPs) are transmembrane proteins responsible for the uptake of a wide range of endogenous compounds and clinically important drugs. The liver-specific OATP1B1 serves crucial roles in the removal of many orally administered drugs. The proper function of the transporter hence is essential for the pharmacokinetics of various therapeutic agents. Membrane proteins tend to form oligomers that are important for their stability, targeting and/or interactions with the substrates. Previous study in our laboratory revealed that OATP1B1 may form homo-oligomers and that a GXXXG motif localized at transmembrane domain 8 (TM8) may affect its oligomerization. In the current study, three short-form leucine heptad repeats within the transmembrane domains of OATP1B1 were investigated. It was found that the disruption of leucine heptad repeats within TM3 dramatically reduced the uptake function and protein-protein association of OATP1B1; while within TM8, only L378 is essential for the function of OATP1B1 and alanine replacement of L378 exhibited no effect on the oligomerization. The fragmental expression of TM3 interfered with the association of OATP1B1 homo-oligomers as well as its association with OATP1B3, which is also selectively expressed at human hepatocytes, suggesting that the region may be shared by both transporters for their protein-protein interactions.

Mechanism of recognition of parallel G-quadruplexes by DEAH/RHAU helicase DHX36 explored by molecular dynamics simulations

Because of high stability and slow unfolding rates of G-quadruplexes (G4), cells have evolved specialized helicases that disrupt these non-canonical DNA and RNA structures in an ATP-dependent manner. One example is DHX36, a DEAH-box helicase, which participates in gene expression and replication by recognizing and unwinding parallel G4s. Here, we studied the molecular basis for the high affinity and specificity of DHX36 for parallel-type G4s using all-atom molecular dynamics simulations. By computing binding free energies, we found that the two main G4-interacting subdomains of DHX36, DSM and OB, separately exhibit high G4 affinity but they act cooperatively to recognize two distinctive features of parallel G4s: the exposed planar face of a guanine tetrad and the unique backbone conformation of a continuous guanine tract, respectively. Our results also show that DSM-mediated interactions are the main contributor to the binding free energy and rely on making extensive van der Waals contacts between the GXXXG motifs and hydrophobic residues of DSM and a flat guanine plane. Accordingly, the sterically more accessible 5′-G-tetrad allows for more favorable van der Waals and hydrophobic interactions which leads to the preferential binding of DSM to the 5′-side. In contrast to DSM, OB binds to G4 mostly through polar interactions by flexibly adapting to the 5′-terminal guanine tract to form a number of strong hydrogen bonds with the backbone phosphate groups. We also identified a third DHX36/G4 interaction site formed by the flexible loop missing in the crystal structure.

Homology-guided identification of a conserved motif linking the antiviral functions of IFITM3 to its oligomeric state.

The interferon-inducible transmembrane (IFITM) proteins belong to the Dispanin/CD225 family and inhibit diverse virus infections. IFITM3 reduces membrane fusion between cells and virions through a poorly characterized mechanism. Mutation of proline-rich transmembrane protein 2 (PRRT2), a regulator of neurotransmitter release, at glycine-305 was previously linked to paroxysmal neurological disorders in humans. Here, we show that glycine-305 and the homologous site in IFITM3, glycine-95, drive protein oligomerization from within a GxxxG motif. Mutation of glycine-95 (and to a lesser extent, glycine-91) disrupted IFITM3 oligomerization and reduced its antiviral activity against Influenza A virus. An oligomerization-defective variant was used to reveal that IFITM3 promotes membrane rigidity in a glycine-95-dependent and amphipathic helix-dependent manner. Furthermore, a compound which counteracts virus inhibition by IFITM3, Amphotericin B, prevented the IFITM3-mediated rigidification of membranes. Overall, these data suggest that IFITM3 oligomers inhibit virus-cell fusion by promoting membrane rigidity.

A Poplar Rust Effector Protein Associates with Protein Disulfide Isomerase and Enhances Plant Susceptibility.

Melampsora larici-populina (Mlp), the causal agent of Populus leaf rust, secretes an array of effectors into the host through the haustorium to gain nutrients and suppress immunity. The precise mechanisms by which these effectors promote virulence remain unclear. To address this question, we developed a transgenic Arabidopsis line expressing a candidate effector, Mlp124357. Constitutive expression of the effector increased plant susceptibility to pathogens. A GxxxG motif present in Mlp124357 is required for its subcellular localization at the vacuolar membrane of the plant cell, as replacement of the glycine residues with alanines led to the delocalization of Mlp124357 to the nucleus and cytoplasm. We used immunoprecipitation and mass spectrometry (MS) to identify Mlp124357 interaction partners. Only one of the putative interaction partners knock-out line caused delocalization of the effector, indicating that Arabidopsis protein disulfide isomerase-11 (AtPDI-11) is required for the effector localization. This interaction was further confirmed by a complementation test, a yeast-two hybrid assay and a molecular modeling experiment. Moreover, localization results and infection assays suggest that AtPDI-11 act as a helper for Mlp124357. In summary, our findings established that one of Mlp effectors resides at the vacuole surface and modulates plant susceptibility.

In silico Analysis of Determinant Factors in Microbial Protease Thermostability

Thermostable proteases are one of the pivotal enzymatic groups which play fundamental roles in biotechnologyrelated industries. The identification of bacterial thermostable enzymes through screening programs is a time and cost consuming process. So, extensive bioinformatics and experimental studies have been conducted to reveal thermo stabilizing factors. The current study was aimed to evaluate distinctive indicators among 33 thermostable and 10 mesostable proteolytic enzymes. The frequency of individual amino acids, aliphatic indexes, melting temperatures, isoelectric points, as well as, the frequency of AXXXA and GXXXG motifs were determined and compared among these enzymes. In addition, types of proteolytic enzymes and their active sites were assigned. Moreover, the frequency of alpha helixes, polar surface regions, and packing volumes of these enzymes with the known structures were characterized. Results showed that the frequency of Ala and AXXXA motifs were significantly higher in thermostable proteolytic enzymes, while they possess lower contents of Met, His, Lys and Leu in comparison to mesostable enzymes (P<0.05). According to statistical analysis, thermostable proteolytic enzymes indicated meaningful lower packing volumes than mesostable enzymes (P<0.05). Findings of the current study in addition to more detailed investigations on the thermostability mechanisms of various protein families are essential for designing more efficient industrial enzymes with functional properties at high temperatures.