FrzS acts as a polar beacon to recruit SgmX, a central activator of type IV pili during Myxococcus xanthus motility.

In rod-shaped bacteria, type IV pili (Tfp) promote twitching motility by assembling and retracting at the cell pole. In Myxococcus xanthus, a bacterium that moves in highly coordinated cell groups, Tfp are activated by a polar activator protein, SgmX. However, while it is known that the Ras-like protein MglA is required for unipolar targeting, how SgmX accesses the cell pole to activate Tfp is unknown. Here, we demonstrate that a polar beacon protein, FrzS, recruits SgmX at the cell pole. We identified two main functional domains, including a Tfp-activating domain and a polar-binding domain. Within the latter, we show that the direct binding of MglA-GTP unveils a hidden motif that binds directly to the FrzS N-terminal response regulator (CheY). Structural analyses reveal that this binding occurs through a novel binding interface for response regulator domains. In conclusion, the findings unveil the protein interaction network leading to the spatial activation of Tfp at the cell pole. This tripartite system is at the root of complex collective behaviours in this predatory bacterium.

A molecular switch controls assembly of bacterial focal adhesions.

Cell motility universally relies on spatial regulation of focal adhesion complexes (FAs) connecting the substrate to cellular motors. In bacterial FAs, the Adventurous gliding motility machinery (Agl-Glt) assembles at the leading cell pole following a Mutual gliding-motility protein (MglA)-guanosine 5'-triphosphate (GTP) gradient along the cell axis. Here, we show that GltJ, a machinery membrane protein, contains cytosolic motifs binding MglA-GTP and AglZ and recruiting the MreB cytoskeleton to initiate movement toward the lagging cell pole. In addition, MglA-GTP binding triggers a conformational shift in an adjacent GltJ zinc-finger domain, facilitating MglB recruitment near the lagging pole. This prompts GTP hydrolysis by MglA, leading to complex disassembly. The GltJ switch thus serves as a sensor for the MglA-GTP gradient, controlling FA activity spatially.

DnaJ (Hsp40 protein) binding to folded substrate impacts KplE1 prophage excision efficiency.

Temperate phages mediate gene transfer and can modify the properties of their host organisms through the acquisition of novel genes, a process called lysogeny. The KplE1 prophage is one of the 10 prophage regions in Escherichia coli K12 MG1655. KplE1 is defective for lysis but fully competent for site-specific recombination. The TorI recombination directionality factor is strictly required for prophage excision from the host genome. We have previously shown that DnaJ promotes KplE1 excision by increasing the affinity of TorI for its site-specific recombination DNA target. Here, we provide evidence of a direct association between TorI and DnaJ using in vitro cross-linking assays and limited proteolysis experiments that show that this interaction allows both proteins to be transiently protected from trypsin digestion. Interestingly, NMR titration experiments showed that binding of DnaJ involves specific regions of the TorI structure. These regions, mainly composed of α-helices, are located on a surface opposite the DNA-binding site. Taken together, we propose that DnaJ, without the aid of DnaK/GrpE, is capable of increasing the efficiency of KplE1 excision by causing a conformational stabilization that allows TorI to adopt a more favorable conformation for binding to its specific DNA target.

Structural and mechanistic insights into unusual thiol disulfide oxidoreductase.

Cytoplasmic desulfothioredoxin (Dtrx) from the anaerobe Desulfovibrio vulgaris Hildenborough has been identified as a new member of the thiol disulfide oxidoreductase family. The active site of Dtrx contains a particular consensus sequence, CPHC, never seen in the cytoplasmic thioredoxins and generally found in periplasmic oxidases. Unlike canonical thioredoxins (Trx), Dtrx does not present any disulfide reductase activity, but it presents instead an unusual disulfide isomerase activity. We have used NMR spectroscopy to gain insights into the structure and the catalytic mechanism of this unusual Dtrx. The redox potential of Dtrx (-181 mV) is significantly less reducing than that of canonical Trx. A pH dependence study allowed the determination of the pK(a) of all protonable residues, including the cysteine and histidine residues. Thus, the pK(a) values for the thiol group of Cys(31) and Cys(34) are 4.8 and 11.3, respectively. The His(33) pK(a) value, experimentally determined for the first time, differs notably as a function of the redox states, 7.2 for the reduced state and 4.6 for the oxidized state. These data suggest an important role for His(33) in the molecular mechanism of Dtrx catalysis that is confirmed by the properties of mutant DtrxH33G protein. The NMR structure of Dtrx shows a different charge repartition compared with canonical Trx. The results presented are likely indicative of the involvement of this protein in the catalysis of substrates specific of the anaerobe cytoplasm of DvH. The study of Dtrx is an important step toward revealing the molecular details of the thiol-disulfide oxidoreductase catalytic mechanism.

Structural basis for galectin-1-dependent pre-B cell receptor (pre-BCR) activation.

During B cell differentiation in the bone marrow, the expression and activation of the pre-B cell receptor (pre-BCR) constitute crucial checkpoints for B cell development. Both constitutive and ligand-dependent pre-BCR activation modes have been described. The pre-BCR constitutes an immunoglobulin heavy chain (Igµ) and a surrogate light chain composed of the invariant λ5 and VpreB proteins. We previously showed that galectin-1 (GAL1), produced by bone marrow stromal cells, is a pre-BCR ligand that induces receptor clustering, leading to efficient pre-BII cell proliferation and differentiation. GAL1 interacts with the pre-BCR via the unique region of λ5 (λ5-UR). Here, we investigated the solution structure of a minimal λ5-UR motif that interacts with GAL1. This motif adopts a stable helical conformation that docks onto a GAL1 hydrophobic surface adjacent to its carbohydrate binding site. We identified key hydrophobic residues from the λ5-UR as crucial for the interaction with GAL1 and for pre-BCR clustering. These residues involved in GAL1-induced pre-BCR activation are different from those essential for autonomous receptor activation. Overall, our results indicate that constitutive and ligand-induced pre-BCR activation could occur in a complementary manner.

1H, 13C and 15N chemical shift assignments of the ZnR and GYF cytoplasmic domains of the GltJ protein from Myxococcus xanthus.

Bacterial cell motility is essential for a range of physiological phenomena such as nutrient sensing, predation, biofilm formation and pathogenesis. One of the most intriguing motilities is bacterial gliding, which is defined as the ability of some bacteria to move across surfaces without an external appendage. In Myxococcus xanthus, gliding motility depends on the assembly of focal adhesion complexes (FAC) which include the Glt mutiprotein complex and allow directional movement of individual cells (A-motility). Within the Glt multiprotein complex, GltJ is one of the key proteins involved in FAC assembly. In this work we report complete backbone and side chain 1H, 13C and 15N chemical shifts of the two cytoplasmic domains of GltJ, GltJ-ZnR (BMRB No. 51104) and GltJ-GYF (BMRB No. 51096). These data provide the first step toward the first high resolution structures of protein domains from the Glt machinery and the atomic level characterization of GltJ cytoplasmic activity during FAC assembly.

Chemokines modulate glycan binding and the immunoregulatory activity of galectins.

Galectins are versatile glycan-binding proteins involved in immunomodulation. Evidence suggests that galectins can control the immunoregulatory function of cytokines and chemokines through direct binding. Here, we report on an inverse mechanism in which chemokines control the immunomodulatory functions of galectins. We show the existence of several specific galectin-chemokine binding pairs, including galectin-1/CXCL4. NMR analyses show that CXCL4 binding induces changes in the galectin-1 carbohydrate binding site. Consequently, CXCL4 alters the glycan-binding affinity and specificity of galectin-1. Regarding immunomodulation, CXCL4 significantly increases the apoptotic activity of galectin-1 on activated CD8+ T cells, while no effect is observed in CD4+ T cells. The opposite is found for another galectin-chemokine pair, i.e., galectin-9/CCL5. This heterodimer significantly reduces the galectin-9 induced apoptosis of CD4+ T cells and not of CD8+ T cells. Collectively, the current study describes an immunomodulatory mechanism in which specific galectin-chemokine interactions control the glycan-binding activity and immunoregulatory function of galectins.

1H, 13C and 15N assignments of the C-terminal intrinsically disordered cytosolic fragment of the receptor tyrosine kinase ErbB2.

ErbB2 (or HER2) is a receptor tyrosine kinase that is involved in signaling pathways controlling cell division, motility and apoptosis. Though important in development and cell growth homeostasis, this protein, when overexpressed, participates in triggering aggressive HER2+ breast cancers. It is composed of an extracellular part and a transmembrane domain, both important for activation by dimerization, and a cytosolic tyrosine kinase, which activates its intrinsically disordered C-terminal end (CtErbB2). Little is known about this C-terminal part of 268 residues, despite its crucial role in interacting with adaptor proteins involved in signaling. Understanding its structural and dynamic characteristics could eventually lead to the design of new interaction inhibitors, and treatments complementary to those already targeting other parts of ErbB2. Here we report backbone and side-chain assignment of CtErbB2, which, together with structural predictions, confirms its intrinsically disordered nature.

The crystal structure of the hexadeca-heme cytochrome Hmc and a structural model of its complex with cytochrome c(3).

Sulfate-reducing bacteria contain a variety of multi-heme c-type cytochromes. The cytochrome of highest molecular weight (Hmc) contains 16 heme groups and is part of a transmembrane complex involved in the sulfate respiration pathway. We present the 2.42 A resolution crystal structure of the Desulfovibrio vulgaris Hildenborough cytochrome Hmc and a structural model of the complex with its physiological electron transfer partner, cytochrome c(3), obtained by NMR restrained soft-docking calculations. The Hmc is composed of three domains, which exist independently in different sulfate-reducing species, namely cytochrome c(3), cytochrome c(7), and Hcc. The complex involves the last heme at the C-terminal region of the V-shaped Hmc and heme 4 of cytochrome c(3), and represents an example for specific cytochrome-cytochrome interaction.

Examination of galectin-induced lattice formation on early B-cell development.

Galectin-1 (GAL1) is a pre-B cell receptor (pre-BCR) ligand that induces pre-BCR clustering and leads to efficient pre-B cell proliferation and differentiation in the bone marrow. To study pre-BCR-GAL1 interactions and its functional consequence on the early steps of the B cell development, we combine structural nuclear magnetic resonance (NMR) approaches and B cell biology techniques. NMR is applied to identify the residues involved in pre-BCR-GAL1 interactions by monitoring chemical shift perturbations when the complex is formed. This structural information is then used at the cellular level to target specifically the complex formation during GAL1-induced pre-BCR clustering and lattice formation, using immunofluorescence techniques. Moreover, an in vivo assay was set up to study the consequence of synapse formation on the early steps of B cell development.

Pre-B cell receptor binding to galectin-1 modifies galectin-1/carbohydrate affinity to modulate specific galectin-1/glycan lattice interactions.

Galectins are glycan-binding proteins involved in various biological processes including cell/cell interactions. During B-cell development, bone marrow stromal cells secreting galectin-1 (GAL1) constitute a specific niche for pre-BII cells. Besides binding glycans, GAL1 is also a pre-B cell receptor (pre-BCR) ligand that induces receptor clustering, the first checkpoint of B-cell differentiation. The GAL1/pre-BCR interaction is the first example of a GAL1/unglycosylated protein interaction in the extracellular compartment. Here we show that GAL1/pre-BCR interaction modifies GAL1/glycan affinity and particularly inhibits binding to LacNAc containing epitopes. GAL1/pre-BCR interaction induces local conformational changes in the GAL1 carbohydrate-binding site generating a reduction in GAL1/glycan affinity. This fine tuning of GAL1/glycan interactions may be a strategic mechanism for allowing pre-BCR clustering and pre-BII cells departure from their niche. Altogether, our data suggest a novel mechanism for a cell to modify the equilibrium of the GAL1/glycan lattice involving GAL1/unglycosylated protein interactions.

The cytochrome c3-[Fe]-hydrogenase electron-transfer complex: structural model by NMR restrained docking.

Cytochrome c(3) (M(r) 13000) is a low redox potential cytochrome specific of the anaerobic metabolism in sulfate-reducing bacteria. This tetrahemic cytochrome is an intermediate between the [Fe]-hydrogenase and the cytochrome Hmc in Desulfovibrio vulgaris Hildenborough strain. The present work describes the structural model of the cytochrome c(3)-[Fe]-hydrogenase complex obtained by nuclear magnetic resonance restrained docking. This model connects the distal cluster of the [Fe]-hydrogenase to heme 4 of the cytochrome, the same heme found in the interaction with cytochrome Hmc. This result gives evidence that cytochrome c(3) is an electron shuttle between the periplasmic hydrogenase and the Hmc membrane-bound complex.

Glycan dependence of Galectin-3 self-association properties.

Human Galectin-3 is found in the nucleus, the cytoplasm and at the cell surface. This lectin is constituted of two domains: an unfolded N-terminal domain and a C-terminal Carbohydrate Recognition Domain (CRD). There are still uncertainties about the relationship between the quaternary structure of Galectin-3 and its carbohydrate binding properties. Two types of self-association have been described for this lectin: a C-type self-association and a N-type self-association. Herein, we have analyzed Galectin-3 oligomerization by Dynamic Light Scattering using both the recombinant CRD and the full length lectin. Our results proved that LNnT induces N-type self-association of full length Galectin-3. Moreover, from Nuclear Magnetic Resonance (NMR) and Surface Plasmon Resonance experiments, we observed no significant specificity or affinity variations for carbohydrates related to the presence of the N-terminal domain of Galectin-3. NMR mapping clearly established that the N-terminal domain interacts with the CRD. We propose that LNnT induces a release of the N-terminal domain resulting in the glycan-dependent self-association of Galectin-3 through N-terminal domain interactions.

¹H, ¹³C and ¹5N backbone and side-chain chemical shift assignments for reduced unusual thioredoxin Patrx2 of Pseudomonas aeruginosa.

The gram-negative organism Pseudomonas aeruginosa is an opportunistic human pathogen and a leading cause of hospital-acquired infections. In P. aeruginosa PAO1, three cytoplasmic thioredoxins have been identified. An unusual thioredoxin (Patrx2) (108 amino acids) encoded by the PA2694 gene, is identified as a new thioredoxin-like protein based on sequence homology. Thioredoxin is a ubiquitous protein, which serves as a general protein disulfide oxidoreductase. Patrx2 present an atypical active site CGHC. We report the nearly complete (1)H, (13)C and (15)N resonance assignments of reduced Patrx2. 2D and 3D heteronuclear NMR experiments were performed with uniformly (15)N-, (13)C-labelled Patrx2, resulting in 97.2% backbone and 92.5% side-chain (1)H, (13)C and (15)N resonance assignments for the reduced form. (BMRB deposits with accession number 18130).

The indispensable N-terminal half of eIF3j/HCR1 cooperates with its structurally conserved binding partner eIF3b/PRT1-RRM and with eIF1A in stringent AUG selection.

Despite recent progress in our understanding of the numerous functions of individual subunits of eukaryotic translation initiation factor (eIF) 3, little is known on the molecular level. Using NMR spectroscopy, we determined the first solution structure of an interaction between eIF3 subunits. We revealed that a conserved tryptophan residue in the human eIF3j N-terminal acidic motif (NTA) is held in the helix alpha1 and loop 5 hydrophobic pocket of the human eIF3b RNA recognition motif (RRM). Mutating the corresponding "pocket" residues in its yeast orthologue reduces cellular growth rate, eliminates eIF3j/HCR1 association with eIF3b/PRT1 in vitro and in vivo, affects 40S occupancy of eIF3, and produces a leaky scanning defect indicative of a deregulation of the AUG selection process. Unexpectedly, we found that the N-terminal half of eIF3j/HCR1 containing the NTA is indispensable and sufficient for wild-type growth of yeast cells. Furthermore, we demonstrate that deletion of either j/HCR1 or its N-terminal half only, or mutation of the key tryptophan residues results in the severe leaky scanning phenotype partially suppressible by overexpressed eIF1A, which is thought to stabilize properly formed preinitiation complexes at the correct start codon. These findings indicate that eIF3j/HCR1 remains associated with the scanning preinitiation complexes and does not dissociate from the small ribosomal subunit upon mRNA recruitment, as previously believed. Finally, we provide further support for earlier mapping of the ribosomal binding site for human eIF3j by identifying specific interactions of eIF3j/HCR1 with small ribosomal proteins RPS2 and RPS23 located in the vicinity of the mRNA entry channel. Taken together, we propose that eIF3j/HCR1 closely cooperates with the eIF3b/PRT1 RRM and eIF1A on the ribosome to ensure proper formation of the scanning-arrested conformation required for stringent AUG recognition.

Structure of eIF3b RNA recognition motif and its interaction with eIF3j: structural insights into the recruitment of eIF3b to the 40 S ribosomal subunit.

Mammalian eIF3 is a 700-kDa multiprotein complex essential for initiation of protein synthesis in eukaryotic cells. It consists of 13 subunits (eIF3a to -m), among which eIF3b serves as a major scaffolding protein. Here we report the solution structure of the N-terminal RNA recognition motif of human eIF3b (eIF3b-RRM) determined by NMR spectroscopy. The structure reveals a noncanonical RRM with a negatively charged surface in the beta-sheet area contradictory with potential RNA binding activity. Instead, eIF3j, which is required for stable 40 S ribosome binding of the eIF3 complex, specifically binds to the rear alpha-helices of the eIF3b-RRM, opposite to its beta-sheet surface. Moreover, we identify that an N-terminal 69-amino acid peptide of eIF3j is sufficient for binding to eIF3b-RRM and that this interaction is essential for eIF3b-RRM recruitment to the 40 S ribosomal subunit. Our results provide the first structure of an important subdomain of a core eIF3 subunit and detailed insights into protein-protein interactions between two eIF3 subunits required for stable eIF3 recruitment to the 40 S subunit.

Role of the tetrahemic subunit in Desulfovibrio vulgaris hildenborough formate dehydrogenase.

In the anaerobic sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough (DvH), the genome sequencing revealed the presence of three operons encoding formate dehydrogenases. fdh1 encodes an alphabetagamma trimeric enzyme containing 11 heme binding sites; fdh2 corresponds to an alphabetagamma trimeric enzyme with a tetrahemic subunit; fdh3 encodes an alphabeta dimeric enzyme. In the present work, spectroscopic measurements demonstrated that the reduction of cytochrome c(553) was obtained in the presence of the trimeric FDH2 and not with the dimeric FDH3, suggesting that the tetrahemic subunit (FDH2C) is essential for the interaction with this physiological electron transfer partner. To further study the role of the tetrahemic subunit, the fdh2C gene was cloned and expressed in Desulfovibrio desulfuricans G201. The recombinant FDH2C was purified and characterized by optical and NMR spectroscopies. The heme redox potentials measured by electrochemistry were found to be identical in the whole enzyme and in the recombinant subunit, indicating a correct folding of the recombinant protein. The mapping of the interacting site by 2D heteronuclear NMR demonstrated a similar interaction of cytochrome c(553) with the native enzyme and the recombinant subunit. The presence of hemes c in the gamma subunit of formate dehydrogenases is specific of these anaerobic sulfate-reducing bacteria and replaces heme b subunit generally found in the enzymes involved in anaerobic metabolisms.

Structural and genetic analyses reveal a key role in prophage excision for the TorI response regulator inhibitor.

TorI (Tor inhibition protein) has been identified in Escherichia coli as a protein inhibitor acting through protein-protein interaction with the TorR response regulator. This interaction, which does not interfere with TorR DNA binding activity, probably prevents the recruitment of RNA polymerase to the torC promoter. In this study we have solved the solution structure of TorI, which adopts a prokaryotic winged-helix arrangement. Despite no primary sequence similarity, the three-dimensional structure of TorI is highly homologous to the (lambda)Xis, Mu bacteriophage repressor (MuR-DBD), and transposase (MuA-DBD) structures. We propose that the TorI protein is the structural missing link between the (lambda)Xis and MuR proteins. Moreover, in vivo assays demonstrated that TorI plays an essential role in prophage excision. Heteronuclear NMR experiments and site-directed mutagenesis studies have pinpointed out key residues involved in the DNA binding activity of TorI. Our findings suggest that TorI-related proteins identified in various pathogenic bacterial genomes define a new family of atypical excisionases.