Design, synthesis, and characterization of protein origami based on self-assembly of a brick and staple artificial protein pair.

A versatile strategy to create an inducible protein assembly with predefined geometry is demonstrated. The assembly is triggered by a binding protein that staples two identical protein bricks together in a predictable spatial conformation. The brick and staple proteins are designed for mutual directional affinity and engineered by directed evolution from a synthetic modular repeat protein library. As a proof of concept, this article reports on the spontaneous, extremely fast and quantitative self-assembly of two designed alpha-repeat (αRep) brick and staple proteins into macroscopic tubular superhelices at room temperature. Small-angle X-ray scattering (SAXS) and transmission electron microscopy (TEM with staining agent and cryoTEM) elucidate the resulting superhelical arrangement that precisely matches the a priori intended 3D assembly. The highly ordered, macroscopic biomolecular construction sustains temperatures as high as 75 °C thanks to the robust αRep building blocks. Since the α-helices of the brick and staple proteins are highly programmable, their design allows encoding the geometry and chemical surfaces of the final supramolecular protein architecture. This work opens routes toward the design and fabrication of multiscale protein origami with arbitrarily programmed shapes and chemical functions.

A new family of structurally conserved fungal effectors displays epistatic interactions with plant resistance proteins.

Recognition of a pathogen avirulence (AVR) effector protein by a cognate plant resistance (R) protein triggers a set of immune responses that render the plant resistant. Pathogens can escape this so-called Effector-Triggered Immunity (ETI) by different mechanisms including the deletion or loss-of-function mutation of the AVR gene, the incorporation of point mutations that allow recognition to be evaded while maintaining virulence function, and the acquisition of new effectors that suppress AVR recognition. The Dothideomycete Leptosphaeria maculans, causal agent of oilseed rape stem canker, is one of the few fungal pathogens where suppression of ETI by an AVR effector has been demonstrated. Indeed, AvrLm4-7 suppresses Rlm3- and Rlm9-mediated resistance triggered by AvrLm3 and AvrLm5-9, respectively. The presence of AvrLm4-7 does not impede AvrLm3 and AvrLm5-9 expression, and the three AVR proteins do not appear to physically interact. To decipher the epistatic interaction between these L. maculans AVR effectors, we determined the crystal structure of AvrLm5-9 and obtained a 3D model of AvrLm3, based on the crystal structure of Ecp11-1, a homologous AVR effector candidate from Fulvia fulva. Despite a lack of sequence similarity, AvrLm5-9 and AvrLm3 are structural analogues of AvrLm4-7 (structure previously characterized). Structure-informed sequence database searches identified a larger number of putative structural analogues among L. maculans effector candidates, including the AVR effector AvrLmS-Lep2, all produced during the early stages of oilseed rape infection, as well as among effector candidates from other phytopathogenic fungi. These structural analogues are named LARS (for Leptosphaeria AviRulence and Suppressing) effectors. Remarkably, transformants of L. maculans expressing one of these structural analogues, Ecp11-1, triggered oilseed rape immunity in several genotypes carrying Rlm3. Furthermore, this resistance could be suppressed by AvrLm4-7. These results suggest that Ecp11-1 shares a common activity with AvrLm3 within the host plant which is detected by Rlm3, or that the Ecp11-1 structure is sufficiently close to that of AvrLm3 to be recognized by Rlm3.

Amino-acyl tXNA as inhibitors or amino acid donors in peptide synthesis.

Xenobiotic nucleic acids (XNAs) offer tremendous potential for synthetic biology, biotechnology, and molecular medicine but their ability to mimic nucleic acids still needs to be explored. Here, to study the ability of XNA oligonucleotides to mimic tRNA, we synthesized three L-Ala-tXNAs analogs. These molecules were used in a non-ribosomal peptide synthesis involving a bacterial Fem transferase. We compared the ability of this enzyme to use amino-acyl tXNAs containing 1',5'-anhydrohexitol (HNA), 2'-fluoro ribose (2'F-RNA) and 2'-fluoro arabinose. L-Ala-tXNA containing HNA or 2'F-RNA were substrates of the Fem enzyme. The synthesis of peptidyl-XNA and the resolution of their structures in complex with the enzyme show the impact of the XNA on protein binding. For the first time we describe functional tXNA in an in vitro assay. These results invite to test tXNA also as substitute for tRNA in translation.

Study of the DnaB:DciA interplay reveals insights into the primary mode of loading of the bacterial replicative helicase.

Replicative helicases are essential proteins that unwind DNA in front of replication forks. Their loading depends on accessory proteins and in bacteria, DnaC and DnaI are well characterized loaders. However, most bacteria do not express either of these two proteins. Instead, they are proposed to rely on DciA, an ancestral protein unrelated to DnaC/I. While the DciA structure from Vibrio cholerae shares no homology with DnaC, it reveals similarities with DnaA and DnaX, two proteins involved during replication initiation. As other bacterial replicative helicases, VcDnaB adopts a toroid-shaped homo-hexameric structure, but with a slightly open dynamic conformation in the free state. We show that VcDnaB can load itself on DNA in vitro and that VcDciA stimulates this function, resulting in an increased DNA unwinding. VcDciA interacts with VcDnaB with a 3/6 stoichiometry and we show that a determinant residue, which discriminates DciA- and DnaC/I-helicases, is critical in vivo. Our work is the first step toward the understanding of the ancestral mode of loading of bacterial replicative helicases on DNA. It sheds light on the strategy employed by phage helicase loaders to hijack bacterial replicative helicases and may explain the recurrent domestication of dnaC/I through evolution in bacteria.

Molecular dissection of pheromone selectivity in the competence signaling system ComRS of streptococci.

Competence allows bacteria to internalize exogenous DNA fragments for the acquisition of new phenotypes such as antibiotic resistance or virulence traits. In most streptococci, competence is regulated by ComRS signaling, a system based on the mature ComS pheromone (XIP), which is internalized to activate the (R)RNPP-type ComR sensor by triggering dimerization and DNA binding. Cross-talk analyses demonstrated major differences of selectivity between ComRS systems and raised questions concerning the mechanism of pheromone-sensor recognition and coevolution. Here, we decipher the molecular determinants of selectivity of the closely related ComRS systems from Streptococcus thermophilus and Streptococcus vestibularis Despite high similarity, we show that the divergence in ComR-XIP interaction does not allow reciprocal activation. We perform the structural analysis of the ComRS system from S. vestibularis. Comparison with its ortholog from S. thermophilus reveals an activation mechanism based on a toggle switch involving the recruitment of a key loop by the XIP C terminus. Together with a broad mutational analysis, we identify essential residues directly involved in peptide binding. Notably, we generate a ComR mutant that displays a fully reversed selectivity toward the heterologous pheromone with only five point mutations, as well as other ComR variants featuring XIP bispecificity and/or neofunctionalization for hybrid XIP peptides. We also reveal that a single XIP mutation relaxes the strictness of ComR activation, suggesting fast adaptability of molecular communication phenotypes. Overall, this study is paving the way toward the rational design or directed evolution of artificial ComRS systems for a range of biotechnological and biomedical applications.

The C-terminal domain of Corynebacterium glutamicum mycoloyltransferase A is composed of five repeated motifs involved in cell wall binding and stability.

The genomes of Corynebacteriales contain several genes encoding mycoloyltransferases (Myt) that are specific cell envelope enzymes essential for the biogenesis of the outer membrane. MytA is a major mycoloyltransferase of Corynebacterium glutamicum, displaying an N-terminal domain with esterase activity and a C-terminal extension containing a conserved repeated Leu-Gly-Phe-Pro (LGFP) sequence motif of unknown function. This motif is highly conserved in Corynebacteriales and found associated with cell wall hydrolases and with proteins of unknown function. In this study, we determined the crystal structure of MytA and found that its C-terminal domain is composed of five LGFP motifs and forms a long stalk perpendicular to the N-terminal catalytic α/ß-hydrolase domain. The LGFP motifs are composed of a 4-stranded ß-fold and occupy alternating orientations along the axis of the stalk. Multiple acetate binding pockets were identified in the stalk, which could correspond to putative ligand-binding sites. By using various MytA mutants and complementary in vitro and in vivo approaches, we provide evidence that the C-terminal LGFP domain interacts with the cell wall peptidoglycan-arabinogalactan polymer. We also show that the C-terminal LGFP domain is not required for the activity of MytA but rather contributes to the overall integrity of the cell envelope.

Structure-function analysis of Sua5 protein reveals novel functional motifs required for the biosynthesis of the universal t6A tRNA modification.

N6-threonyl-carbamoyl adenosine (t6A) is a universal tRNA modification found at position 37, next to the anticodon, in almost all tRNAs decoding ANN codons (where N = A, U, G, or C). t6A stabilizes the codon-anticodon interaction and hence promotes translation fidelity. The first step of the biosynthesis of t6A, the production of threonyl-carbamoyl adenylate (TC-AMP), is catalyzed by the Sua5/TsaC family of enzymes. While TsaC is a single domain protein, Sua5 enzymes are composed of the TsaC-like domain, a linker and an extra domain called SUA5 of unknown function. In the present study, we report structure-function analysis of Pyrococcus abyssi Sua5 (Pa-Sua5). Crystallographic data revealed binding sites for bicarbonate substrate and pyrophosphate product. The linker of Pa-Sua5 forms a loop structure that folds into the active site gorge and closes it. Using structure-guided mutational analysis, we established that the conserved sequence motifs in the linker and the domain-domain interface are essential for the function of Pa-Sua5. We propose that the linker participates actively in the biosynthesis of TC-AMP by binding to ATP/PPi and by stabilizing the N-carboxy-l-threonine intermediate. Hence, TsaC orthologs which lack such a linker and SUA5 domain use a different mechanism for TC-AMP synthesis.

Novel N-substituted 5-phosphate-d-arabinonamide derivatives as strong inhibitors of phosphoglucose isomerases: Synthesis, structure-activity relationship and crystallographic studies.

Phosphoglucose isomerase (PGI) is a cytosolic enzyme that catalyzes the reversible interconversion of d-glucose 6-phosphate and d-fructose 6-phosphate in glycolysis. Outside the cell, PGI is also known as autocrine motility factor (AMF), a cytokine secreted by a large variety of tumor cells that stimulates motility of cancer cells in vitro and metastases development in vivo. Human PGI and AMF are strictly identical proteins both in terms of sequence and 3D structure, and AMF activity is known to involve, at least in part, the enzymatic active site. Hence, with the purpose of finding new strong AMF-PGI inhibitors that could be potentially used as anticancer agents and/or as bioreceptors for carbohydrate-based electrochemical biosensors, we report in this study the synthesis and kinetic evaluation of several new human PGI inhibitors derived from the synthon 5-phospho-d-arabinono-1,4-lactone. Although not designed as high-energy intermediate analogue inhibitors of the enzyme catalyzed isomerization reaction, several of these N-substituted 5-phosphate-d-arabinonamide derivatives appears as new strong PGI inhibitors. For one of them, we report its crystal structure in complex with human PGI at 2.38 Å. Detailed analysis of its interactions at the active site reveals a new binding mode and shows that human PGI is relatively tolerant for modified inhibitors at the "head" C-1 part, offering promising perspectives for the future design of carbohydrate-based biosensors.

The structure of the TsaB/TsaD/TsaE complex reveals an unexpected mechanism for the bacterial t6A tRNA-modification.

The universal N6-threonylcarbamoyladenosine (t6A) modification at position A37 of ANN-decoding tRNAs is essential for translational fidelity. In bacteria the TsaC enzyme first synthesizes an l-threonylcarbamoyladenylate (TC-AMP) intermediate. In cooperation with TsaB and TsaE, TsaD then transfers the l-threonylcarbamoyl-moiety from TC-AMP onto tRNA. We determined the crystal structure of the TsaB-TsaE-TsaD (TsaBDE) complex of Thermotoga maritima in presence of a non-hydrolysable AMPCPP. TsaE is positioned at the entrance of the active site pocket of TsaD, contacting both the TsaB and TsaD subunits and prohibiting simultaneous tRNA binding. AMPCPP occupies the ATP binding site of TsaE and is sandwiched between TsaE and TsaD. Unexpectedly, the binding of TsaE partially denatures the active site of TsaD causing loss of its essential metal binding sites. TsaE interferes in a pre- or post-catalytic step and its binding to TsaBD is regulated by ATP hydrolysis. This novel binding mode and activation mechanism of TsaE offers good opportunities for antimicrobial drug development.

Correction: Structural Insights into Streptococcal Competence Regulation by the Cell-to-Cell Communication System ComRS.

[This corrects the article DOI: 10.1371/journal.ppat.1005980.].

The crystal structure of Trz1, the long form RNase Z from yeast.

tRNAs are synthesized as precursor RNAs that have to undergo processing steps to become functional. Yeast Trz1 is a key endoribonuclease involved in the 3΄ maturation of tRNAs in all domains of life. It is a member of the ß-lactamase family of RNases, characterized by an HxHxDH sequence motif involved in coordination of catalytic Zn-ions. The RNase Z family consists of two subfamilies: the short (250-400 residues) and the long forms (about double in size). Short form RNase Z enzymes act as homodimers: one subunit embraces tRNA with a protruding arm, while the other provides the catalytic site. The long form is thought to contain two fused ß-lactamase domains within a single polypeptide. Only structures of short form RNase Z enzymes are known. Here we present the 3.1 Å crystal structure of the long-form Trz1 from Saccharomyces cerevisiae. Trz1 is organized into two ß-lactamase domains connected by a long linker. The N-terminal domain has lost its catalytic residues, but retains the long flexible arm that is important for tRNA binding, while it is the other way around in the C-terminal domain. Trz1 likely evolved from a duplication and fusion of the gene encoding the monomeric short form RNase Z.

Alpha repeat proteins (αRep) as expression and crystallization helpers.

We have previously described a highly diverse library of artificial repeat proteins based on thermostable HEAT-like repeats, named αRep. αReps binding specifically to proteins difficult to crystallize have been selected and in several examples, they made possible the crystallization of these proteins. To further simplify the production and crystallization experiments we have explored the production of chimeric proteins corresponding to covalent association between the targets and their specific binders strengthened by a linker. Although chimeric proteins with expression partners are classically used to enhance expression, these fusions cannot usually be used for crystallization. With specific expression partners like a cognate αRep this is no longer true, and chimeric proteins can be expressed purified and crystallized. αRep selection by phage display suppose that at least a small amount of the target protein should be produced to be used as a bait for selection and this might, in some cases, be difficult. We have therefore transferred the αRep library in a new construction adapted to selection by protein complementation assay (PCA). This new procedure allows to select specific binders by direct interaction with the target in the cytoplasm of the bacteria and consequently does not require preliminary purification of target protein. αRep binders selected by PCA or by phage display can be used to enhance expression, stability, solubility and crystallogenesis of proteins that are otherwise difficult to express, purify and/or crystallize.

Correction: How Quorum Sensing Connects Sporulation to Necrotrophism in Bacillus thuringiensis.

[This corrects the article DOI: 10.1371/journal.ppat.1005779.].

How Quorum Sensing Connects Sporulation to Necrotrophism in Bacillus thuringiensis.

Bacteria use quorum sensing to coordinate adaptation properties, cell fate or commitment to sporulation. The infectious cycle of Bacillus thuringiensis in the insect host is a powerful model to investigate the role of quorum sensing in natural conditions. It is tuned by communication systems regulators belonging to the RNPP family and directly regulated by re-internalized signaling peptides. One such RNPP regulator, NprR, acts in the presence of its cognate signaling peptide NprX as a transcription factor, regulating a set of genes involved in the survival of these bacteria in the insect cadaver. Here, we demonstrate that, in the absence of NprX and independently of its transcriptional activator function, NprR negatively controls sporulation. NprR inhibits expression of Spo0A-regulated genes by preventing the KinA-dependent phosphorylation of the phosphotransferase Spo0F, thus delaying initiation of the sporulation process. This NprR function displays striking similarities with the Rap proteins, which also belong to the RNPP family, but are devoid of DNA-binding domain and indirectly control gene expression via protein-protein interactions in Bacilli. Conservation of the Rap residues directly interacting with Spo0F further suggests a common inhibition of the sporulation phosphorelay. The crystal structure of apo NprR confirms that NprR displays a highly flexible Rap-like structure. We propose a molecular regulatory mechanism in which key residues of the bifunctional regulator NprR are directly and alternatively involved in its two functions. NprX binding switches NprR from a dimeric inhibitor of sporulation to a tetrameric transcriptional activator involved in the necrotrophic lifestyle of B. thuringiensis. NprR thus tightly coordinates sporulation and necrotrophism, ensuring survival and dissemination of the bacteria during host infection.

Structural Insights into Streptococcal Competence Regulation by the Cell-to-Cell Communication System ComRS.

In Gram-positive bacteria, cell-to-cell communication mainly relies on extracellular signaling peptides, which elicit a response either indirectly, by triggering a two-component phosphorelay, or directly, by binding to cytoplasmic effectors. The latter comprise the RNPP family (Rgg and original regulators Rap, NprR, PrgX and PlcR), whose members regulate important bacterial processes such as sporulation, conjugation, and virulence. RNPP proteins are increasingly considered as interesting targets for the development of new antibacterial agents. These proteins are characterized by a TPR-type peptide-binding domain, and except for Rap proteins, also contain an N-terminal HTH-type DNA-binding domain and display a transcriptional activity. Here, we elucidate the structure-function relationship of the transcription factor ComR, a new member of the RNPP family, which positively controls competence for natural DNA transformation in streptococci. ComR is directly activated by the binding of its associated pheromone XIP, the mature form of the comX/sigX-inducing-peptide ComS. The crystal structure analysis of ComR from Streptococcus thermophilus combined with a mutational analysis and in vivo assays allows us to propose an original molecular mechanism of the ComR regulation mode. XIP-binding induces release of the sequestered HTH domain and ComR dimerization to allow DNA binding. Importantly, we bring evidence that this activation mechanism is conserved and specific to ComR orthologues, demonstrating that ComR is not an Rgg protein as initially proposed, but instead constitutes a new member of the RNPP family. In addition, identification of XIP and ComR residues important for competence activation constitutes a crucial step towards the design of antagonistic strategies to control gene exchanges among streptococci.

Synthesis of Avibactam Derivatives and Activity on ß-Lactamases and Peptidoglycan Biosynthesis Enzymes of Mycobacteria.

There is a renewed interest for ß-lactams for treating infections due to Mycobacterium tuberculosis and M. abscessus because their ß-lactamases are inhibited by classical (clavulanate) or new generation (avibactam) inhibitors, respectively. Here, access to an azido derivative of the diazabicyclooctane (DBO) scaffold of avibactam for functionalization by the Huisgen-Sharpless cycloaddition reaction is reported. The amoxicillin-DBO combinations were active, indicating that the triazole ring is compatible with drug penetration (minimal inhibitory concentration of 16 µg mL-1 for both species). Mechanistically, ß-lactamase inhibition was not sufficient to account for the potentiation of amoxicillin by DBOs. Thus, the latter compounds were investigated as inhibitors of l,d-transpeptidases (Ldts), which are the main peptidoglycan polymerases in mycobacteria. The DBOs acted as slow-binding inhibitors of Ldts by S-carbamoylation indicating that optimization of DBOs for Ldt inhibition is an attractive strategy to obtain drugs selectively active on mycobacteria.

Trz1, the long form RNase Z from yeast, forms a stable heterohexamer with endonuclease Nuc1 and mutarotase.

Proteomic studies have established that Trz1, Nuc1 and mutarotase form a complex in yeast. Trz1 is a ß-lactamase-type RNase composed of two ß-lactamase-type domains connected by a long linker that is responsible for the endonucleolytic cleavage at the 3'-end of tRNAs during the maturation process (RNase Z activity); Nuc1 is a dimeric mitochondrial nuclease involved in apoptosis, while mutarotase (encoded by YMR099C) catalyzes the conversion between the α- and ß-configuration of glucose-6-phosphate. Using gel filtration, small angle X-ray scattering and electron microscopy, we demonstrated that Trz1, Nuc1 and mutarotase form a very stable heterohexamer, composed of two copies of each of the three subunits. A Nuc1 homodimer is at the center of the complex, creating a two-fold symmetry and interacting with both Trz1 and mutarotase. Enzymatic characterization of the ternary complex revealed that the activities of Trz1 and mutarotase are not affected by complex formation, but that the Nuc1 activity is completely inhibited by mutarotase and partially by Trz1. This suggests that mutarotase and Trz1 might be regulators of the Nuc1 apoptotic nuclease activity.

Insights into Brain Glycogen Metabolism: THE STRUCTURE OF HUMAN BRAIN GLYCOGEN PHOSPHORYLASE.

Brain glycogen metabolism plays a critical role in major brain functions such as learning or memory consolidation. However, alteration of glycogen metabolism and glycogen accumulation in the brain contributes to neurodegeneration as observed in Lafora disease. Glycogen phosphorylase (GP), a key enzyme in glycogen metabolism, catalyzes the rate-limiting step of glycogen mobilization. Moreover, the allosteric regulation of the three GP isozymes (muscle, liver, and brain) by metabolites and phosphorylation, in response to hormonal signaling, fine-tunes glycogenolysis to fulfill energetic and metabolic requirements. Whereas the structures of muscle and liver GPs have been known for decades, the structure of brain GP (bGP) has remained elusive despite its critical role in brain glycogen metabolism. Here, we report the crystal structure of human bGP in complex with PEG 400 (2.5 Å) and in complex with its allosteric activator AMP (3.4 Å). These structures demonstrate that bGP has a closer structural relationship with muscle GP, which is also activated by AMP, contrary to liver GP, which is not. Importantly, despite the structural similarities between human bGP and the two other mammalian isozymes, the bGP structures reveal molecular features unique to the brain isozyme that provide a deeper understanding of the differences in the activation properties of these allosteric enzymes by the allosteric effector AMP. Overall, our study further supports that the distinct structural and regulatory properties of GP isozymes contribute to the different functions of muscle, liver, and brain glycogen.

Mycoloyltransferases: A large and major family of enzymes shaping the cell envelope of Corynebacteriales.

Mycobacterium and Corynebacterium are important genera of the Corynebacteriales order, the members of which are characterized by an atypical diderm cell envelope. Indeed the cytoplasmic membrane of these bacteria is surrounded by a thick mycolic acid-arabinogalactan-peptidoglycan (mAGP) covalent polymer. The mycolic acid-containing part of this complex associates with other lipids (mainly trehalose monomycolate (TMM) and trehalose dimycolate (TDM)) to form an outer membrane. The metabolism of mycolates in the cell envelope is governed by esterases called mycoloyltransferases that catalyze the transfer of mycoloyl chains from TMM to another TMM molecule or to other acceptors such as the terminal arabinoses of arabinogalactan or specific polypeptides. In this review we present an overview of this family of Corynebacteriales enzymes, starting with their expression, localization, structure and activity to finally discuss their putative functions in the cell. In addition, we show that Corynebacteriales possess multiple mycoloyltransferases encoding genes in their genome. The reason for this multiplicity is not known, as their function in mycolates biogenesis appear to be only partially redundant. It is thus possible that, in some species living in specific environments, some mycoloyltransferases have evolved to gain some new functions. In any case, the few characterized mycoloyltransferases are very important for the bacterial physiology and are also involved in adaptation in the host where they constitute major secreted antigens. Although not discussed in this review, all these functions make them interesting targets for the discovery of new antibiotics and promising vaccines candidates. This article is part of a Special Issue entitled "Science for Life" Guest Editor: Dr. Austen Angell, Dr. Salvatore Magazù and Dr. Federica Migliardo.