Christine Kellenberger

How much can a T-cell antigen receptor adapt to structurally distinct antigenic peptides?

Binding degeneracy is thought to constitute a fundamental property of the T‐cell antigen receptor (TCR), yet its structural basis is poorly understood. We determined the crystal structure of a complex involving the BM3.3 TCR and a peptide (pBM8) bound to the H‐2Kbm8 major histocompatibility complex (MHC) molecule, and compared it with the structures of the BM3.3 TCR bound to H‐2Kb molecules loaded with two peptides that had a minimal level of primary sequence identity with pBM8. Our findings provide a refined structural view of the basis of BM3.3 TCR cross‐reactivity and a structural explanation for the long‐standing paradox that a TCR antigen‐binding site can be both specific and degenerate. We also measured the thermodynamic features and biological penalties that incurred during cross‐recognition. Our data illustrate the difficulty for a given TCR in adapting to distinct peptide‐MHC surfaces while still maintaining affinities that result in functional in vivo responses. Therefore, when induction of protective effector T cells is used as the ultimate criteria for adaptive immunity, TCRs are probably much less degenerate than initially assumed.

SERINE PROTEASE INHIBITION BY INSECT PEPTIDES CONTAINING A CYSTEINE KNOT AND A TRIPLE-STRANDED BETA -SHEET

Three insect peptides showing high sequence similarity and belonging to the same structural family incorporating a cysteine knot and a short three-stranded antiparallel β-sheet were studied. Their inhibitory effect on two serine proteases (bovine α-chymotrypsin and human leukocyte elastase) is reported. One of them, PMP-C, is a strong α-chymotrypsin inhibitor (Ki = 0.2 nM) and interacts with leukocyte elastase with a Ki of 0.12 μM. The other two peptides, PMP-D2 and HI, interact only weakly with α-chymotrypsin and do not inhibit leukocyte elastase. Synthetic variants of these peptides were prepared by solid-phase synthesis, and their action toward serine proteases was evaluated. This enabled us to locate the P1 residues within the reactive sites (Leu-30 for PMP-C and Arg-29 for PMP-D2 and HI), and, interestingly, variants of PMP-D2 and HI were converted into powerful inhibitors of both α-chymotrypsin and leukocyte elastase, the most potent elastase inhibitor obtained in this study having a Ki of 3 nM.

Monalysin, a novel ß-pore-forming toxin from the Drosophila pathogen Pseudomonas entomophila, contributes to host intestinal damage and lethality

Pseudomonas entomophila is an entomopathogenic bacterium that infects and kills Drosophila. P. entomophila pathogenicity is linked to its ability to cause irreversible damages to the Drosophila gut, preventing epithelium renewal and repair. Here we report the identification of a novel pore-forming toxin (PFT), Monalysin, which contributes to the virulence of P. entomophila against Drosophila. Our data show that Monalysin requires N-terminal cleavage to become fully active, forms oligomers in vitro, and induces pore-formation in artificial lipid membranes. The prediction of the secondary structure of the membrane-spanning domain indicates that Monalysin is a PFT of the ß-type. The expression of Monalysin is regulated by both the GacS/GacA two-component system and the Pvf regulator, two signaling systems that control P. entomophila pathogenicity. In addition, AprA, a metallo-protease secreted by P. entomophila, can induce the rapid cleavage of pro-Monalysin into its active form. Reduced cell death is observed upon infection with a mutant deficient in Monalysin production showing that Monalysin plays a role in P. entomophila ability to induce intestinal cell damages, which is consistent with its activity as a PFT. Our study together with the well-established action of Bacillus thuringiensis Cry toxins suggests that production of PFTs is a common strategy of entomopathogens to disrupt insect gut homeostasis.

Crystal structure of Drosophila PGRP-SD suggests binding to DAP-type but not lysine-type peptidoglycan.

In Drosophila the synthesis of antimicrobial peptides in response to microbial infections is under the control of the Toll and immune deficiency (Imd) signaling pathways. The Toll signaling pathway responds mainly to Gram-positive bacterial and fungal infection while the Imd pathway mediates the response to Gram-negative bacteria. Microbial recognition upstream of Toll involves, at least in part, peptidoglycan recognition proteins (PGRPs). The sensing of Gram-positive bacteria is mediated by the pattern recognition receptors PGRP-SA and Gram-negative binding protein 1 (GNBP1) that cooperate to detect the presence of lysine-type peptidoglycan in the host. Recently it has been shown that a loss-of-function mutation in peptidoglycan recognition protein SD (PGRP-SD) severely exacerbates the PGRP-SA and GNBP1 mutant phenotypes. Here we have solved the crystal structure of PGRP-SD at 1.5A resolution. Comparison with available structures of PGRPs in complex with their peptidoglycan (PGN) ligand strongly suggests a diaminopimelic acid (DAP) specificity for PGRP-SD. This result is supported by pull-down assays with insoluble PGNs. In addition we show that Toll pathway activation after infection by DAP-type PGN containing bacteria is clearly reduced in PGRP-SD mutant flies. Our hypothesis is that the role of PGRP-SD is the recognition of DAP-type PGNs responsible for the activation of the Toll pathway by Gram-negative bacteria.

Complexation of Two Proteic Insect Inhibitors to the Active Site of Chymotrypsin Suggests Decoupled Roles for Binding and Selectivity

The crystal structures of two homologous inhibitors (PMP-C and PMP-D2v) from the insect Locusta migratoria have been determined in complex with bovine α-chymotrypsin at 2.1- and 3.0-Å resolution, respectively. PMP-C is a potent bovine α-chymotrypsin inhibitor whereas native PMP-D2 is a weak inhibitor of bovine trypsin. One unique mutation at the P1 position converts PMP-D2 into a potent bovine α-chymotrypsin inhibitor. The two peptides have a similar overall conformation, which consists of a triple-stranded antiparallel β-sheet connected by three disulfide bridges, thus defining a novel family of serine protease inhibitors. They have in common the protease interaction site, which is composed of the classical protease binding loop (position P5 to P′4, corresponding to residues 26–34) and of an internal segment (residues 15–18), held together by two disulfide bridges. Structural divergences between the two inhibitors result in an additional interaction site between PMP-D2v (position P10 to P6, residues 21–25) and the residues 172–175 of α-chymotrypsin. This unusual interaction may be responsible for species selectivity. A careful comparison of data on bound and free inhibitors (from this study and previous NMR studies, respectively) suggests that complexation to the protease stabilizes the flexible binding loop (from P5 to P′4).

The N-terminal domain of Drosophila Gram-negative binding protein 3 (GNBP3) defines a novel family of fungal pattern recognition receptors.

Gram-negative binding protein 3 (GNBP3), a pattern recognition receptor that circulates in the hemolymph of Drosophila, is responsible for sensing fungal infection and triggering Toll pathway activation. Here, we report that GNBP3 N-terminal domain binds to fungi upon identifying long chains of β-1,3-glucans in the fungal cell wall as a major ligand. Interestingly, this domain fails to interact strongly with short oligosaccharides. The crystal structure of GNBP3-Nter reveals an immunoglobulin-like fold in which the glucan binding site is masked by a loop that is highly conserved among glucan-binding proteins identified in several insect orders. Structure-based mutagenesis experiments reveal an essential role for this occluding loop in discriminating between short and long polysaccharides. The displacement of the occluding loop is necessary for binding and could explain the specificity of the interaction with long chain structured polysaccharides. This represents a novel mechanism for β-glucan recognition.

The Drosophila Peptidoglycan-Recognition Protein Lf Interacts with Peptidoglycan-Recognition Protein Lc to Downregulate the Imd Pathway.

The peptidoglycan (PGN)‐recognition protein LF (PGRP‐LF) is a specific negative regulator of the immune deficiency (Imd) pathway in Drosophila. We determine the crystal structure of the two PGRP domains constituting the ectodomain of PGRP‐LF at 1.72 and 1.94 Å resolution. The structures show that the LFz and LFw domains do not have a PGN‐docking groove that is found in other PGRP domains, and they cannot directly interact with PGN, as confirmed by biochemical‐binding assays. By using surface plasmon resonance analysis, we show that the PGRP‐LF ectodomain interacts with the PGRP‐LCx ectodomain in the absence and presence of tracheal cytotoxin. Our results suggest a mechanism for downregulation of the Imd pathway on the basis of the competition between PRGP‐LCa and PGRP‐LF to bind to PGRP‐LCx.

A phospholipase A1 antibacterial Type VI secretion effector interacts directly with the C-terminal domain of the VgrG spike protein for delivery.

The Type VI secretion system (T6SS) is a multiprotein machine that delivers protein effectors in both prokaryotic and eukaryotic cells, allowing interbacterial competition and virulence. The mechanism of action of the T6SS requires the contraction of a sheath‐like structure that propels a needle towards target cells, allowing the delivery of protein effectors. Here, we provide evidence that the entero‐aggregative Escherichia coli Sci‐1 T6SS is required to eliminate competitor bacteria. We further identify Tle1, a toxin effector encoded by this cluster and showed that Tle1 possesses phospholipase A1 and A2 activities required for the interbacterial competition. Self‐protection of the attacker cell is secured by an outer membrane lipoprotein, Tli1, which binds Tle1 in a 1:1 stoichiometric ratio with nanomolar affinity, and inhibits its phospholipase activity. Tle1 is delivered into the periplasm of the prey cells using the VgrG1 needle spike protein as carrier. Further analyses demonstrate that the C‐terminal extension domain of VgrG1, including a transthyretin‐like domain, is responsible for the interaction with Tle1 and its subsequent delivery into target cells. Based on these results, we propose an additional mechanism of transport of T6SS effectors in which cognate effectors are selected by specific motifs located at the C‐terminus of VgrG proteins.

A phospholipase A1 anti-bacterial T6SS effector interacts directly with the C-terminal domain of the VgrG spike protein for delivery

The Type VI secretion system (T6SS) is a multiprotein machine that delivers protein effectors in both prokaryotic and eukaryotic cells, allowing interbacterial competition and virulence. The mechanism of action of the T6SS requires the contraction of a sheath‐like structure that propels a needle towards target cells, allowing the delivery of protein effectors. Here, we provide evidence that the entero‐aggregative Escherichia coli Sci‐1 T6SS is required to eliminate competitor bacteria. We further identify Tle1, a toxin effector encoded by this cluster and showed that Tle1 possesses phospholipase A1 and A2 activities required for the interbacterial competition. Self‐protection of the attacker cell is secured by an outer membrane lipoprotein, Tli1, which binds Tle1 in a 1:1 stoichiometric ratio with nanomolar affinity, and inhibits its phospholipase activity. Tle1 is delivered into the periplasm of the prey cells using the VgrG1 needle spike protein as carrier. Further analyses demonstrate that the C‐terminal extension domain of VgrG1, including a transthyretin‐like domain, is responsible for the interaction with Tle1 and its subsequent delivery into target cells. Based on these results, we propose an additional mechanism of transport of T6SS effectors in which cognate effectors are selected by specific motifs located at the C‐terminus of VgrG proteins.

Cytokine Diedel and a viral homologue suppress the IMD pathway in Drosophila

Significance We report the identification and characterization of a family of proteins encoded by insect DNA viruses and present in the venom of parasitic wasps. These molecules are homologous to the product of the uncharacterized Drosophila gene diedel (die). We show that Diedel is an immunomodulatory cytokine, which down-regulates the evolutionarily conserved immune deficiency (IMD) pathway of host defense in flies. The importance of this factor is highlighted by the fact that die mutant flies, which express high levels of IMD-regulated immunity genes, have reduced viability. Our work provides the first characterization of virokines in insects to our knowledge, and reveals that besides RNA interference and apoptosis, two well-characterized antiviral responses, insect viruses can also suppress a major signaling pathway of the innate immune response. Viruses are obligatory intracellular parasites that suffer strong evolutionary pressure from the host immune system. Rapidly evolving viral genomes can adapt to this pressure by acquiring genes that counteract host defense mechanisms. For example, many vertebrate DNA viruses have hijacked cellular genes encoding cytokines or cytokine receptors to disrupt host cell communication. Insect viruses express suppressors of RNA interference or apoptosis, highlighting the importance of these cell intrinsic antiviral mechanisms in invertebrates. Here, we report the identification and characterization of a family of proteins encoded by insect DNA viruses that are homologous to a 12-kDa circulating protein encoded by the virus-induced Drosophila gene diedel (die). We show that die mutant flies have shortened lifespan and succumb more rapidly than controls when infected with Sindbis virus. This reduced viability is associated with deregulated activation of the immune deficiency (IMD) pathway of host defense and can be rescued by mutations in the genes encoding the homolog of IKKγ or IMD itself. Our results reveal an endogenous pathway that is exploited by insect viruses to modulate NF-κB signaling and promote fly survival during the antiviral response.