Pierre Legrand

Pentameric ligand-gated ion channel ELIC is activated by GABA and modulated by benzodiazepines

GABAA receptors are pentameric ligand-gated ion channels involved in fast inhibitory neurotransmission and are allosterically modulated by the anxiolytic, anticonvulsant, and sedative-hypnotic benzodiazepines. Here we show that the prokaryotic homolog ELIC also is activated by GABA and is modulated by benzodiazepines with effects comparable to those at GABAA receptors. Crystal structures reveal important features of GABA recognition and indicate that benzodiazepines, depending on their concentration, occupy two possible sites in ELIC. An intrasubunit site is adjacent to the GABA-recognition site but faces the channel vestibule. A second intersubunit site partially overlaps with the GABA site and likely corresponds to a low-affinity benzodiazepine-binding site in GABAA receptors that mediates inhibitory effects of the benzodiazepine flurazepam. Our study offers a structural view how GABA and benzodiazepines are recognized at a GABA-activated ion channel.

Crystal structure of the 14-subunit RNA polymerase I

Protein biosynthesis depends on the availability of ribosomes, which in turn relies on ribosomal RNA production. In eukaryotes, this process is carried out by RNA polymerase I (Pol I), a 14-subunit enzyme, the activity of which is a major determinant of cell growth. Here we present the crystal structure of Pol I from Saccharomyces cerevisiae at 3.0 Å resolution. The Pol I structure shows a compact core with a wide DNA-binding cleft and a tightly anchored stalk. An extended loop mimics the DNA backbone in the cleft and may be involved in regulating Pol I transcription. Subunit A12.2 extends from the A190 jaw to the active site and inserts a transcription elongation factor TFIIS-like zinc ribbon into the nucleotide triphosphate entry pore, providing insight into the role of A12.2 in RNA cleavage and Pol I insensitivity to α-amanitin. The A49–A34.5 heterodimer embraces subunit A135 through extended arms, thereby contacting and potentially regulating subunit A12.2.

Structural characterization of filaments formed by human Xrcc4–Cernunnos/XLF complex involved in nonhomologous DNA end-joining

Cernunnos/XLF is a core protein of the nonhomologous DNA end-joining (NHEJ) pathway that processes the majority of DNA double-strand breaks in mammals. Cernunnos stimulates the final ligation step catalyzed by the complex between DNA ligase IV and Xrcc4 (X4). Here we present the crystal structure of the X41–157-Cernunnos1–224 complex at 5.5-Å resolution and identify the relative positions of the two factors and their binding sites. The X-ray structure reveals a filament arrangement for X41–157 and Cernunnos1–224 homodimers mediated by repeated interactions through their N-terminal head domains. A filament arrangement of the X4–Cernunnos complex was confirmed by transmission electron microscopy analyses both with truncated and full-length proteins. We further modeled the interface and used structure-based site-directed mutagenesis and calorimetry to characterize the roles of various residues at the X4–Cernunnos interface. We identified four X4 residues (Glu55, Asp58, Met61, and Phe106) essential for the interaction with Cernunnos. These findings provide new insights into the molecular bases for stimulatory and bridging roles of Cernunnos in the final DNA ligation step.

Structural insight into magnetochrome-mediated magnetite biomineralization

Magnetotactic bacteria align along the Earth’s magnetic field using an organelle called the magnetosome, a biomineralized magnetite (Fe(ii)Fe(iii)2O4) or greigite (Fe(ii)Fe(iii)2S4) crystal embedded in a lipid vesicle. Although the need for both iron(ii) and iron(iii) is clear, little is known about the biological mechanisms controlling their ratio. Here we present the structure of the magnetosome-associated protein MamP and find that it is built on a unique arrangement of a self-plugged PDZ domain fused to two magnetochrome domains, defining a new class of c-type cytochrome exclusively found in magnetotactic bacteria. Mutational analysis, enzyme kinetics, co-crystallization with iron(ii) and an in vitro MamP-assisted magnetite production assay establish MamP as an iron oxidase that contributes to the formation of iron(iii) ferrihydrite eventually required for magnetite crystal growth in vivo. These results demonstrate the molecular mechanisms of iron management taking place inside the magnetosome and highlight the role of magnetochrome in iron biomineralization.

Structure of the phage TP901-1 1.8 MDa baseplate suggests an alternative host adhesion mechanism

Phages of the Caudovirales order possess a tail that recognizes the host and ensures genome delivery upon infection. The X-ray structure of the approximately 1.8 MDa host adsorption device (baseplate) from the lactococcal phage TP901-1 shows that the receptor-binding proteins are pointing in the direction of the host, suggesting that this organelle is in a conformation ready for host adhesion. This result is in marked contrast with the lactococcal phage p2 situation, whose baseplate is known to undergo huge conformational changes in the presence of Ca2+ to reach its active state. In vivo infection experiments confirmed these structural observations by demonstrating that Ca2+ ions are required for host adhesion among p2-like phages (936-species) but have no influence on TP901-1-like phages (P335-species). These data suggest that these two families rely on diverse adhesion strategies which may lead to different signaling for genome release.

Crystal structure of Apis mellifera OBP14, a C-minus odorant-binding protein, and its complexes with odorant molecules.

Apis mellifera (Amel) relies on its olfactory system to detect and identify new-sources of floral food. The Odorant-Binding Proteins (OBPs) are the first proteins involved in odorant recognition and interaction, before activation of the olfactory receptors. The Amel genome possess a set of 21 OBPs, much fewer compared to the 60-70 OBPs found in Diptera genomes. We have undertaken a structural proteomics study of Amel OBPs, alone or in complex with odorant or model compounds. We report here the first 3D structure of a member of the C-minus class OBPs, AmelOBP14, characterized by only two disulfide bridges of the three typical of classical OBPs. We show that AmelOBP14 possesses a core of 6 α-helices comparable to that of classical OBPs, and an extra exposed C-terminal helix. Its binding site is located within this core and is completely closed. Fluorescent experiments using 1-NPN displacement demonstrate that AmelOBP14 is able to bind several compounds with sub micromolar dissociation constants, among which citralva and eugenol exhibit the highest affinities. We have determined the structures of AmelOBP14 in complex with 1-NPN, eugenol and citralva, explaining their strong binding. Finally, by introducing a double cysteine mutant at positions 44 and 97, we show that a third disulfide bridge was formed in the same position as in classical OBPs without disturbing the fold of AmelOBP14.

Priming and polymerization of a bacterial contractile tail structure

Contractile tails are composed of an inner tube wrapped by an outer sheath assembled in an extended, metastable conformation that stores mechanical energy necessary for its contraction. Contraction is used to propel the rigid inner tube towards target cells for DNA or toxin delivery. Although recent studies have revealed the structure of the contractile sheath of the type VI secretion system, the mechanisms by which its polymerization is controlled and coordinated with the assembly of the inner tube remain unknown. Here we show that the starfish-like TssA dodecameric complex interacts with tube and sheath components. Fluorescence microscopy experiments in enteroaggregative Escherichia coli reveal that TssA binds first to the type VI secretion system membrane core complex and then initiates tail polymerization. TssA remains at the tip of the growing structure and incorporates new tube and sheath blocks. On the basis of these results, we propose that TssA primes and coordinates tail tube and sheath biogenesis.

Dual functions of the Hsm3 protein in chaperoning and scaffolding regulatory particle subunits during the proteasome assembly

The 26S proteasome, a molecular machine responsible for regulated protein degradation, consists of a proteolytic core particle (20S CP) associated with 19S regulatory particles (19S RPs) subdivided into base and lid subcomplexes. The assembly of 19S RP base subcomplex is mediated by multiple dedicated chaperones. Among these, Hsm3 is important for normal growth and directly targets the carboxyl-terminal (C-terminal) domain of Rpt1 of the Rpt1–Rpt2–Rpn1 assembly intermediate. Here, we report crystal structures of the yeast Hsm3 chaperone free and bound to the C-terminal domain of Rpt1. Unexpectedly, the structure of the complex suggests that within the Hsm3–Rpt1–Rpt2 module, Hsm3 also contacts Rpt2. We show that in both yeast and mammals, Hsm3 actually directly binds the AAA domain of Rpt2. The Hsm3 C-terminal region involved in this interaction is required in vivo for base assembly, although it is dispensable for binding Rpt1. Although Rpt1 and Rpt2 exhibit weak affinity for each other, Hsm3 unexpectedly acts as an essential matchmaker for the Rpt1-Rpt2-Rpn1 assembly by bridging both Rpt1 and Rpt2. In addition, we provide structural and biochemical evidence on how Hsm3/S5b may regulate the 19S RP association to the 20S CP proteasome. Our data point out the diverse functions of assembly chaperones.

Structure and allosteric inhibition of excitatory amino acid transporter 1

Human members of the solute carrier 1 (SLC1) family of transporters take up excitatory neurotransmitters in the brain and amino acids in peripheral organs. Dysregulation of their functions is associated to neurodegenerative disorders and cancer. Here we present the first crystal structures of a thermostabilized human SLC1 transporter, the excitatory amino acid transporter 1 (EAAT1), with and without allosteric and competitive inhibitors bound. The structures show novel architectural features of the human transporters, including intra- and extracellular domains with potential roles in transport function, as well as regulation by lipids and post-translational modifications. The coordination of the inhibitor in the structures and the change in the transporter dynamics measured by hydrogen-deuterium exchange mass spectrometry, reveal an allosteric mechanism of inhibition, whereby the transporter is locked in the outward-facing states of the transport cycle. Our results provide unprecedented insights into the molecular mechanisms of function and pharmacology of human SLC1 transporters.

Type VI secretion TssK baseplate protein exhibits structural similarity with phage receptor-binding proteins and evolved to bind the membrane complex

The type VI secretion system (T6SS) is a multiprotein machine widespread in Gram-negative bacteria that delivers toxins into both eukaryotic and prokaryotic cells. The mechanism of action of the T6SS is comparable to that of contractile myophages. The T6SS builds a tail-like structure made of an inner tube wrapped by a sheath, assembled under an extended conformation. Contraction of the sheath propels the inner tube towards the target cell. The T6SS tail is assembled on a platform—the baseplate—which is functionally similar to bacteriophage baseplates. In addition, the baseplate docks the tail to a trans-envelope membrane complex that orients the tail towards the target. Here, we report the crystal structure of TssK, a central component of the T6SS baseplate. We show that TssK is composed of three domains, and establish the contribution of each domain to the interaction with TssK partners. Importantly, this study reveals that the N-terminal domain of TssK is structurally homologous to the shoulder domain of phage receptor-binding proteins, and the C-terminal domain binds the membrane complex. We propose that TssK has conserved the domain of attachment to the virion particle but has evolved the reception domain to use the T6SS membrane complex as receptor.