Institut de Biologie StructuraleGrenoble / France

2018

Les opsonines C1q et MBL utilisent un site d’ancrage commun sur le récepteur cr1

CR1 (Complement receptor 1) est une glycoprotéine membranaire polymorphique impliquée dans l’élimination des agents nocifs pour l’organisme. En fixant des cibles étiquetées par des opsonines du système du complément, le récepteur CR1 à la surface des érythrocytes contribue à leur élimination par transport vers le foie puis phagocytose par des monocytes, macrophages ou neutrophiles. CR1 est un récepteur de type I de 30 modules CCP homologues qui servent de station d’ancrage des opsonines C3b et C4b du complément. Si C3b et C4b sont connues depuis longtemps comme ligands de CR1, ce n’est que plus récemment qu’il a été montré que les collagènes de défense C1q et MBL ont également un rôle d’opsonine.
Dans cette étude, l’équipe IRPAS a localisé le site de fixation de C1q et de la MBL à CR1 grâce à une stratégie de dissection moléculaire particulièrement adaptée à l’étude des protéines multi-modulaires. Le site de fixation identifié est commun pour C1q et MBL et se réduit à une paire de modules CCP24 et 25 parmi les 30 de CR1. Cette étude contribue à élargir les connaissances sur le rôle multifonctionnel des collagènes de défense du complément humain et plus particulièrement sur cette nouvelle fonction d’opsonine qui permet le transfert d’agents étrangers ou du soi modifié sur divers récepteurs cellulaires en vue de leur élimination.

C1q and MBL interact with CR1 in the same region on CCP24-25 modules. Jacquet M, Cioci G, Fouët G, Bally I, Thielens NM, Gaboriaud C, Rossi V. Frontiers in Immunology ;9, 453

C1q and MBL opsonins use a common anchor site on the CR1 receptor

Complement receptor type 1 (CR1) is a multi modular membrane receptor involved in the clearance of complement opsonized components from the blood stream. By biding binding targets tagged with complement-opsonins, the CR1 receptor on the surface of erythrocytes contributes to their elimination by transport to the liver, then phagocytosis by monocytes, macrophages or neutrophils.
CR1 is composed of 30 homologous complement control protein (CCP) modules and is a receptor for complement-opsonins C3b and C4b. While C3b and C4b have long been known as ligands of CR1, it is only recently that defense collagens such as mannose-binding lectin (MBL), ficolin-2, and C1q have also been shown to act as opsonins.
In this study, the IRPAS group located the attachment site of C1q and MBL to CR1 thanks to a molecular dissection strategy particularly adapted to the study of multi-modular proteins. The interaction site for both MBL and C1q is located on the same pair of modules CCP24-25 out of the 30 modules in CR1. This study contributes to enlarge knowledge on the multifunctional role of complement defense collagens and more especially on this new opsonin function that allows the transfer of foreign agents or altereded self on various receptors for clearance.

C1q and MBL interact with CR1 in the same region on CCP24-25 modules. Jacquet M, Cioci G, Fouët G, Bally I, Thielens NM, Gaboriaud C, Rossi V. Frontiers in Immunology;9, 453

How Detergent Impacts Membrane Proteins

Many cellular processes involve membrane proteins (MPs) and characterization of their structure, interactions and dynamics remains a challenge for structural biology. The difficulty is related to the need to extract these proteins from their biological membrane in order to study them. Detergents are frequently used but their physical properties differ from those of lipids and could alter the structural organization of MPs.
In this study, a family of membrane proteins, mitochondrial transporters, were analyzed in detail in a common detergent, dodecylphosphocholine (DPC). Several studies on these mitochondrial carriers in DPC had already proposed details on structure, dynamics and interactions and interpreted these observations from a biological perspective, but their biological relevance has been questionned. The NMR and MEMBRANE group studies, in collaboration with researchers at Nancy and Cambridge, combine NMR, biochemistry and MD simulation methods to resolve unambiguously this controversy. The results show a subtle balance of interactions between protein and detergent, which induces a significant disruption of the protein; the specificity of interaction with substrates is strongly impacted, and the protein samples partially unfolded conformations. A review of a number of structures obtained in DPC illustrates that these effects are relatively general, and helps clarify a debate on the impact of detergents.

1) How Detergent Impacts Membrane Proteins: Atomic-Level Views of Mitochondrial Carriers in Dodecylphosphocholine. Kurauskas V, Hessel A, Ma P, Lunetti P, Weinhäupl K, Imbert L, Brutscher B, King MS, Sounier R4, Dolce V, Kunji ERS, Capobianco L, Chipot C, Dehez F, Bersch B, Schanda P. Journal of Physical Chemistry Letters;9(5):933-938
2) Perturbations of Native Membrane Protein Structure in Alkyl Phosphocholine Detergents: A Critical Assessment of NMR and Biophysical Studies. Chipot C, Dehez F, Schnell JR, Zitzmann N, Pebay-Peyroula E, Catoire L, Miroux B, Kunji ERS, Veglia G, Cross TA, Schanda P. Chemical Reviews;118(7):3559-3607
3) Dynamics and interactions of ADP/ATP transporter AAC3 in DPC detergent are not functionally relevant. Kurauskas V, Hessel A, Dehez F, Chipot F, Bersch B, Schanda P. Nature Structural & Molecular Biology;doi: https://doi.org/10.1101/317669

New light on the mevalonate bioynthetic reaction in archaea

Mevalonate is a starting material to synthesize many chemicals in industry; it is also the building block of the lipids from all archaea. Scientists at the IBS and and collaborators at the Max Planck Institute for Terrestrial Microbiology in Marburg and ENS Lyon discovered a coupling between the two enzymes responsible for the first step in mevalonate biosynthesis in archaea. This finding explain how archaea can produce mevalonate at high rate to support their growth, and can be applied in industry to optimize mevalonate production. Details

Archaeal acetoacetyl-CoA thiolase/HMG-CoA synthase complex channels the intermediate via a fused CoA-binding site. Vögeli, B., Engilberge, S., Girard, E., Riobé, F., Maury, O., Erb, T.J., Shima, S., Wagner, T. PNAS, 115(13): 3380-3385

Deciphering the Dynamic Interaction Profile of an Intrinsically Disordered Protein by NMR Exchange Spectroscopy

Intrinsically Disordered Proteins (IPDs) are functionally active despite lacking a well-defined three-dimensional structure. Their great flexibility allows them to easily adapt to the surface of their partners and they are able to fold during interaction. In some cases, they may even form a so-called fuzzy complex, in which the IPD does not adopt a single conformation defined on the partner’s surface, but continues to sample multiple conformations in a highly dynamic complex.
The FDP group of the IBS, in collaboration with researchers from IAB Grenoble and the ENS Paris, succeeded in obtaining an atomic resolution description of the dynamics of an IDP in complex with its partner using nuclear magnetic resonance spectroscopy. Researchers applied this approach to the dynamic signaling complex formed between the mitogen-activated protein kinase (MAPK) p38α and the intrinsically disordered regulatory domain of the MAPK kinase MKK4.. The study shows that MKK4 uses a combination of interaction modes to link to p38α, leading to a complex displaying significantly different dynamics between linked regions. The results show how IPDs can engage in very specific interactions without having a strong binding affinity.

Deciphering the Dynamic Interaction Profile of an Intrinsically Disordered Protein by NMR Exchange Spectroscopy. Delaforge E, Kragelj J, Tengo L, Palencia A, Milles S, Bouvignies G, Salvi N, Blackledge M, Jensen MR. Journal of the American Chemical Society;140(3):1148-1158

Capture of a «phantom » state of green fluorescent proteins

Green fluorescent proteins (GFPs) are genetically encoded markers allowing the localization of down to individual proteins by optical microscopy. Their fluorophore is formed from three amino acids and is located at the center of a beta barrel that protects it from the environment. GFPs have high absorption coefficient and fluorescence yield, but only moderate photostability. They can reversibly form temporary nonfluorescent “dark” states before definitely losing fluorescence after about 100000 excitation/emission cycles.
At the IBS, the Pixel team has previously solved the structures of some of such dark states. All of them showed considerable chemical modifications which raised the question as to their irreversibility. Therefore, it was suspected that a reversible gateway state should exist the knowledge of which would allow to improve the GFP’s behavior. But little was known on this putative « phantom » state: its chemical character (triplet, radical, biradical ?), its yield (percents, permills ?), its lifetime (nano-, micro-, millisecondes ?), its reactivity (oxidative, reductive, both ?), its absorption spectrum (UV, VIS, IR ?).
Thanks to a collaboration with the team of Klaus BRETTEL at I2BC (CEA Saclay), the Pixel team of the DYNAMOP group at the IBS exposed the paradigmatic fluorescent protein EGFP to a study by transient absorption spectroscopy. This technique has previously been applied to fluorescent proteins very little. Pushing it to its resolution limits allowed the detection of the spectral signature of the « phantom » dark state and its characterization by answering all of the above questions. These findings open the way for a better understanding of the photophysical functioning of fluorescent proteins and fuel the hope for finding keys to their further optimization.

A Long-Lived Triplet State Is the Entrance Gateway to Oxidative Photochemistry in Green Fluorescent Proteins. Byrdin M, Duan C, Bourgeois D, Brettel K. Journal of the American Chemical Society;. doi: 10.1021/jacs.7b12755

How bacteria converse in floating biofilms

Biofilms are bacterial communities with high antibiotic resistance. Within biofilms, bacteria exchange information chemically - a mechanism called quorum-sensing. Researchers at the Institute de Biologie Structurale in Grenoble, the University of the Mediterranean in Marseille and the Jacobs University in Bremen have shown that the gram-negative pathogen Providencia stuartii forms floating communities within which adjacent cells are in apparent contact,before depositing as canonical surface-attached biofilms. Because porins are the most abundant proteins in the outer membrane of gram-negative bacteria, they hypothesized that they could be involved in cell-to-cell contact and undertook a structure-function relationship study on the two porins of P. stuartii, Omp-Pst1 and Omp-Pst2. The crystal structures reveal that these porins can selfassociate through their extracellular loops, forming dimers of trimers (DOTs) that could enable cell-to-cell contact within floating communities. Support for this hypothesis was obtained by studying the porin-dependent aggregation of liposomes and model cells. The observation that facing channels are open in the two porin structures suggests that DOTs could not only promote cell-to-cell contact but also contribute to intercellular communication. Proteins involved in this interaction could be new targets in the fight against biofilms.

Porin self-association enables cell-to-cell contact in Providencia stuartii floating communities. El-Khatib M, Nasrallah C, Lopes J, Tran QT, Tetreau G, Basbous H, Fenel D, Gallet B, Lethier M, Bolla JM, Pagès JM, Vivaudou M, Weik M, Winterhalter M, Colletier JP. Proceedings of the National Academy of Sciences of the United States of America. 2018 Feb 23. pii: 201714582. doi: 10.1073/pnas.1714582115.

Bacterial pathogens can reprogram target cells by influencing epigenetic factors

The type III secretion system (T3SS) is a complex nanomachine used by numerous Gram-negative bacteria to inject toxins directly into target cells. Its architecture resembles a syringe, and toxins are believe to travel through its interior. One key aspect of the T3SS is the translocon, a complex of two membrane proteins that are synthesized within the bacterial cytoplasm, transported through the interior of the needle, and subsequently inserted directly into the membrane of the eukaryotic cell, allowing toxin passage. In this work IBS scientists and their collaborators from BIG and London Imperial College showed that insertion of the translocon proteins (PopB and PopD) by the human pathogen Pseudomonas aeruginosa into target membranes engenders epigenetic modifications on histone H3 as a consequence of ion exchange through the formed pore. This thus indicates, for the first time, that the translocon acts not only as a pore, but also as a bona fide virulence factor.

Pore-forming activity of the Pseudomonas aeruginosa type III secretion system translocon alters the host epigenome. Laurent Dortet, Charlotte Lombardi, François Cretin, Andréa Dessen, Alain Filloux. Nature Microbiology 2018 Feb 5. doi: 10.1038/s41564-018-0109-7

Analytical Description of NMR Relaxation Highlights Correlated Dynamics in Intrinsically Disordered Proteins

The dynamic fluctuations of intrinsically disordered proteins (IDPs) define their function. Although experimental nuclear magnetic resonance (NMR) relaxation reveals the motional complexity of these highly flexible proteins, the absence of physical models describing IDP dynamics hinders their mechanistic interpretation. Combining molecular dynamics simulation and NMR, the researchers of the FDP group (Protein Dynaics and Flexibility by NMR) introduce a framework in which distinct motions are attributed to local libration, backbone dihedral angle dynamics and longer-range tumbling of one or more peptide planes. This model provides unique insight into segmental organization of dynamics in IDPs and allows them to investigate the presence and extent of the correlated motions that are essential for function.

Analytical Description of NMR Relaxation Highlights Correlated Dynamics in Intrinsically Disordered Proteins. Salvi N, Abyzov A, Blackledge M. Angewandte Chemie International Edition England ;56(45):14020-14024.

Antibiotics and radical-based chemistry: the 1,2-diol dehydratase AprD4 from the inside

New sources of antibiotics are required to fight against multidrug-resistant pathogens. Natural product biosynthetic pathways are a vast source of inspiration to develop new efficient and environment-friendly chemical synthesis processes. Radical-based chemistry, using high-energy intermediates can afford difficult reactions in water. ‘Radical SAM’ enzymes control such intermediates to perform regio- and stereo-specific reactions. The crystal structure of the radical SAM 1,2-diol dehydratase AprD4, determined by the Metalloproteins unit at IBS in collaboration with the group of Pr. Qi Zhang at Fudan University (Shanghai, China), has revealed that the remarkable tridimensional arrangement at its active site, while keeping substrate-specificity, gives the radical intermediate enough freedom to adopt different conformations, in order to release a specific water molecule. This modification makes certain aminoglycoside antibiotics insensitive to the most common mechanisms of resistance toward this family of antimicrobial agents.

1,2-diol dehydration by the radical SAM enzyme AprD4 - a matter of proton circulation and substrate flexibility. Liu WQ, Amara P, Mouesca JM, Ji X, Renoux O, Martin L, Zhang C, Zhang Q, Nicolet Y. Journal of the American Chemical Society 2018 Jan 4. doi: 10.1021/jacs.7b10501. [Epub ahead of print]