Christophe Briand

Insights into the Regulatory Mechanism for Caspase-8 Activation

In the death receptor induced apoptotic pathway, caspase-8 autocatalytically cleaves itself at specific cleavage sites. To better understand the regulatory mechanisms behind caspase-8 activation, we compared active wild-type caspase-8 (wtC8) and an uncleavable form of procaspase-8 (uncleavable C8). We demonstrate that wtC8 predominantly exists as a monomer and dimerizes in a concentration and inhibitor binding-dependent fashion. The K(D) for dimeric wtC8 is approximately 50 micro M and decreases when inhibitor bound. Uncleavable C8 is mainly monomeric, but a small amount that dimerizes is as active as wtC8. Inhibitor binding does not favor dimerization but induces active site rearrangements in uncleavable C8. Our findings suggest that dimerization is the crucial factor for caspase-8 activation.

Drug Export Pathway of Multidrug Exporter AcrB Revealed by DARPin Inhibitors

The multidrug exporter AcrB is the inner membrane component of the AcrAB-TolC drug efflux system in Escherichia coli and is responsible for the resistance of this organism to a wide range of drugs. Here we describe the crystal structure of the trimeric AcrB in complex with a designed ankyrin-repeat protein (DARPin) inhibitor at 2.5-Å resolution. The three subunits of AcrB are locked in different conformations revealing distinct channels in each subunit. There seems to be remote conformational coupling between the channel access, exit, and the putative proton-translocation site, explaining how the proton motive force is used for drug export. Thus our structure suggests a transport pathway not through the central pore but through the identified channels in the individual subunits, which greatly advances our understanding of the multidrug export mechanism.

Crystal structure of a heterodimeric ABC transporter in its inward-facing conformation.

ATP-binding cassette (ABC) transporters shuttle a wide variety of molecules across cell membranes by alternating between inward- and outward-facing conformations, harnessing the energy of ATP binding and hydrolysis at their nucleotide binding domains (NBDs). Here we present the 2.9-Å crystal structure of the heterodimeric ABC transporter TM287–TM288 (TM287/288) from Thermotoga maritima in its inward-facing state. In contrast to previous studies, we found that the NBDs only partially separate, remaining in contact through an interface involving conserved motifs that connect the two ATP hydrolysis sites. We observed AMP-PNP binding to the degenerate catalytic site, which deviates from the consensus sequence in the same positions as the eukaryotic homologs CFTR and TAP1–TAP2 (TAP1/2). The TM287/288 structure provides unprecedented insights into the mechanism of heterodimeric ABC exporters and will enable future studies on this large transporter superfamily.

Crystal Structure of the Multidrug Exporter Mexb from Pseudomonas Aeruginosa.

We report here the crystal structure of the Pseudomonas aeruginosa multidrug exporter MexB, an intensively studied member of the resistance-nodulation-cell division family of secondary active transporters, at 3.0 A. MexB forms an asymmetric homotrimer where each subunit adopts a different conformation representing three snapshots of the transport cycle similar to the recently determined structures of its close homologue AcrB from Escherichia coli, so far the sole structurally characterized member of the superfamily. As for AcrB, the conformations of two subunits can be clearly assigned to either the binding step or the extrusion step in the transport process. Unexpectedly, a remarkable conformational shift in the third subunit is observed in MexB, which has potential implications for the assembly of the tripartite MexAB-OprM drug efflux system. Furthermore, an n-dodecyl-d-maltoside molecule was found bound to the internal multidrug-binding cavity, which might indicate that MexB binds and transports detergent molecules as substrates. As the only missing piece of the puzzle in the MexAB-OprM system, the X-ray structure of MexB completes the molecular picture of the major pump mediating intrinsic and acquired multidrug resistance in P. aeruginosa.

Structure of the PRYSPRY-domain: Implications for autoinflammatory diseases

We determined the first structure of PRYSPRY, a domain found in over 500 different proteins, involved in innate immune signaling, cytokine signaling suppression, development, cell growth and retroviral restriction. The fold encompasses a 7‐stranded and a 6‐stranded antiparallel β‐sheet, arranged in a β‐sandwich. In the crystal, PRYSPRY forms a dimer where the C‐terminus of an acceptor molecule binds to the concave surface of a donor molecule, which represents a putative interaction site. Mutations in the PRYSPRY domains of Pyrin, which are responsible for familial Mediterranean fever, map on the putative PRYSPRY interaction site.

The Crystal Structure of Caspase-6, a Selective Effector of Axonal Degeneration.

Neurodegenerative diseases pose one of the most pressing unmet medical needs today. It has long been recognized that caspase-6 may play a role in several neurodegenerative diseases for which there are currently no disease-modifying therapies. Thus it is a potential target for neurodegenerative drug development. In the present study we report on the biochemistry and structure of caspase-6. As an effector caspase, caspase-6 is a constitutive dimer independent of the maturation state of the enzyme. The ligand-free structure shows caspase-6 in a partially mature but latent conformation. The cleaved inter-domain linker remains partially inserted in the central groove of the dimer, as observed in other caspases. However, in contrast with the structures of other caspases, not only is the catalytic machinery misaligned, but several structural elements required for substrate recognition are missing. Most importantly, residues forming a short anti-parallel beta-sheet abutting the substrate in other caspase structures are part of an elongation of the central alpha-helix. Despite the dramatic structural changes that are required to adopt a canonical catalytically competent conformation, the pre-steady-state kinetics exhibit no lag phase in substrate turnover. This suggests that the observed conformation does not play a regulatory role in caspase-6 activity. However, targeting the latent conformation in search for specific and bio-available caspase-6 inhibitors might offer an alternative to active-site-directed approaches.

Identification of a basic surface area of the FADD death effector domain critical for apoptotic signaling

Death effector domains (DEDs) are protein–protein interaction domains found in the death inducing signaling complex (DISC). Performing a structure‐based alignment of all DED sequences we identified a region of high diversity in α‐helix 3 and propose a classification of DEDs into class I DEDs typically containing a stretch of basic residues in the α‐helix 3 region whereas DEDs of class II do not. Functional assays using mutants of Fas‐associated death domain revealed that this basic region influences binding and recruitment of caspase‐8 and cellular FLICE inhibitor protein to the DISC.

A series of Fas receptor agonist antibodies that demonstrate an inverse correlation between affinity and potency

Receptor agonism remains poorly understood at the molecular and mechanistic level. In this study, we identified a fully human anti-Fas antibody that could efficiently trigger apoptosis and therefore function as a potent agonist. Protein engineering and crystallography were used to mechanistically understand the agonistic activity of the antibody. The crystal structure of the complex was determined at 1.9 Å resolution and provided insights into epitope recognition and comparisons with the natural ligand FasL (Fas ligand). When we affinity-matured the agonist antibody, we observed that, surprisingly, the higher-affinity antibodies demonstrated a significant reduction, rather than an increase, in agonist activity at the Fas receptor. We propose and experimentally demonstrate a model to explain this non-intuitive impact of affinity on agonist antibody signalling and explore the implications for the discovery of therapeutic agonists in general.

Crystal structure of neuroserpin: a neuronal serpin involved in a conformational disease

The protease inhibitor neuroserpin regulates the development of the nervous system and its plasticity in the adult. Neuroserpins carrying the Ser53Pro or Ser56Arg mutation form polymers in neuronal cells. We describe here the structure of wild‐type neuroserpin in a cleaved form. The structure provides a basis to understand the role of the mutations in the polymerization process. We propose that these mutations could delay the insertion of the reactive center loop into the central β‐sheet A, an essential step in the inhibition and possibly in the polymerization of neuroserpin.

Structure and oligomeric state of the mammalian tumour-associated antigen UK114.

The tumour-associated antigen UK114, isolated from goat liver, belongs to the YER057c/YIL051c/YjgF protein family, which has members in both the prokaryotes and eukaryotes. The crystal structure of a mammalian representative, goat UK114, was determined, revealing a trimeric arrangement in the crystal. It was confirmed by ultracentrifugation that UK114 is a trimer in solution. These results are in agreement with the published structures of homologues from unicellular organisms, but contrast with those reported for the rat homologue of UK114, for which a dimeric quaternary structure was proposed.