Biomedical subjects

Christophe Briand

Publications and source records attributed to Christophe Briand.

10 recordsLinked to original sources

PubMed2007

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-A 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.

Ankyrin Repeat↗

PubMed2006

Structure of the mammalian NOS regulator dimethylarginine dimethylaminohydrolase: A basis for the design of specific inhibitors.

Dimethylarginine dimethylaminohydrolase (DDAH) is involved in the regulation of nitric oxide synthase (NOS) by metabolizing the free endogenous arginine derivatives N(omega)-methyl-L-arginine (MMA) and N(omega),N(omega)-dimethyl-L-arginine (ADMA), which are competitive inhibitors of NOS. Here, we present high-resolution crystal structures of DDAH isoform 1 (DDAH-1) isolated from bovine brain in complex with different inhibitors, including S-nitroso-L-homocysteine and Zn2+, a regulator of this mammalian enzyme. The structure of DDAH-1 consists of a propeller-like fold similar to other arginine-modifying enzymes and a flexible loop, which adopts different conformations and acts as a lid at the entrance of the active site. The orientation and interaction mode of inhibitors in the active site give insight into the regulation and the molecular mechanism of the enzyme. The presented structures provide a basis for the structure-based development of specific DDAH-1 inhibitors that might be useful in the therapeutic treatment of NOS dysfunction-related diseases.

Amidohydrolases↗

PubMed2005

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 beta-sheet, arranged in a beta-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.

Amino Acid Motifs↗

PubMed2005

Allosteric inhibition of aminoglycoside phosphotransferase by a designed ankyrin repeat protein.

Aminoglycoside phosphotransferase (3')-IIIa (APH) is a bacterial kinase that confers antibiotic resistance to many pathogenic bacteria and shares structural homology with eukaryotic protein kinases. We report here the crystal structure of APH, trapped in an inactive conformation by a tailor-made inhibitory ankyrin repeat (AR) protein, at 2.15 A resolution. The inhibitor was selected from a combinatorial library of designed AR proteins. The AR protein binds the C-terminal lobe of APH and thereby stabilizes three alpha helices, which are necessary for substrate binding, in a significantly displaced conformation. BIAcore analysis and kinetic enzyme inhibition experiments are consistent with the proposed allosteric inhibition mechanism. In contrast to most small-molecule kinase inhibitors, the AR proteins are not restricted to active site binding, allowing for higher specificity. Inactive conformations of pharmaceutically relevant enzymes, as can be elucidated with the approach presented here, represent powerful starting points for rational drug design.

Allosteric Regulation↗

PubMed2004

High-affinity binders selected from designed ankyrin repeat protein libraries.

We report here the evolution of ankyrin repeat (AR) proteins in vitro for specific, high-affinity target binding. Using a consensus design strategy, we generated combinatorial libraries of AR proteins of varying repeat numbers with diversified binding surfaces. Libraries of two and three repeats, flanked by 'capping repeats,' were used in ribosome-display selections against maltose binding protein (MBP) and two eukaryotic kinases. We rapidly enriched target-specific binders with affinities in the low nanomolar range and determined the crystal structure of one of the selected AR proteins in complex with MBP at 2.3 A resolution. The interaction relies on the randomized positions of the designed AR protein and is comparable to natural, heterodimeric protein-protein interactions. Thus, our AR protein libraries are valuable sources for binding molecules and, because of the very favorable biophysical properties of the designed AR proteins, an attractive alternative to antibody libraries.

Amino Acid Sequence↗

PubMed2003

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.

Animals↗

PubMed2003

Crystal structure of caspase-2, apical initiator of the intrinsic apoptotic pathway.

The cell death protease caspase-2 has recently been recognized as the most apical caspase in the apoptotic cascade ignited during cell stress signaling. Cytotoxic stress, such as that caused by cancer therapies, leads to activation of caspase-2, which acts as a direct effector of the mitochondrion-dependent apoptotic pathway resulting in programmed cell death. Here we report the x-ray structure of caspase-2 in complex with the inhibitor acetyl-Leu-Asp-Glu-Ser-Asp-aldehyde at 1.65-A resolution. Compared with other caspases, significant structural differences prevail in the active site region and the dimer interface. The structure reveals the hydrophobic properties of the S5 specificity pocket, which is unique to caspase-2, and provides the details of the inhibitor-protein interactions in subsites S1-S4. These features form the basis of caspase-2 specificity and allow the design of caspase-2-directed ligands for medical and analytical use. Another unique feature of caspase-2 is a disulfide bridge at the dimer interface, which covalently links the two monomers. Consistent with this finding, caspase-2 exists as a (p19/p12)2 dimer in solution, even in the absence of substrates or inhibitors. The intersubunit disulfide bridge stabilizes the dimeric form of caspase-2, whereas all other long prodomain caspases exist as monomers in solution, and dimer formation is driven by ligand binding. Therefore, the central disulfide bridge appears to represent a novel way of dimer stabilization in caspases.

Apoptosis↗

PubMed2003

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.

Amino Acid Chloromethyl Ketones↗

PubMed2002

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 alpha-helix 3 and propose a classification of DEDs into class I DEDs typically containing a stretch of basic residues in the alpha-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.

Adaptor Proteins, Signal Transducing↗

PubMed2002

The long form of FLIP is an activator of caspase-8 at the Fas death-inducing signaling complex.

Death receptors, such as Fas and tumor necrosis factor-related apoptosis-inducing ligand receptors, recruit Fas-associated death domain and pro-caspase-8 homodimers, which are then autoproteolytically activated. Active caspase-8 is released into the cytoplasm, where it cleaves various proteins including pro-caspase-3, resulting in apoptosis. The cellular Fas-associated death domain-like interleukin-1-beta-converting enzyme-inhibitory protein long form (FLIP(L)), a structural homologue of caspase-8 lacking caspase activity because of several mutations in the active site, is a potent inhibitor of death receptor-induced apoptosis. FLIP(L) is proposed to block caspase-8 activity by forming a proteolytically inactive heterodimer with caspase-8. In contrast, we propose that FLIP(L)-bound caspase-8 is an active protease. Upon heterocomplex formation, a limited caspase-8 autoprocessing occurs resulting in the generation of the p43/41 and the p12 subunits. This partially processed form but also the non-cleaved FLIP(L)-caspase-8 heterocomplex are proteolytically active because they both bind synthetic substrates efficiently. Moreover, FLIP(L) expression favors receptor-interacting kinase (RIP) processing within the Fas-signaling complex. We propose that FLIP(L) inhibits caspase-8 release-dependent pro-apoptotic signals, whereas the single, membrane-restricted active site of the FLIP(L)-caspase-8 heterocomplex is proteolytically active and acts on local substrates such as RIP.

Amino Acid Sequence↗