Bernard Gilquin

On the Convergent Evolution of Animal Toxins CONSERVATION OF A DIAD OF FUNCTIONAL RESIDUES IN POTASSIUM CHANNEL-BLOCKING TOXINS WITH UNRELATED STRUCTURES

BgK is a K+ channel-blocking toxin from the sea anemone Bunodosoma granulifera It is a 37-residue protein that adopts a novel fold, as determined by NMR and modeling. An alanine-scanning-based analysis revealed the functional importance of five residues, which include a critical lysine and an aromatic residue separated by 6.6 ± 1.0 Å. The same diad is found in the three known homologous toxins from sea anemones. More strikingly, a similar functional diad is present in all K+ channel-blocking toxins from scorpions, although these toxins adopt a distinct scaffold. Moreover, the functional diads of potassium channel-blocking toxins from sea anemone and scorpions superimpose in the three-dimensional structures. Therefore, toxins that have unrelated structures but similar functions possess conserved key functional residues, organized in an identical topology, suggesting a convergent functional evolution for these small proteins.

Crystal structure of the human urokinase plasminogen activator receptor bound to an antagonist peptide

We report the crystal structure of a soluble form of human urokinase‐type plasminogen activator receptor (uPAR/CD87), which is expressed at the invasive areas of the tumor‐stromal microenvironment in many human cancers. The structure was solved at 2.7 Å in association with a competitive peptide inhibitor of the urokinase‐type plasminogen activator (uPA)–uPAR interaction. uPAR is composed of three consecutive three‐finger domains organized in an almost circular manner, which generates both a deep internal cavity where the peptide binds in a helical conformation, and a large external surface. This knowledge combined with the discovery of a convergent binding motif shared by the antagonist peptide and uPA allowed us to build a model of the human uPA–uPAR complex. This model reveals that the receptor‐binding module of uPA engages the uPAR central cavity, thus leaving the external receptor surface accessible for other protein interactions (vitronectin and integrins). By this unique structural assembly, uPAR can orchestrate the fine interplay with the partners that are required to guide uPA‐focalized proteolysis on the cell surface and control cell adhesion and migration.

Experimentally based model of a complex between a snake toxin and the α7 nicotinic receptor

To understand how snake neurotoxins interact with nicotinic acetylcholine receptors, we have elaborated an experimentally based model of the α–cobratoxin–α7 receptor complex. This model was achieved by using (i) a three-dimensional model of the α7 extracellular domain derived from the crystallographic structure of the homologous acetylcholine-binding protein, (ii) the previously solved x-ray structure of the toxin, and (iii) nine pairs of residues identified by cycle-mutant experiments to make contacts between the α-cobratoxin and α7 receptor. Because the receptor loop F occludes entrance of the toxin binding pocket, we submitted this loop to a dynamics simulation and selected a conformation that allowed the toxin to reach its binding site. The three-dimensional structure of the toxin–receptor complex model was validated a posteriori by an additional double-mutant experiment. The model shows that the toxin interacts perpendicularly to the receptor axis, in an equatorial position of the extracellular domain. The tip of the toxin central loop plugs into the receptor between two subunits, just below the functional receptor loop C, the C-terminal tail of the toxin making adjacent additional interactions at the receptor surface. The receptor establishes major contacts with the toxin by its loop C, which is assisted by principal (loops A and B) and complementary (loops D, F, and 1) functional regions. This model explains the antagonistic properties of the toxin toward the neuronal receptor and opens the way to the design of new antagonists.

Structural Characterization of the LEM Motif Common to Three Human Inner Nuclear Membrane Proteins

BACKGROUND Integral membrane proteins of the inner nuclear membrane are involved in chromatin organization and postmitotic reassembly of the nucleus. The discovery that mutations in the gene encoding emerin causes X-linked Emery-Dreifuss muscular dystrophy has enhanced interest in such proteins. A common structural domain of 50 residues, called the LEM domain, has been identified in emerin MAN1, and lamina-associated polypeptide (LAP) 2. In particular, all LAP2 isoforms share an N-terminal segment composed of such a LEM domain that is connected to a highly divergent LEM-like domain by a linker that is probably unstructured. RESULTS We have determined the three-dimensional structures of the LEM and LEM-like domains of LAP2 using nuclear magnetic resonance and molecular modeling. Both domains adopt the same fold, mainly composed of two large parallel alpha helices. CONCLUSIONS The structural LEM motif is found in human inner nuclear membrane proteins and in protein-protein interaction domains from bacterial multienzyme complexes. This suggests that LEM and LEM-like domains are protein-protein interaction domains. A region conserved in all LEM domains, at the surface of helix 2, could mediate interaction between LEM domains and a common protein partner.

Structure of bacteriophage SPP1 head-to-tail connection reveals mechanism for viral DNA gating

In many bacterial viruses and in certain animal viruses, the double-stranded DNA genome enters and exits the capsid through a portal gatekeeper. We report a pseudoatomic structure of a complete portal system. The bacteriophage SPP1 gatekeeper is composed of dodecamers of the portal protein gp6, the adaptor gp15, and the stopper gp16. The solution structures of gp15 and gp16 were determined by NMR. They were then docked together with the X-ray structure of gp6 into the electron density of the ≈1-MDa SPP1 portal complex purified from DNA-filled capsids. The resulting structure reveals that gatekeeper assembly is accompanied by a large rearrangement of the gp15 structure and by folding of a flexible loop of gp16 to form an intersubunit parallel β-sheet that closes the portal channel. This stopper system prevents release of packaged DNA. Disulfide cross-linking between β-strands of the stopper blocks the key conformational changes that control genome ejection from the virus at the beginning of host infection.

Novel miniproteins engineered by the transfer of active sites to small natural scaffolds.

Small multidisulfide-containing proteins are attractive structural templates to produce a biologically active conformation that mimics the binding surface of natural large proteins. In particular, the structural motif that is evolutionary conserved in all scorpion toxins has a small size (30-40 amino acid residues), a great structural stability, and high permissiveness for sequence mutation. This motif is composed of a beta-sheet and an alpha-helix bridged in the interior core by three disulfides. We have used this motif successfully to transfer within its beta-sheet new functional sites, including the curaremimetic loop of a snake neurotoxin and the CDR2-like site of human CD4. Accumulated evidence indicated that the two miniproteins produced, the curaremimetic miniprotein and the CD4 mimetic, contain the alpha/beta fold that is characteristic of the scaffold used and bind respectively to the acetylcholine receptor and to the envelope gp120 of HIV-1. Furthermore, the latter was shown to prevent viral infection of lymphocytes. These examples illustrate that, by the transfer of active sites to small and stable natural scaffolds, it is possible to engineer miniproteins reproducing, in part, the function of much larger proteins. Such miniproteins may be of great utility as tools in structure-function studies and as leads in drug design.

Structural analysis of emerin, an inner nuclear membrane protein mutated in X-linked Emery-Dreifuss muscular dystrophy

Like Duchenne and Becker muscular dystrophies, Emery–Dreifuss muscular dystrophy (EDMD) is characterized by myopathic and cardiomyopathic abnormalities. EDMD has the particularity of being linked to mutations in nuclear proteins. The X‐linked form of EDMD is caused by mutations in the emerin gene, whereas autosomal dominant EDMD is caused by mutations in the lamin A/C gene. Emerin colocalizes with lamin A/C in interphase cells, and binds in vitro to lamin A/C. Recent work suggests that lamin A/C might serve as a receptor for emerin. We have undertaken a structural analysis of emerin, and in particular of its N‐terminal domain, which is comprised in the emerin segment critical for binding to lamin A/C. We show that region 2–54 of emerin adopts the LEM fold. This fold was originally described in the two N‐terminal domains of another inner nuclear membrane protein called lamina‐associated protein 2 (LAP2). The existence of a conserved solvent‐exposed surface on the LEM domains of LAP2 and emerin is discussed, as well as the nature of a possible common target.

Characterization of the functional epitope on the urokinase receptor. Complete alanine scanning mutagenesis supplemented by chemical cross-linking.

The high affinity interaction between the serine protease urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR) represents one of the key regulatory steps in cell surface-associated plasminogen activation. On the basis on our crystal structure solved for uPAR in complex with a peptide antagonist, we recently proposed a model for the corresponding complex with the growth factor-like domain of uPA (Llinas et al. (2005) EMBO J. 24, 1655-1663). In the present study, we provide experimental evidence that consolidates and further develops this model using data from a comprehensive alanine scanning mutagenesis of uPAR combined with low resolution distance constraints defined within the complex using chemical cross-linkers as molecular rulers. The kinetic rate constants for the interaction between pro-uPA and 244 purified uPAR mutants with single-site replacements were determined by surface plasmon resonance. This complete alanine scanning of uPAR highlighted the involvement of 20 surface-exposed side chains in this interaction. Mutations causing ΔΔG ≥ 1 kcal/mol for the uPA interaction are all located within or at the rim of the central cavity uniquely formed by the assembly of all three domains in uPAR, whereas none are found outside this crevice. Identification of specific cross-linking sites in uPAR and pro-uPA enabled us to build a model of the uPAR·uPA complex in which the kringle domain of uPA was positioned by the constraints established by the range of these cross-linkers. The nature of this interaction is predominantly hydrophobic and highly asymmetric, thus emphasizing the importance of the shape and size of the central cavity when designing low molecular mass antagonists of the uPAR/uPA interaction.

Changing the Structural Context of a Functional -Hairpin SYNTHESIS AND CHARACTERIZATION OF A CHIMERA CONTAINING THE CURAREMIMETIC LOOP OF A SNAKE TOXIN IN THE SCORPION α/β SCAFFOLD

An approach to obtain new active proteins is the incorporation of all or a part of a well defined active site onto a natural structure acting as a structural scaffold. According to this strategy we tentatively engineered a new curaremimetic molecule by transferring the functional central loop of a snake toxin, sequence 26-37, sandwiched between two hairpins, onto the structurally similar β-hairpin of the scorpion toxin charybdotoxin, stabilized by a short helix. The resulting chimeric molecule, only 31 amino acids long, was produced by solid phase synthesis, refolded, and purified to homogeneity. As shown by structural analysis performed by CD and NMR spectroscopy, the chimera maintained the expected α/β fold characteristic of scorpion toxins and presented a remarkable structural stability. The chimera competitively displaces the snake curaremimetic toxin α from the acetylcholine receptor at 10M concentrations. Antibodies, elicited in rabbits against the chimera, recognize the parent snake toxin and prevent its binding to the acetylcholine receptor, thus neutralizing its toxic function. All these data demonstrate that the strategy of active site transfer to the charybdotoxin scaffold has general applications in the engineering of novel ligands for membrane receptors and in vaccine design.

The carboxyl-terminal nucleoplasmic region of MAN1 exhibits a DNA binding winged helix domain.

MAN1 is an integral protein of the inner nuclear membrane that interacts with nuclear lamins and emerin, thus playing a role in nuclear organization. It also binds to chromatin-associated proteins and transcriptional regulators, including the R-Smads, Smad1, Smad2, and Smad3. Mutations in the human gene encoding MAN1 cause sclerosing bone dysplasias, which sometimes have associated skin abnormalities. At the molecular level, these mutations lead to loss of the MAN1-R-Smads interaction, thus perturbing transforming growth factor β superfamily signaling pathway. As a first step to understanding the physical basis of MAN1 interaction with R-Smads, we here report the structural characterization of the carboxyl-terminal nucleoplasmic region of MAN1, which is responsible for Smad binding. This region exhibits an amino-terminal globular domain adopting a winged helix fold, as found in several Smad-associated sequence-specific DNA binding factors. Consistently, it binds to DNA through the positively charged recognition helix H3 of its winged helix motif. However, it does not show the predicted carboxyl-terminal U2AF homology domain in solution, suggesting that the folding and stability of such a domain in MAN1 depend upon binding to an unidentified partner. Modeling the complex between DNA and the winged helix domain shows that the regions involved in DNA binding are essentially distinct from those reported to be involved in Smad binding. This suggests that MAN1 binds simultaneously to R-Smads and their targeted DNA sequences.