Nicolas Wolff

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.

Attenuation of Rabies Virulence: Takeover by the Cytoplasmic Domain of Its Envelope Protein

Survival of rabies virus–infected neurons depends on a single amino acid in the PDZ-binding site of a viral protein. Tipping the Balance Strains of rabies virus, which infects neurons, may be virulent, in which case the cells survive long enough for the virus to replicate and spread, or they may be attenuated, in which case the infected cells die by apoptosis. Préhaud et al. compared one attenuated and one virulent viral strain and found that a single amino acid change in a region of a viral envelope protein that binds to host cell proteins was sufficient to account for the death or survival of infected cells. The binding properties of the attenuated virus protein were expanded, thereby affecting the balance in the activities of host kinases and phosphatases sufficiently to trigger cell death. These findings may inform strategies to engineer attenuated viruses, which are often used in live vaccines. The capacity of a rabies virus to promote neuronal survival (a signature of virulence) or death (a marker of attenuation) depends on the cellular partners recruited by the PDZ-binding site (PDZ-BS) of its envelope glycoprotein (G). Neuronal survival requires the selective association of the PDZ-BS of G with the PDZ domains of two closely related serine-threonine kinases, MAST1 and MAST2. Here, we found that a single amino acid change in the PDZ-BS triggered the apoptotic death of infected neurons and enabled G to interact with additional PDZ partners, in particular the tyrosine phosphatase PTPN4. Knockdown of PTPN4 abrogated virus-mediated apoptosis. Thus, we propose that attenuation of rabies virus requires expansion of the set of host PDZ proteins with which G interacts, which interferes with the finely tuned homeostasis required for survival of the infected neuron.

Haemophore‐mediated bacterial haem transport: evidence for a common or overlapping site for haem‐free and haem‐loaded haemophore on its specific outer membrane receptor

Bacterial extracellular haemophores also named HasA for haem acquisition system form an independent family of haemoproteins that take up haem from host haeme carriers and shuttle it to specific receptors (HasR). Haemophore receptors are required for the haemophore‐dependent haem acquisition pathway and alone allow free or haemoglobin‐bound haem uptake, but the synergy between the haemophore and its receptor greatly facilitates this uptake. The three‐dimensional structure of the Serratia marcescens holo‐haemophore (HasASM) has been determined previously and revealed that the haem iron atom is ligated by tyrosine 75 and histidine 32. The phenolate of tyrosine 75 is also tightly hydrogen bonded to the Nδ atom of histidine 83. Alanine mutagenesis of these three HasASM residues was performed, and haem‐binding constants of the wild‐type protein, the three single mutant proteins, the three double mutant proteins and the triple mutant protein were compared by absorption spectrometry to probe the roles of H32, Y75 and H83 in haem binding. We show that one axial iron ligand is sufficient to ligate haem efficiently and that H83 may become an alternative iron ligand in the absence of Y75 or both H32 and Y75. All the single mutant proteins retained the ability to stimulate haemophore‐dependent haem uptake in vivo. Thus, the residues H32, Y75 and H83 are not individually necessary for haem delivery to the receptor. The binding of haem‐free and haem‐loaded HasASM proteins to HasRSM‐producing strains was studied. Both proteins bind to HasRSM with similar apparent Kd. The double mutant H32A‐Y75A competitively inhibits binding to the receptor of both holo‐HasASM and apo‐HasASM, showing that there is a unique or overlapping site on HasRSM for the apo‐ and holo‐haemophores. Thus, we propose a new mechanism for haem uptake, in which haem is exchanged between haem‐loaded haemophores and unloaded haemophores bound to the receptor without swapping of haemophores on the receptor.

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.

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Bacterial extracellular haemophores also named HasA for haem acquisition system form an independent family of haemoproteins that take up haem from host haeme carriers and shuttle it to specific receptors (HasR). Haemophore receptors are required for the haemophore‐dependent haem acquisition pathway and alone allow free or haemoglobin‐bound haem uptake, but the synergy between the haemophore and its receptor greatly facilitates this uptake. The three‐dimensional structure of the Serratia marcescens holo‐haemophore (HasASM) has been determined previously and revealed that the haem iron atom is ligated by tyrosine 75 and histidine 32. The phenolate of tyrosine 75 is also tightly hydrogen bonded to the Nδ atom of histidine 83. Alanine mutagenesis of these three HasASM residues was performed, and haem‐binding constants of the wild‐type protein, the three single mutant proteins, the three double mutant proteins and the triple mutant protein were compared by absorption spectrometry to probe the roles of H32, Y75 and H83 in haem binding. We show that one axial iron ligand is sufficient to ligate haem efficiently and that H83 may become an alternative iron ligand in the absence of Y75 or both H32 and Y75. All the single mutant proteins retained the ability to stimulate haemophore‐dependent haem uptake in vivo. Thus, the residues H32, Y75 and H83 are not individually necessary for haem delivery to the receptor. The binding of haem‐free and haem‐loaded HasASM proteins to HasRSM‐producing strains was studied. Both proteins bind to HasRSM with similar apparent Kd. The double mutant H32A‐Y75A competitively inhibits binding to the receptor of both holo‐HasASM and apo‐HasASM, showing that there is a unique or overlapping site on HasRSM for the apo‐ and holo‐haemophores. Thus, we propose a new mechanism for haem uptake, in which haem is exchanged between haem‐loaded haemophores and unloaded haemophores bound to the receptor without swapping of haemophores on the receptor.

Histidine pKa shifts and changes of tautomeric states induced by the binding of gallium-protoporphyrin IX in the hemophore HasASM

The HasASM hemophore, secreted by Serratia marcescens, binds free or hemoprotein bound heme with high affinity and delivers it to a specific outer membrane receptor, HasR. In HasASM, heme is held by two loops and coordinated to iron by two residues, His 32 and Tyr 75. A third residue His 83 was shown recently to play a crucial role in heme ligation. To address the mechanistic issues of the heme capture and release processes, the histidine protonation states were studied in both apo‐ and holo‐forms of HasASM in solution. Holo‐HasASM was formed with gallium‐protoporphyrin IX (GaPPIX), giving rise to a diamagnetic protein. By use of heteronuclear correlation NMR spectroscopy, the imidazole side‐chain 15N and 1H resonances of the six HasASM histidines were assigned and their pKa values and predominant tautomeric states according to pH were determined. We show that protonation states of the heme pocket histidines can modulate the nucleophilic character of the two axial ligands and, consequently, control the heme binding. In particular, the essential role of the His 83 is emphasized according to its direct interaction with Tyr 75.

Comparative analysis of structural and dynamic properties of the loaded and unloaded hemophore HasA: functional implications.

A heme-acquisition system present in several Gram-negative bacteria requires the secretion of hemophores. These extracellular carrier proteins capture heme and deliver it to specific outer membrane receptors. The Serratia marcescens HasA hemophore is a monodomain protein that binds heme with a very high affinity. Its alpha/beta structure, as that of its binding pocket, has no common features with other iron- or heme-binding proteins. Heme is held by two loops L1 and L2 and coordinated to iron by an unusual ligand pair, H32/Y75. Two independent regions of the hemophore beta-sheet are involved in HasA-HasR receptor interaction. Here, we report the 3-D NMR structure of apoHasA and the backbone dynamics of both loaded and unloaded hemophore. While the overall structure of HasA is very similar in the apo and holo forms, the hemophore presents a transition from an open to a closed form upon ligand binding, through a large movement, of up to 30 A, of loop L1 bearing H32. Comparison of loaded and unloaded HasA dynamics on different time scales reveals striking flexibility changes in the binding pocket. We propose a mechanism by which these structural and dynamic features provide the dual function of heme binding and release to the HasR receptor.

Antifolding Activity of the SecB Chaperone Is Essential for Secretion of HasA, a Quickly Folding ABC Pathway Substrate

We have previously shown that SecB, the ATP-independent chaperone of the Sec pathway, is required for the secretion of the HasA hemophore from Serratia marcescens via its type I secretion pathway, both in the reconstituted system in Escherichia coli and in the original host. The refolding of apo-HasA after denaturation with guanidine HCl was followed by stopped-flow measurements of fluorescence of its single tryptophan, both in the absence and presence of SecB. In the absence of SecB, HasA folds very quickly with one main phase (45 s–1) accounting for 92% of the signal. SecB considerably slows down HasA folding. At stoichiometric amounts of SecB and HasA, a single phase (0.014 s–1) of refolding is observed. Two double point mutants of HasA were made, abolishing two hydrogen bonds between N-terminal and C-terminal side chain residues. In both cases, the mutants essentially maintained the same secondary and tertiary structure as wild-type HasA and were fully functional. Refolding of both mutants was much slower than that of wild-type HasA and they were secreted essentially independently of SecB. We conclude that SecB has mainly an antifolding function in the HasA ABC secretion pathway.

A Unified Nomenclature and Amino Acid Numbering for Human PTEN

With the discovery of an isoform based on an alternative translation start site, PTEN nomenclature needs an update. The tumor suppressor PTEN is a major brake for cell transformation, mainly due to its phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] phosphatase activity that directly counteracts the oncogenicity of phosphoinositide 3-kinase (PI3K). PTEN mutations are frequent in tumors and in the germ line of patients with tumor predisposition or with neurological or cognitive disorders, which makes the PTEN gene and protein a major focus of interest in current biomedical research. After almost two decades of intense investigation on the 403-residue-long PTEN protein, a previously uncharacterized form of PTEN has been discovered that contains 173 amino-terminal extra amino acids, as a result of an alternate translation initiation site. To facilitate research in the field and to avoid ambiguities in the naming and identification of PTEN amino acids from publications and databases, we propose here a unifying nomenclature and amino acid numbering for this longer form of PTEN.

Interference with the PTEN-MAST2 Interaction by a Viral Protein Leads to Cellular Relocalization of PTEN

The G protein of rabies virus manipulates the cellular localization of PTEN, which may promote cell survival. Rabies Virus Relocalizes PTEN Virulent strains of rabies virus infect neurons and promote survival of the infected cells to favor viral replication. Among the host factors that inhibit neuronal survival are the phosphatase PTEN and one of its binding partners, the kinase MAST2. PTEN and MAST2 interact through the PDZ domain of MAST2 and the PDZ domain–binding site (PDZ-BS) of PTEN. Terrien et al. found that the rabies virus glycoprotein (G protein), which contains a PDZ-BS, disrupted the MAST2-PTEN complex in infected cells. Structural analysis showed that the surfaces of PTEN and G protein that interacted with MAST2 were similar and contained previously uncharacterized PDZ-binding regions. Finally, disruption of the MAST2-PTEN complex by viral G protein resulted in the relocalization of PTEN from the nucleus to the cytoplasm. Together, these data suggest that competition between viral G protein and MAST2 for binding to PTEN plays a role in the survival of infected cells. PTEN (phosphatase and tensin homolog deleted on chromosome 10) and MAST2 (microtubule-associated serine and threonine kinase 2) interact with each other through the PDZ domain of MAST2 (MAST2-PDZ) and the carboxyl-terminal (C-terminal) PDZ domain–binding site (PDZ-BS) of PTEN. These two proteins function as negative regulators of cell survival pathways, and silencing of either one promotes neuronal survival. In human neuroblastoma cells infected with rabies virus (RABV), the C-terminal PDZ domain of the viral glycoprotein (G protein) can target MAST2-PDZ, and RABV infection triggers neuronal survival in a PDZ-BS–dependent fashion. These findings suggest that the PTEN-MAST2 complex inhibits neuronal survival and that viral G protein disrupts this complex through competition with PTEN for binding to MAST2-PDZ. We showed that the C-terminal sequences of PTEN and the viral G protein bound to MAST2-PDZ with similar affinities. Nuclear magnetic resonance structures of these complexes exhibited similar large interaction surfaces, providing a structural basis for their binding specificities. Additionally, the viral G protein promoted the nuclear exclusion of PTEN in infected neuroblastoma cells in a PDZ-BS–dependent manner without altering total PTEN abundance. These findings suggest that formation of the PTEN-MAST2 complex is specifically affected by the viral G protein and emphasize how disruption of a critical protein-protein interaction regulates intracellular PTEN trafficking. In turn, the data show how the viral protein might be used to decipher the underlying molecular mechanisms and to clarify how the subcellular localization of PTEN regulates neuronal survival.