Aggregation and Amyloidogenicity of the Nuclear Coactivator Binding Domain of CREB-Binding Protein.

The nuclear coactivator binding domain (NCBD) of transcriptional co-regulator CREB-binding protein (CBP) is an example of conformationally malleable proteins that can bind to structurally unrelated protein targets and adopt distinct folds in the respective protein complexes. Here, we show that the folding landscape of NCBD contains an alternative pathway that results in protein aggregation and self-assembly into amyloid fibers. The initial steps of such protein misfolding are driven by intermolecular interactions of its N-terminal α-helix bringing multiple NCBD molecules into contact. These oligomers then undergo slow but progressive interconversion into ß-sheet-containing aggregates. To reveal the concealed aggregation potential of NCBD we used a chemically synthesized mirror-image d-NCBD form. The addition of d-NCBD promoted self-assembly into amyloid precipitates presumably due to formation of thermodynamically more stable racemic ß-sheet structures. The unexpected aggregation of NCBD needs to be taken into consideration given the multitude of protein-protein interactions and resulting biological functions mediated by CBP.

Structural insight into negative DNA supercoiling by DNA gyrase, a bacterial type 2A DNA topoisomerase.

Type 2A DNA topoisomerases (Topo2A) remodel DNA topology during replication, transcription and chromosome segregation. These multisubunit enzymes catalyze the transport of a double-stranded DNA through a transient break formed in another duplex. The bacterial DNA gyrase, a target for broad-spectrum antibiotics, is the sole Topo2A enzyme able to introduce negative supercoils. We reveal here for the first time the architecture of the full-length Thermus thermophilus DNA gyrase alone and in a cleavage complex with a 155 bp DNA duplex in the presence of the antibiotic ciprofloxacin, using cryo-electron microscopy. The structural organization of the subunits of the full-length DNA gyrase points to a central role of the ATPase domain acting like a 'crossover trap' that may help to sequester the DNA positive crossover before strand passage. Our structural data unveil how DNA is asymmetrically wrapped around the gyrase-specific C-terminal ß-pinwheel domains and guided to introduce negative supercoils through cooperativity between the ATPase and ß-pinwheel domains. The overall conformation of the drug-induced DNA binding-cleavage complex also suggests that ciprofloxacin traps a DNA pre-transport conformation.

Power and limitations of electrophoretic separations in proteomics strategies.

Proteomics can be defined as the large-scale analysis of proteins. Due to the complexity of biological systems, it is required to concatenate various separation techniques prior to mass spectrometry. These techniques, dealing with proteins or peptides, can rely on chromatography or electrophoresis. In this review, the electrophoretic techniques are under scrutiny. Their principles are recalled, and their applications for peptide and protein separations are presented and critically discussed. In addition, the features that are specific to gel electrophoresis and that interplay with mass spectrometry (i.e., protein detection after electrophoresis, and the process leading from a gel piece to a solution of peptides) are also discussed.

A novel, generic and effective method for the rapid purification of G protein-coupled receptors.

G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors and are of major therapeutic importance. Structure determination of G protein-coupled receptors and other applications require milligram quantities of purified receptor proteins on a regular basis. Recombinant GPCRs fused to a heterologous biotinylation domain were produced in the yeast Pichia pastoris. We describe an efficient method for their rapid purification that relies on the capture of these receptors with streptavidin immobilized on agarose beads, and their subsequent release by enzymatic digestion with TEV protease. This method has been applied to several GPCRs belonging to the class A rhodopsin subfamily, leading to high yields of purified proteins; it represents a method of choice for biochemical and biophysical studies when large quantities of purified GPCRs are needed.

Structural Basis for DNA Gyrase Interaction with Coumermycin A1.

Coumermycin A1 is a natural aminocoumarin that inhibits bacterial DNA gyrase, a member of the GHKL proteins superfamily. We report here the first cocrystal structures of gyrase B bound to coumermycin A1, revealing that one coumermycin A1 molecule traps simultaneously two ATP-binding sites. The inhibited dimers from different species adopt distinct sequence-dependent conformations, alternative to the ATP-bound form. These structures provide a basis for the rational development of coumermycin A1 derivatives for antibiotherapy and biotechnology applications.

Mass spectrometry studies of demetallation of haemin by recombinant horse L chain apoferritin and its mutant (E 53,56,57,60 Q).

An essential difference between eukaryotic ferritins and bacterioferritins is that the latter contain naturally, in vivo haem as Fe-protoporphyrin IX. This haem is located in a hydrophobic pocket along the 2-fold symmetry axes and is liganded by two Met 52. However, in in vivo studies, a cofactor has been isolated in horse spleen apoferritin similar to protoporphyrin IX; in in vitro experiments, it has been shown that horse spleen apoferritin is able to interact with haem. Studies of haemin (Fe(III)-PPIX) incorporation into horse spleen apoferritin have been carried out, which show that the metal free porphyrin is found in a corresponding pocket to haem in bacterioferritins [Précigoux, G., Yariv, J., Gallois, B., Dautant, A., Courseille, C. and Langlois, d'Estaintot B. (1994) A crystallographic study of haem binding to ferritin. Acta Cryst. D 50, 739-743]. A mechanism of demetallation of haemin by L-chain apoferritin was proposed [Crichton, R.R., Soruco, J.A., Roland, F., Michaux, M.A., Gallois, B., Précigoux, G., Mahy, J.P. and Mansuy. (1997) Remarkable ability of horse spleen apoferritin to demetallate hemin and to metallate protoporphyrin IX as a function of pH. J. P. Biochem. 36, 49, 15049-15054]: this involved four Glu residues (53,56,57,60) situated at the entrance of the hydrophobic pocket and appeared to be favoured by acidic conditions. To verify this mechanism, we have mutated these four Glu to Gln and examined demetallation in both acidic and basic conditions. In this paper, we report the mass spectrometry studies of L-chain apoferritin and its mutant incubated with haemin and analysed after different times of incubation: 15 days, 2 months, 6 months, 9 months and 12 months. These studies show that the recombinant L-chain apoferritin and its mutant are able to demetallate haemin to give a hydroxyethyl protoporphyrin IX derivative in a dimeric form [Macieira, S., Martins, B. M. and Huber, R. (2003) Oxygen-dependent coproporphyrinogen IX oxidase from Escherichia coli: one-step purification and biochemical characterization. FEMS. Microbiology Letters 226, 31-37].

Monitoring of the retinoic acid receptor-retinoid X receptor dimerization upon DNA binding by native mass spectrometry.

Identifying protein-DNA interactions is essential to understand the regulatory networks of cells and their influence on gene expression. In this study, we use native electrospray mass spectrometry (ESI-MS) to investigate how the heterodimerization of retinoic acid receptor-retinoid X receptor (RAR-RXR) is mediated by DNA sequence. In presence of various RAR response elements (RAREs), three oligomeric states of RAR-RXR DNA binding domains (DBDs) bound to RAREs (monomer, homo- or heterodimers) were detected and individually monitored to follow subunit assembly and disassembly upon RAREs' abundancy or sequence. In particular, a cooperative heterodimerization was shown with RARb2 DR5 (5 base pair spaced direct repeat) while a high heterogeneity reflecting random complex formation could be observed with the DR0 response elements, in agreement with native gel electrophoresis data or molecular modeling. Such MS information will help to identify the composition of species formed in solution and to define which DR sequence is specific for RAR-RXR heterodimerization.

Ligand-metal ion binding to proteins: investigation by ESI mass spectrometry.

The objective of this chapter is to show the general mass spectrometry (MS)-based strategies that can be used to retrieve information regarding protein-metal and protein-ligand noncovalent complexes. Indeed, when using carefully controlled conditions in the atmospheric pressure-vacuum interface of the mass spectrometer, and when sample preparation is optimized, it is possible to preserve large specific multiprotein-metal-ligand noncovalent complexes during MS analysis. Examples describing the possibilities of electrospray ionization MS (ESI-MS) are shown. For instance, it can be used to probe cooperativity in the binding of a ligand or a metal to a protein or may constitute a new methodology for a more rational approach for drug discovery and for human genome annotation. Thanks to its ability to directly give information on stoichiometry or dynamics of the interactions formed in solution, MS offers new possibilities to tackle more and more various applications.

Combining inducible protein overexpression with NMR-grade triple isotope labeling in the cyanobacterium Anabaena sp. PCC 7120.

The difficulty and expense of preparing protein samples highly enriched in stable isotopes is a bottleneck for structural studies by nuclear magnetic resonance (NMR) spectroscopy. We have developed a new regulatable expression/labeling vector system in the cyanobacterium Anabaena sp. PCC 7120 using the endogenous promoter of the nitrate assimilation nir operon. Standard proteins were overexpressed upon induction with NaNO3, yielding up to 250 mg/L of culture. When the cyanobacteria were grown in the presence of inexpensive 15N-, 13C-labeled mineral salts and 2H2O, the expressed polypeptides were highly (>90%) enriched in stable isotopes. Furthermore, the tight repression of the nir promoter upon induction allowed the production of the toxic oncoprotein E6. In addition, under these conditions, the malE31 protein, while insoluble in Escherichia coli, was found to be soluble in Anabaena. Together, these properties render the described system especially suitable for the production and/or triple labeling of recombinant protein samples. It represents an interesting alternative to conventional protein expression systems used in structural genomics.

Electrospray ionization mass spectrometry studies of noncovalent myosin VI complexes reveal a new specific calmodulin binding site.

Among the myosin superfamily, myosin VI differs from all others by a reverse directionality and a particular motility. Little structural information is available for myosin VI. It is known that it binds one calmodulin (CaM) by means of a single "IQ motif" and that myosin VI contains a specific insert located at the junction between the motor domain (MD) and the lever arm, likely to play a critical role for the unusual motility previously observed. Electrospray ionization mass spectrometry (MS) was used to determine the CaM and Ca2+ stoichiometries in several myosin VI constructs. In particular, the experimental conditions required for the observation of multiprotein/Ca2+ noncovalent assemblies are detailed for two truncated MD constructs (less than 20 kDa) and for three full MD constructs (more than 90 KDa). The specificity of the detected stoichiometries is discussed for each construct and the resolving power of Time of Flight mass spectrometry is stressed, in particular for the detection of metal ions binding to high molecular weight complexes. MS reveals a new CaM binding site for myosin VI and highlights a different behavior for the five myosin VI constructs versus Ca2+ binding. In addition to these stoichiometry based experiments, gas-phase dissociation analyses on intact complexes are described. They reveal that Ca2+ transfer between protein partners occurs during the dissociation process for one construct with a full MD. Charge-transfer and dissociation behavior has allowed to draw structural assumptions for the interaction of the MD with the CaM N-terminal lobe.

A vitamin D receptor selectively activated by gemini analogs reveals ligand dependent and independent effects.

The bioactive form of vitamin D [1,25(OH)2D3] regulates mineral and bone homeostasis and exerts potent anti-inflammatory and antiproliferative properties through binding to the vitamin D receptor (VDR). The 3D structures of the VDR ligand-binding domain with 1,25(OH)2D3 or gemini analogs unveiled the molecular mechanism underlying ligand recognition. On the basis of structure-function correlations, we generated a point-mutated VDR (VDR(gem)) that is unresponsive to 1,25(OH)2D3, but the activity of which is efficiently induced by the gemini ligands. Moreover, we show that many VDR target genes are repressed by unliganded VDR(gem) and that mineral ion and bone homeostasis are more impaired in VDR(gem) mice than in VDR null mice, demonstrating that mutations abolishing VDR ligand binding result in more severe skeletal defects than VDR null mutations. As gemini ligands induce VDR(gem) transcriptional activity in mice and normalize their serum calcium levels, VDR(gem) is a powerful tool to further unravel both liganded and unliganded VDR signaling.

Chemical cross-linking and mass spectrometry to determine the subunit interaction network in a recombinant human SAGA HAT subcomplex.

Understanding the way how proteins interact with each other to form transient or stable protein complexes is a key aspect in structural biology. In this study, we combined chemical cross-linking with mass spectrometry to determine the binding stoichiometry and map the protein-protein interaction network of a human SAGA HAT subcomplex. MALDI-MS equipped with high mass detection was used to follow the cross-linking reaction using bis[sulfosuccinimidyl] suberate (BS3) and confirm the heterotetrameric stoichiometry of the specific stabilized subcomplex. Cross-linking with isotopically labeled BS3 d0-d4 followed by trypsin digestion allowed the identification of intra- and intercross-linked peptides using two dedicated search engines: pLink and xQuest. The identified interlinked peptides suggest a strong network of interaction between GCN5, ADA2B and ADA3 subunits; SGF29 is interacting with GCN5 and ADA3 but not with ADA2B. These restraint data were combined to molecular modeling and a low-resolution interacting model for the human SAGA HAT subcomplex could be proposed, illustrating the potential of an integrative strategy using cross-linking and mass spectrometry for addressing the structural architecture of multiprotein complexes.

Using nondenaturing mass spectrometry to detect fortuitous ligands in orphan nuclear receptors.

Nondenaturing electrospray mass spectrometry (ESI-MS) has been used to reveal the presence of potential ligands in the ligand-binding domain (LBD) of orphan nuclear receptors. This new approach, based on supramolecular mass spectrometry, allowed the detection and identification of fortuitous ligands for the retinoic acid-related orphan receptor beta (RORbeta) and the ultraspiracle protein (USP). These fortuitous ligands were specifically captured from the host cell with the proper stoichiometry. After organic extraction, these molecules have been characterized by classic analytical methods and identified as stearic acid for RORbeta and a phosphatidylethanolamine (PE) for USP, as confirmed by crystallography. These molecules act as "fillers" and may not be the physiological ligands, but they prove to be essential to stabilize the active conformation of the LBD, enabling its crystallization. The resulting crystal structures provide a detailed picture of the ligand-binding pocket, allowing the design of highly specific synthetic ligands that can be used to characterize the function of orphan nuclear receptors. An additional advantage of this new method is that it is not based on a functional test and that it can detect low-affinity ligands.

Structural insights into the molecular mechanism of vitamin D receptor activation by lithocholic acid involving a new mode of ligand recognition.

The vitamin D receptor (VDR), an endocrine nuclear receptor for 1α,25-dihydroxyvitamin D3, acts also as a bile acid sensor by binding lithocholic acid (LCA). The crystal structure of the zebrafish VDR ligand binding domain in complex with LCA and the SRC-2 coactivator peptide reveals the binding of two LCA molecules by VDR. One LCA binds to the canonical ligand-binding pocket, and the second one, which is not fully buried, is anchored to a site located on the VDR surface. Despite the low affinity of the alternative site, the binding of the second molecule promotes stabilization of the active receptor conformation. Biological activity assays, structural analysis, and molecular dynamics simulations indicate that the recognition of two ligand molecules is crucial for VDR agonism by LCA. The unique binding mode of LCA provides clues for the development of new chemical compounds that target alternative binding sites for therapeutic applications.

The asymmetric binding of PGC-1α to the ERRα and ERRγ nuclear receptor homodimers involves a similar recognition mechanism.

BACKGROUND: PGC-1α is a crucial regulator of cellular metabolism and energy homeostasis that functionally acts together with the estrogen-related receptors (ERRα and ERRγ) in the regulation of mitochondrial and metabolic gene networks. Dimerization of the ERRs is a pre-requisite for interactions with PGC-1α and other coactivators, eventually leading to transactivation. It was suggested recently (Devarakonda et al) that PGC-1α binds in a strikingly different manner to ERRγ ligand-binding domains (LBDs) compared to its mode of binding to ERRα and other nuclear receptors (NRs), where it interacts directly with the two ERRγ homodimer subunits. METHODS/PRINCIPAL FINDINGS: Here, we show that PGC-1α receptor interacting domain (RID) binds in an almost identical manner to ERRα and ERRγ homodimers. Microscale thermophoresis demonstrated that the interactions between PGC-1α RID and ERR LBDs involve a single receptor subunit through high-affinity, ERR-specific L3 and low-affinity L2 interactions. NMR studies further defined the limits of PGC-1α RID that interacts with ERRs. Consistent with these findings, the solution structures of PGC-1α/ERRα LBDs and PGC-1α/ERRγ LBDs complexes share an identical architecture with an asymmetric binding of PGC-1α to homodimeric ERR. CONCLUSIONS/SIGNIFICANCE: These studies provide the molecular determinants for the specificity of interactions between PGC-1α and the ERRs, whereby negative cooperativity prevails in the binding of the coactivators to these receptors. Our work indicates that allosteric regulation may be a general mechanism controlling the binding of the coactivators to homodimers.

The "Phantom Effect" of the Rexinoid LG100754: structural and functional insights.

Retinoic acid receptors (RARs) and Retinoid X nuclear receptors (RXRs) are ligand-dependent transcriptional modulators that execute their biological action through the generation of functional heterodimers. RXR acts as an obligate dimer partner in many signalling pathways, gene regulation by rexinoids depending on the liganded state of the specific heterodimeric partner. To address the question of the effect of rexinoid antagonists on RAR/RXR function, we solved the crystal structure of the heterodimer formed by the ligand binding domain (LBD) of the RARα bound to its natural agonist ligand (all-trans retinoic acid, atRA) and RXRα bound to a rexinoid antagonist (LG100754). We observed that RARα exhibits the canonical agonist conformation and RXRα an antagonist one with the C-terminal H12 flipping out to the solvent. Examination of the protein-LG100754 interactions reveals that its propoxy group sterically prevents the H12 associating with the LBD, without affecting the dimerization or the active conformation of RAR. Although LG100754 has been reported to act as a 'phantom ligand' activating RAR in a cellular context, our structural data and biochemical assays demonstrate that LG100754 mediates its effect as a full RXR antagonist. Finally we show that the 'phantom ligand effect' of the LG100754 is due to a direct binding of the ligand to RAR that stabilizes coactivator interactions thus accounting for the observed transcriptional activation of RAR/RXR.

Crystal growth of proteins, nucleic acids, and viruses in gels.

Medium-sized single crystals with perfect habits and no defect producing intense and well-resolved diffraction patterns are the dream of every protein crystallographer. Crystals of biological macromolecules possessing these characteristics can be prepared within a medium in which mass transport is restricted to diffusion. Chemical gels (like polysiloxane) and physical gels (such as agarose) provide such an environment and are therefore suitable for the crystallisation of biological macromolecules. Instructions for the preparation of each type of gel are given to urge crystal growers to apply diffusive media for enhancing crystallographic quality of their crystals. Examples of quality enhancement achieved with silica and agarose gels are given. Results obtained with other substances forming gel-like media (such as lipidic phases and cellulose derivatives) are presented. Finally, the use of gels in combination with capillary tubes for counter-diffusion experiments is discussed. Methods and techniques implemented with proteins can also be applied to nucleic acids and nucleoprotein assemblies such as viruses.

Modulation of E-cadherin monomer folding by cooperative binding of calcium ions.

Classical cadherins are transmembrane glycoproteins involved in calcium-dependent cell-cell adhesion. Calcium ions are coordinated at the interface between successive modules of the cadherin ectodomain and are thought to regulate the adhesive interactions of cadherins when present at millimolar concentrations. It is widely accepted that calcium plays a critical role in cadherin-mediated cell-cell adhesion, but the nature of cadherin-calcium binding remains a matter of debate. We investigated the parameters of noncovalent cadherin-calcium binding, using the two N-terminal modules of E-cadherin (E/EC12) with a native N-terminal end and nondenaturing electrospray ionization mass spectrometry. By directly visualizing the molecular complexes, we demonstrated that E/EC12 binds three calcium ions, with an average KD of 20 +/- 0.7 microM. These calcium ions bound cooperatively to E/EC12 in its monomeric state, and these properties were not modified by an N-terminal extension consisting of a single methionine residue. This binding induced specific structural changes, as shown by assessments of protease sensitivity, circular dichroism, and mass spectrometry. Furthermore, the D103A mutation (a residue involved in E-cadherin adhesive function) modified calcium binding and led to a loss of cooperativity and the absence of structural changes, despite calcium binding. As the amino acids involved in calcium binding are found within the cadherin consensus motif, our findings may be relevant to other members of the cadherin family.