The Tumor Suppressor ING5 Is a Dimeric, Bivalent Recognition Molecule of the Histone H3K4me3 Mark.

The INhibitor of Growth (ING) family of tumor suppressors regulates the transcriptional state of chromatin by recruiting remodeling complexes to sites with histone H3 trimethylated at lysine 4 (H3K4me3). This modification is recognized by the plant homeodomain (PHD) present at the C-terminus of the five ING proteins. ING5 facilitates histone H3 acetylation by the HBO1 complex, and also H4 acetylation by the MOZ/MORF complex.

Molecular mechanism of Gαi activation by non-GPCR proteins with a Gα-Binding and Activating motif.

Heterotrimeric G proteins are quintessential signalling switches activated by nucleotide exchange on Gα. Although activation is predominantly carried out by G-protein-coupled receptors (GPCRs), non-receptor guanine-nucleotide exchange factors (GEFs) have emerged as critical signalling molecules and therapeutic targets. Here we characterize the molecular mechanism of G-protein activation by a family of non-receptor GEFs containing a Gα-binding and -activating (GBA) motif.

The metastasis suppressor KISS1 is an intrinsically disordered protein slightly more extended than a random coil.

The metastasis suppressor KISS1 is reported to be involved in the progression of several solid neoplasias, making it a promising molecular target for controlling their metastasis. The KISS1 sequence contains an N-terminal secretion signal and several dibasic sequences that are proposed to be the proteolytic cleavage sites. We present the first structural characterization of KISS1 by circular dichroism, multi-angle light scattering, small angle X-Ray scattering and NMR spectroscopy.

The tumor suppressor inhibitor of growth 4 binds double-stranded DNA through its disordered central region.

The tumor suppressor inhibitor of growth 4 (ING4) regulates chromatin structure by recruiting the histone acetyl transferase complex HBO1 to sites with histone H3 trimethylated at K4. ING4 dimerizes through its N-terminal domain and recognizes H3K4me3 by the C-terminal plant homeodomain (PHD). The central region of ING4 is disordered and contains the nuclear localization signal.

Intramolecular trimerization, a novel strategy for making multispecific antibodies with controlled orientation of the antigen binding domains.

Here, we describe a new strategy that allows the rapid and efficient engineering of mono and multispecific trivalent antibodies. By fusing single-domain antibodies from camelid heavy-chain-only immunoglobulins (VHHs) to the N-terminus of a human collagen XVIII trimerization domain (TIE(XVIII)) we produced monospecific trimerbodies that were efficiently secreted as soluble functional proteins by mammalian cells. The purified VHH-TIE(XVIII) trimerbodies were trimeric in solution and exhibited excellent antigen binding capacity.

Structure of the I-SceI nuclease complexed with its dsDNA target and three catalytic metal ions.

Homing endonucleases are highly specific DNA-cleaving enzymes that recognize and cleave long stretches of DNA. The engineering of these enzymes provides instruments for genome modification in a wide range of fields, including gene targeting. The homing endonuclease I-SceI from the yeast Saccharomyces cerevisiae has been purified after overexpression in Escherichia coli and its crystal structure has been determined in complex with its target DNA.

Crystal Structure of the Homing Endonuclease I-CvuI Provides a New Template for Genome Modification.

Homing endonucleases recognize and generate a DNA double-strand break, which has been used to promote gene targeting. These enzymes recognize long DNA stretches; they are highly sequence-specific enzymes and display a very low frequency of cleavage even in complete genomes. Although a large number of homing endonucleases have been identified, the landscape of possible target sequences is still very limited to cover the complexity of the whole eukaryotic genome.

Structure of p15(PAF)-PCNA complex and implications for clamp sliding during DNA replication and repair.

The intrinsically disordered protein p15(PAF) regulates DNA replication and repair by binding to the proliferating cell nuclear antigen (PCNA) sliding clamp. We present the structure of the human p15(PAF)-PCNA complex. Crystallography and NMR show the central PCNA-interacting protein motif (PIP-box) of p15(PAF) tightly bound to the front-face of PCNA.

Efficient production of single-chain fragment variable-based N-terminal trimerbodies in Pichia pastoris.

Background: Recombinant antibodies are highly successful in many different pathological conditions and currently enjoy overwhelming recognition of their potential. There are a wide variety of protein expression systems available, but almost all therapeutic antibodies are produced in mammalian cell lines, which mimic human glycosylation. The production of clinical-grade antibodies in mammalian cells is, however, extremely expensive.

Crystallization and preliminary X-ray diffraction analysis of the homing endonuclease I-CvuI from Chlorella vulgaris in complex with its target DNA.

Homing endonucleases are highly specific DNA-cleaving enzymes that recognize long stretches of DNA. The engineering of these enzymes provides novel instruments for genome modification in a wide range of fields, including gene targeting, by inducing specific double-strand breaks. I-CvuI is a homing endonuclease from the green alga Chlorella vulgaris.

p15PAF is an intrinsically disordered protein with nonrandom structural preferences at sites of interaction with other proteins.

We present to our knowledge the first structural characterization of the proliferating-cell-nuclear-antigen-associated factor p15(PAF), showing that it is monomeric and intrinsically disordered in solution but has nonrandom conformational preferences at sites of protein-protein interactions. p15(PAF) is a 12 kDa nuclear protein that acts as a regulator of DNA repair during DNA replication. The p15(PAF) gene is overexpressed in several types of human cancer.

Proliferating cell nuclear antigen (PCNA) interactions in solution studied by NMR.

PCNA is an essential factor for DNA replication and repair. It forms a ring shaped structure of 86 kDa by the symmetric association of three identical protomers. The ring encircles the DNA and acts as a docking platform for other proteins, most of them containing the PCNA Interaction Protein sequence (PIP-box).

Production of meganucleases by cell-free protein synthesis for functional and structural studies.

Meganucleases are highly specific endonucleases that recognize and cleave long DNA sequences, making them powerful tools for gene targeting. We describe the production of active recombinant meganucleases suitable for functional and structural studies using a batch-based cell-free protein synthesis method. Isotopic labeling of the I-CreI meganuclease is demonstrated opening the way for structural and ligand binding studies in solution by nuclear magnetic resonance (NMR)(2) which was previously hampered by the problems associated with the toxicity of the enzyme for Escherichia coli limiting its growth.

The heterotrimeric laminin coiled-coil domain exerts anti-adhesive effects and induces a pro-invasive phenotype.

Laminins are large heterotrimeric cross-shaped extracellular matrix glycoproteins with terminal globular domains and a coiled-coil region through which the three chains are assembled and covalently linked. Laminins are key components of basement membranes, and they serve as attachment sites for cell adhesion, migration and proliferation. In this work, we produced a recombinant fragment comprising the entire laminin coiled-coil of the α1-, β1-, and γ1-chains that assemble into a stable heterotrimeric coiled-coil structure independently of the rest of the molecule.

Improved stability of multivalent antibodies containing the human collagen XV trimerization domain.

We recently described the in vitro and in vivo properties of an engineered homotrimeric antibody made by fusing the N-terminal trimerization region of collagen XVIII NC1 domain to the C-terminus of a scFv fragment [trimerbody (scFv-NC1) 3; 110 kDa]. Here, we demonstrated the utility of the N-terminal trimerization region of collagen XV NC1 domain in the engineering of trivalent antibodies. We constructed several scFv-based trimerbodies containing the human type XV trimerization domain and demonstrated that all the purified trimerbodies were trimeric in solution and exhibited excellent antigen binding capacity.

Crystal structure of inhibitor of growth 4 (ING4) dimerization domain reveals functional organization of ING family of chromatin-binding proteins.

The protein ING4 binds to histone H3 trimethylated at Lys-4 (H3K4me3) through its C-terminal plant homeodomain, thus recruiting the HBO1 histone acetyltransferase complex to target promoters. The structure of the plant homeodomain finger bound to an H3K4me3 peptide has been described, as well as the disorder and flexibility in the ING4 central region. We report the crystal structure of the ING4 N-terminal domain, which shows an antiparallel coiled-coil homodimer with each protomer folded into a helix-loop-helix structure.

Reduced stability and increased dynamics in the human proliferating cell nuclear antigen (PCNA) relative to the yeast homolog.

Proliferating Cell Nuclear Antigen (PCNA) is an essential factor for DNA replication and repair. PCNA forms a toroidal, ring shaped structure of 90 kDa by the symmetric association of three identical monomers. The ring encircles the DNA and acts as a platform where polymerases and other proteins dock to carry out different DNA metabolic processes.

Molecular basis of engineered meganuclease targeting of the endogenous human RAG1 locus.

Homing endonucleases recognize long target DNA sequences generating an accurate double-strand break that promotes gene targeting through homologous recombination. We have modified the homodimeric I-CreI endonuclease through protein engineering to target a specific DNA sequence within the human RAG1 gene. Mutations in RAG1 produce severe combined immunodeficiency (SCID), a monogenic disease leading to defective immune response in the individuals, leaving them vulnerable to infectious diseases.

Inositol 1,3,4,5,6-pentakisphosphate 2-kinase is a distant IPK member with a singular inositide binding site for axial 2-OH recognition.

Inositol phosphates (InsPs) are signaling molecules with multiple roles in cells. In particular (InsP(6)) is involved in mRNA export and editing or chromatin remodeling among other events. InsP(6) accumulates as mixed salts (phytate) in storage tissues of plants and plays a key role in their physiology.

Crystallization and preliminary X-ray diffraction analysis of inositol 1,3,4,5,6-pentakisphosphate kinase from Arabidopsis thaliana.

Inositol 1,3,4,5,6-pentakisphosphate kinase (IP(5) 2-K) is an enzyme involved in inositol metabolism that synthesizes IP(6) (inositol 1,2,3,4,5,6-hexakisphosphate) from inositol 1,3,4,5,6-pentakisphosphate (IP(5)) and ATP. IP(6) is the major phosphorus reserve in plants, while in mammals it is involved in multiple cellular events such as DNA editing and chromatin remodelling. In addition, IP(6) is the precursor of other highly phosphorylated inositols which also play highly relevant roles.

Efficient targeting of a SCID gene by an engineered single-chain homing endonuclease.

Sequence-specific endonucleases recognizing long target sequences are emerging as powerful tools for genome engineering. These endonucleases could be used to correct deleterious mutations or to inactivate viruses, in a new approach to molecular medicine. However, such applications are highly demanding in terms of safety.