ABSTRACT
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ABSTRACT
Autophagy is a degradation pathway important for cellular homeostasis. The E1-like enzyme ATG7 is a key component of the autophagy machinery, with the main function of mediating the lipidation of LC3/GABARAP during autophagosome formation. By analysing mRNA-sequencing data we found that in addition to the full-length ATG7 isoform, various tissues express a shorter isoform lacking an exon of 27 amino acids in the C-terminal part of the protein, termed ATG7(2). We further show that ATG7(2) does not bind LC3B and fails to mediate the lipidation of members of the LC3/GABARAP family. We have thus identified an isoform of ATG7 that is unable to carry out the best characterized function of the protein during the autophagic response. This short isoform will have to be taken into consideration when further studying the role of ATG7.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Autophagy-Related Protein 7/metabolism , Microtubule-Associated Proteins/metabolism , Amino Acid Sequence , Animals , Apoptosis Regulatory Proteins , Autophagy , Autophagy-Related Protein 7/chemistry , HEK293 Cells , Humans , Lipid Metabolism , Mice , Models, Molecular , Protein Binding , Protein Isoforms/chemistry , Protein Isoforms/metabolismABSTRACT
It is proposed to perform quantum mechanical/molecular dynamics calculations of chemical reactions that are planned to be catalyzed by antibodies and then conduct a virtual screening of the library of potential antibody mutants to select an optimal biocatalyst. We tested the effectiveness of this approach by the example of hydrolysis of organophosphorus toxicant paraoxon using kinetic approaches and X-ray analysis of the antibody biocatalyst designed de novo.
Subject(s)
Antibodies/genetics , Antibodies/metabolism , Biocatalysis , Computational Biology/instrumentation , Mutation , Antibodies/chemistry , Models, Molecular , Protein ConformationABSTRACT
Symmetric DNA sequence motifs allow the formation of palindromic protein/DNA complexes. Although symmetric protein sequence motifs are less common, recent structural discoveries have unraveled a few protein/protein complexes with palindromic symmetry. Remarkably, symmetric protein/protein complexes can be generated either by adjacent or remote sequence motifs, which may be repeated or inverted. This contribution reflects and comments on recent findings of palindromic protein/protein complexes.
Subject(s)
Amino Acid Motifs , Macromolecular Substances , Proteins/chemistry , Proteins/genetics , Models, Molecular , Protein Conformation , Proteins/metabolismABSTRACT
The Structural Proteomics In Europe (SPINE) programme is aimed at the development and implementation of high-throughput technologies for the efficient structure determination of proteins of biomedical importance, such as those of bacterial and viral pathogens linked to human health. Despite the challenging nature of some of these targets, 175 novel pathogen protein structures (approximately 220 including complexes) have been determined to date. Here the impact of several technologies on the structural determination of proteins from human pathogens is illustrated with selected examples, including the parallel expression of multiple constructs, the use of standardized refolding protocols and optimized crystallization screens.
Subject(s)
Bacterial Infections/metabolism , Bacterial Proteins/chemistry , Proteomics/methods , Viral Proteins/chemistry , Virus Diseases/metabolism , Animals , Bacterial Infections/microbiology , Humans , Protein Folding , Virus Diseases/virologyABSTRACT
Circular dichroism spectropolarimetry and X-ray scattering data, obtained using synchrotron radiation, can yield information about the secondary and tertiary structure of proteins in solution. These techniques have been used to analyse the architecture and shape of a complex of two proteins in solution. The crystal structures of two separate proteins, the C-terminal domain of Pex5p and SCP2, are available but their complex has not previously been structurally characterized. Circular dichroism spectropolarimetry indicated that complex formation requires little secondary structure rearrangement. X-ray scattering data fit an elongated irregular 'shoe'-shaped particle of the complex of the two proteins, with dimensions of the order of 30 A x 40 A x 90 A. Comparison with the known crystal structures suggests that this 'shoe' shape requires a conformational change of the C-terminus of SCP2 to appropriately locate its peroxisomal targeting signal type-1 recognition motif into the binding pocket of the Pex5p receptor. Implications of the combined use of synchrotron-based circular dichroism spectropolarimetry and X-ray scattering in structural biology and proteomics are discussed.
Subject(s)
Carrier Proteins/chemistry , Circular Dichroism/methods , Models, Molecular , Receptors, Cytoplasmic and Nuclear/chemistry , Synchrotrons , X-Ray Diffraction/methods , Binding Sites , Carrier Proteins/analysis , Computer Simulation , Ligands , Multiprotein Complexes/analysis , Multiprotein Complexes/chemistry , Peroxisome-Targeting Signal 1 Receptor , Protein Binding , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , Receptors, Cytoplasmic and Nuclear/analysis , Solutions , Systems IntegrationABSTRACT
An effort to combine theoretical analyses and protein engineering methods has been made to probe the folding mechanism of SH3 by using Energy Landscape Theory and a phi-value analysis. Particular emphasis was given to core residues and the effect of desolvation during the folding event by replacing the core valines with isosteric threonines. These mutations have the advantage of keeping the core structurally invariant while affecting core stability relative to the unfolded state. Although the valines that form the core appear spatially invariant, the folding kinetics of their threonine mutants varies, indicating their different extent of solvation in the transition-state ensemble. Theoretical studies predicted the distribution of folding kinetics of threonine mutants without previous knowledge of the measured rates. This initial success encourages further investigations of the molecular details behind these macroscopic phenomena and of the role of solvation in the folding mechanism.
Subject(s)
Protein Conformation , Protein Folding , Proteins/chemistry , Amino Acid Sequence , Amino Acid Substitution , Models, Biological , Models, Molecular , Mutagenesis, Site-Directed , Protein Denaturation , Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , SolutionsABSTRACT
The TB Structural Genomics Consortium is an organization devoted to encouraging, coordinating, and facilitating the determination and analysis of structures of proteins from Mycobacterium tuberculosis. The Consortium members hope to work together with other M. tuberculosis researchers to identify M. tuberculosis proteins for which structural information could provide important biological information, to analyze and interpret structures of M. tuberculosis proteins, and to work collaboratively to test ideas about M. tuberculosis protein function that are suggested by structure or related to structural information. This review describes the TB Structural Genomics Consortium and some of the proteins for which the Consortium is in the progress of determining three-dimensional structures.
Subject(s)
Genomics/organization & administration , Mycobacterium tuberculosis/genetics , Amino Acid Sequence , Bacterial Proteins/genetics , Genome, Bacterial , Humans , International Cooperation , Molecular Sequence Data , Mycobacterium tuberculosis/metabolism , Protein Conformation , Sequence AlignmentABSTRACT
The (betaalpha)8-barrel is the most versatile and most frequently encountered fold among enzymes. It is an interesting question how the contemporary (betaalpha)8-barrels are evolutionarily related and by which mechanisms they evolved from more simple precursors. Comprehensive comparisons of amino acid sequences and three-dimensional structures suggest that a large fraction of the known (betaalpha)8-barrels have divergently evolved from a common ancestor. The mutational interconversion of enzymatic activities of several (betaalpha)8-barrels further supports their common evolutionary origin. Moreover, the high structural similarity between the N- and C-terminal (betaalpha)4 units of two (betaalpha)8-barrel enzymes from histidine biosynthesis indicates that the contemporary proteins evolved by tandem duplication and fusion of the gene of an ancestral 'half-barrel' precursor. In support of this hypothesis, recombinantly produced 'half-barrels' were shown to be folded, dimeric proteins.
Subject(s)
Biological Evolution , Enzymes/genetics , Enzymes/chemistry , Enzymes/metabolism , Phosphates/metabolism , Protein ConformationABSTRACT
The POU domain of the human Oct1 transcription factor has been crystallized with two different POU-dimer-binding DNA elements. Protein-DNA cocrystals suitable for structural analysis could be obtained only with a redox-insensitive version of the POU domain. The recombinant protein expression in a prokaryotic host was adjusted for fast purification. Optimized crystals were obtained by systematically varying the length of the oligonucleotide and by modifying cryofreezing procedures. These steps are generally applicable to the preparation of protein-DNA complexes for structural studies.
Subject(s)
DNA/chemistry , Organic Cation Transporter 1/chemistry , Crystallization , Crystallography, X-Ray , DNA/metabolism , Humans , Models, Molecular , Nucleic Acid Conformation , Oxidation-Reduction , Protein Conformation , Protein Structure, Tertiary , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistryABSTRACT
Two crystal structures of Oct-1 POU domain bound to DNA provide a rationale for differential, conformation-dependent recruitment of transcription cofactors. The POU-homeo and POU-specific subdomains of Oct-1 contain two different nonoverlapping pairs of surface patches that are capable of forming unrelated protein-protein interfaces. Members of the POU factor family contain one or two conserved sequence motifs in the interface that are known to be phosphorylated, as noted for Oct-1 and Pit-1. Modeling of Oct-4 reveals the unique case where the same conserved sequence is located in both interfaces. Our studies provide the basis for two distinct dimeric POU factor arrangements that are dictated by the architecture of each DNA response element. We suggest interface swapping in dimers could be a general mechanism of modulating the activity of transcription factors.
Subject(s)
DNA-Binding Proteins/metabolism , DNA/metabolism , Protein Structure, Tertiary , Transcription Factors/metabolism , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , DNA-Binding Proteins/chemistry , Dimerization , Host Cell Factor C1 , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Nucleic Acid Conformation , Octamer Transcription Factor-1 , Octamer Transcription Factor-3 , Protein Binding , Protein Conformation , Sequence Alignment , Transcription Factors/chemistryABSTRACT
The (beta alpha)(8)-barrel is a versatile single-domain protein fold that is adopted by a large number of enzymes. The (beta alpha)(8)-barrel fold has been used as a model to elucidate the structural basis of protein thermostability and in studies to interconvert catalytic activities or substrate specificities by rational design or directed evolution. Recently, the (beta alpha)(4)-half-barrel was identified as a possible structural subdomain.
Subject(s)
Directed Molecular Evolution/methods , Phosphopyruvate Hydratase/chemistry , Protein Folding , Thermodynamics , Catalysis , Evolution, Molecular , Phosphopyruvate Hydratase/metabolism , Protein Structure, Secondary , Substrate SpecificityABSTRACT
BACKGROUND: The giant muscle protein titin contributes to the filament system in skeletal and cardiac muscle cells by connecting the Z disk and the central M line of the sarcomere. One of the physiological functions of titin is to act as a passive spring in the sarcomere, which is achieved by the elastic properties of its central I band region. Titin contains about 300 domains of which more than half are folded as immunoglobulin-like (Ig) domains. Ig domain segments of the I band of titin have been extensively used as templates to investigate the molecular basis of protein elasticity. RESULTS: The structure of the Ig domain I1 from the I band of titin has been determined to 2.1 A resolution. It reveals a novel, reversible disulphide bridge, which is neither required for correct folding nor changes the chemical stability of I1, but it is predicted to contribute mechanically to the elastic properties of titin in active sarcomeres. From the 92 Ig domains in the longest isoform of titin, at least 40 domains have a potential for disulphide bridge formation. CONCLUSIONS: We propose a model where the formation of disulphide bridges under oxidative stress conditions could regulate the elasticity of the I band in titin by increasing sarcomeric resistance. In this model, the formation of the disulphide bridge could refrain a possible directed motion of the two beta sheets or other mechanically stable entities of the I1 Ig domain with respect to each other when exposed to mechanical forces.
Subject(s)
Disulfides/chemistry , Disulfides/metabolism , Muscle Proteins/chemistry , Muscle Proteins/metabolism , Muscles/chemistry , Protein Kinases/chemistry , Protein Kinases/metabolism , Amino Acid Sequence , Cells, Cultured , Connectin , Crystallography, X-Ray , Elasticity , Humans , Immunoglobulins/chemistry , Models, Molecular , Molecular Sequence Data , Protein Denaturation , Protein Folding , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Alignment , Spectrometry, Fluorescence , Static ElectricityABSTRACT
Anthranilate phosphoribosyltransferase (TrpD; EC 2.4.2.18) from the hyperthermophilic archaeon Sulfolobus solfataricus (ssTrpD) was expressed in Escherichia coli, purified and crystallized. Analytical gel permeation chromatography revealed a homodimeric composition of the enzyme. The steady-state kinetic characteristics suggest tight binding of the substrate anthranilic acid and efficient catalysis at the physiological growth temperature of S. solfataricus. Crystals of ssTrpD diffract to better than 2.6 A resolution and preliminary X-ray characterization was carried out. The crystals are suitable for structure determination.
Subject(s)
Anthranilate Phosphoribosyltransferase/chemistry , Anthranilate Phosphoribosyltransferase/isolation & purification , Sulfolobus/enzymology , Catalysis , Chromatography, Gel , Crystallography , Crystallography, X-Ray , Electrophoresis, Polyacrylamide Gel , Escherichia coli/metabolism , Kinetics , Methionine/chemistry , Models, Chemical , Protein Binding , Temperature , Time Factors , ortho-Aminobenzoates/chemistryABSTRACT
The crystal structure of an alpha-spectrin Src-homology 3 (SH3) domain mutant has been refined at 1.12 A resolution. This X-ray structure is at the highest resolution achieved so far for an SH3 domain. The structure allows the identification of a complete set of specific interactions and is useful for the elucidation of relations between structure and pH-dependent thermodynamic stability in a series of SH3 domain mutants.
Subject(s)
Spectrin/chemistry , src Homology Domains , Crystallization , Crystallography, X-Ray , Hydrogen Bonding , Models, Molecular , Mutation , Protein Structure, Secondary , Sensitivity and Specificity , Spectrin/genetics , Spectrin/isolation & purificationABSTRACT
The atomic structures of two proteins in the histidine biosynthesis pathway consist of beta/alpha barrels with a twofold repeat pattern. It is likely that these proteins evolved by twofold gene duplication and gene fusion from a common half-barrel ancestor. These ancestral domains are not visible as independent domains in the extant proteins but can be inferred from a combination of sequence and structural analysis. The detection of subdomain structures may be useful in efforts to search genome sequences for functionally and structurally related proteins.
Subject(s)
Aldose-Ketose Isomerases/chemistry , Aminohydrolases/chemistry , Evolution, Molecular , Gene Duplication , Protein Structure, Tertiary , Recombination, Genetic , Aldose-Ketose Isomerases/genetics , Aldose-Ketose Isomerases/metabolism , Amino Acid Motifs , Amino Acid Sequence , Aminohydrolases/genetics , Aminohydrolases/metabolism , Binding Sites , Catalysis , Crystallography, X-Ray , Histidine/biosynthesis , Models, Molecular , Molecular Sequence Data , Protein Folding , Sequence Alignment , Thermotoga maritima/enzymologyABSTRACT
Enzymes participating in different metabolic pathways often have similar catalytic mechanisms and structures, suggesting their evolution from a common ancestral precursor enzyme. We sought to create a precursor-like enzyme for N'-[(5'-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (ProFAR) isomerase (HisA; EC ) and phosphoribosylanthranilate (PRA) isomerase (TrpF; EC ), which catalyze similar reactions in the biosynthesis of the amino acids histidine and tryptophan and have a similar (betaalpha)(8)-barrel structure. Using random mutagenesis and selection, we generated several HisA variants that catalyze the TrpF reaction both in vivo and in vitro, and one of these variants retained significant HisA activity. A more detailed analysis revealed that a single amino acid exchange could establish TrpF activity on the HisA scaffold. These findings suggest that HisA and TrpF may have evolved from an ancestral enzyme of broader substrate specificity and underscore that (betaalpha)(8)-barrel enzymes are very suitable for the design of new catalytic activities.
Subject(s)
Aldose-Ketose Isomerases/chemistry , Aldose-Ketose Isomerases/genetics , Aldose-Ketose Isomerases/metabolism , Directed Molecular Evolution , Amino Acid Sequence , Amino Acid Substitution , Catalysis , Cloning, Molecular , Escherichia coli/genetics , Evolution, Molecular , Gene Library , Kinetics , Molecular Sequence Data , Mutagenesis , Plasmids , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence AlignmentABSTRACT
Recombinant non-phosphorylating NAD(+)-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN) of the hyperthermophilic crenarchaeote Thermoproteus tenax has been overexpressed, purified and crystallized using the hanging-drop vapour-diffusion technique. Crystals of different habits were obtained from several precipitant solutions (salts and polyethylene glycols). Preliminary X-ray analysis was performed with crystals grown in ammonium formate, which belonged to the primitive hexagonal space group P622, and had unit-cell parameters a = b = 184.8, c = 133.0 A, gamma = 120 degrees. Assuming a molecular weight of 55 kDa, a Matthews parameter of 3.3 A(3) Da(-1) is calculated assuming two molecules per asymmetric unit. The diffraction limit of these crystals is 2.5 A resolution.
Subject(s)
Glyceraldehyde-3-Phosphate Dehydrogenases/chemistry , Glyceraldehyde-3-Phosphate Dehydrogenases/isolation & purification , Thermoproteaceae/enzymology , Base Sequence , Crystallization , Crystallography, X-Ray , DNA Primers/genetics , Escherichia coli/genetics , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Thermoproteaceae/geneticsABSTRACT
POU domain proteins contain a bipartite DNA binding domain divided by a flexible linker that enables them to adopt various monomer configurations on DNA. The versatility of POU protein operation is additionally conferred at the dimerization level. The POU dimer formed on the PORE (ATTTGAAATGCAAAT) can recruit the transcriptional coactivator OBF-1, whereas POU dimers formed on the consensus MORE (ATGCATATGCAT) or on MOREs from immunoglobulin heavy chain promoters (AT[G/A][C/A]ATATGCAA) fail to interact. An interaction with OBF-1 is precluded since the same Oct-1 residues that form the MORE dimerization interface are also used for OBF-1/Oct-1 interactions on the PORE. Our findings provide a paradigm of how specific POU dimer assemblies can differentially recruit a coregulatory activity with distinct transcriptional readouts.
