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Nature Oct 2017Ubiquitination controls the stability of most cellular proteins, and its deregulation contributes to human diseases including cancer. Deubiquitinases remove ubiquitin...
Ubiquitination controls the stability of most cellular proteins, and its deregulation contributes to human diseases including cancer. Deubiquitinases remove ubiquitin from proteins, and their inhibition can induce the degradation of selected proteins, potentially including otherwise 'undruggable' targets. For example, the inhibition of ubiquitin-specific protease 7 (USP7) results in the degradation of the oncogenic E3 ligase MDM2, and leads to re-activation of the tumour suppressor p53 in various cancers. Here we report that two compounds, FT671 and FT827, inhibit USP7 with high affinity and specificity in vitro and within human cells. Co-crystal structures reveal that both compounds target a dynamic pocket near the catalytic centre of the auto-inhibited apo form of USP7, which differs from other USP deubiquitinases. Consistent with USP7 target engagement in cells, FT671 destabilizes USP7 substrates including MDM2, increases levels of p53, and results in the transcription of p53 target genes, induction of the tumour suppressor p21, and inhibition of tumour growth in mice.
Topics: Animals; Apoenzymes; Cell Line, Tumor; Cell Proliferation; Crystallography, X-Ray; Female; Humans; Mice; Models, Molecular; Neoplasms; Piperidines; Proto-Oncogene Proteins c-mdm2; Pyrazoles; Pyrimidines; Substrate Specificity; Transcription, Genetic; Tumor Suppressor Protein p53; Ubiquitin-Specific Peptidase 7; Ubiquitination; Xenograft Model Antitumor Assays
PubMed: 29045389
DOI: 10.1038/nature24451 -
Nature Aug 2021Single-particle cryogenic electron microscopy (cryo-EM) has become a standard technique for determining protein structures at atomic resolution. However, cryo-EM studies...
Single-particle cryogenic electron microscopy (cryo-EM) has become a standard technique for determining protein structures at atomic resolution. However, cryo-EM studies of protein-free RNA are in their early days. The Tetrahymena thermophila group I self-splicing intron was the first ribozyme to be discovered and has been a prominent model system for the study of RNA catalysis and structure-function relationships, but its full structure remains unknown. Here we report cryo-EM structures of the full-length Tetrahymena ribozyme in substrate-free and bound states at a resolution of 3.1 Å. Newly resolved peripheral regions form two coaxially stacked helices; these are interconnected by two kissing loop pseudoknots that wrap around the catalytic core and include two previously unforeseen (to our knowledge) tertiary interactions. The global architecture is nearly identical in both states; only the internal guide sequence and guanosine binding site undergo a large conformational change and a localized shift, respectively, upon binding of RNA substrates. These results provide a long-sought structural view of a paradigmatic RNA enzyme and signal a new era for the cryo-EM-based study of structure-function relationships in ribozymes.
Topics: Apoenzymes; Cryoelectron Microscopy; Holoenzymes; Models, Molecular; Nucleic Acid Conformation; RNA, Catalytic; Tetrahymena thermophila
PubMed: 34381213
DOI: 10.1038/s41586-021-03803-w -
The Journal of Biological Chemistry Aug 2017The biogenesis of iron-sulfur (Fe/S) proteins in eukaryotes is a multistage, multicompartment process that is essential for a broad range of cellular functions,... (Review)
Review
The biogenesis of iron-sulfur (Fe/S) proteins in eukaryotes is a multistage, multicompartment process that is essential for a broad range of cellular functions, including genome maintenance, protein translation, energy conversion, and the antiviral response. Genetic and cell biological studies over almost 2 decades have revealed some 30 proteins involved in the synthesis of cellular [2Fe-2S] and [4Fe-4S] clusters and their incorporation into numerous apoproteins. Mechanistic aspects of Fe/S protein biogenesis continue to be elucidated by biochemical and ultrastructural investigations. Here, we review recent developments in the pursuit of constructing a comprehensive model of Fe/S protein assembly in the mitochondrion.
Topics: Acyl Carrier Protein; Adrenodoxin; Animals; Apoenzymes; Gene Expression Regulation, Enzymologic; Humans; Iron-Binding Proteins; Iron-Sulfur Proteins; Mitochondria; Mitochondrial Proteins; Models, Biological; Models, Molecular; Protein Conformation; Protein Folding; Protein Multimerization; Protein Transport; Saccharomyces cerevisiae Proteins; Species Specificity; Sulfurtransferases; Frataxin
PubMed: 28615445
DOI: 10.1074/jbc.R117.787101 -
Biochemistry. Biokhimiia Oct 2002Modern approaches for developing antibodies with coenzyme-dependent activities are discussed for pyridoxal 5'-phosphate dependent transformation of amino acid as an... (Review)
Review
Modern approaches for developing antibodies with coenzyme-dependent activities are discussed for pyridoxal 5'-phosphate dependent transformation of amino acid as an example. A new type of antigens analogous to enzyme--substrate compounds is suggested for the production of such antibodies. Approaches for the development of pyridoxal antiidiotypic antibody using analogs of coenzyme--substrate compounds and corresponding apoenzyme complexes are reviewed.
Topics: Antibodies, Anti-Idiotypic; Antibodies, Catalytic; Antigens; Apoenzymes; Protein Conformation; Protein Engineering; Pyridoxal Phosphate
PubMed: 12460106
DOI: 10.1023/a:1020955005503 -
The Journal of Biological Chemistry May 2013The iron-molybdenum cofactor (the M-cluster) serves as the active site of molybdenum nitrogenase. Arguably one of the most complex metal cofactors in biological systems,... (Review)
Review
The iron-molybdenum cofactor (the M-cluster) serves as the active site of molybdenum nitrogenase. Arguably one of the most complex metal cofactors in biological systems, the M-cluster is assembled through the formation of an 8Fe core prior to the insertion of molybdenum and homocitrate into this core. Here, we review the recent progress in the research area of M-cluster assembly, with an emphasis on our work that provides useful insights into the mechanistic details of this process.
Topics: Apoenzymes; Bacterial Proteins; Catalytic Domain; Coenzymes; Models, Molecular; Molybdoferredoxin; Nitrogenase; Protein Structure, Tertiary; Protein Transport
PubMed: 23539617
DOI: 10.1074/jbc.R113.454041 -
Protein Science : a Publication of the... Jan 2003L-Aspartate-beta-semialdehyde dehydrogenase (ASADH) catalyzes the reductive dephosphorylation of beta-aspartyl phosphate to L-aspartate-beta-semialdehyde in the... (Comparative Study)
Comparative Study
L-Aspartate-beta-semialdehyde dehydrogenase (ASADH) catalyzes the reductive dephosphorylation of beta-aspartyl phosphate to L-aspartate-beta-semialdehyde in the aspartate biosynthetic pathway of plants and micro-organisms. The aspartate pathway produces fully one-quarter of the naturally occurring amino acids, but is not found in humans or other eukaryotic organisms, making ASADH an attractive target for the development of new antibacterial, fungicidal, or herbicidal compounds. We have determined the structure of ASADH from Vibrio cholerae in two states; the apoenzyme and a complex with NADP, and a covalently bound active site inhibitor, S-methyl-L-cysteine sulfoxide. Upon binding the inhibitor undergoes an enzyme-catalyzed reductive demethylation leading to a covalently bound cysteine that is observed in the complex structure. The enzyme is a functional homodimer, with extensive intersubunit contacts and a symmetrical 4-amino acid bridge linking the active site residues in adjacent subunits that could serve as a communication channel. The active site is essentially preformed, with minimal differences in active site conformation in the apoenzyme relative to the ternary inhibitor complex. The conformational changes that do occur result primarily from NADP binding, and are localized to the repositioning of two surface loops located on the rim at opposite sides of the NADP cleft.
Topics: Apoenzymes; Aspartate-Semialdehyde Dehydrogenase; Binding Sites; Cysteine; Enzyme Inhibitors; Escherichia coli; Methylation; Models, Molecular; NADP; Protein Conformation; Vibrio cholerae
PubMed: 12493825
DOI: 10.1110/ps.0230803 -
Proceedings of the National Academy of... Sep 2008Several general mechanisms of metallocenter biosynthesis have been reported and reviewed, and in all cases, the components or subunits of an apoprotein remain in the...
Several general mechanisms of metallocenter biosynthesis have been reported and reviewed, and in all cases, the components or subunits of an apoprotein remain in the final holoprotein. Here, we first discovered that one subunit of an apoenzyme did not remain in the functional holoenzyme. The cobalt-containing low-molecular-mass nitrile hydratase (L-NHase) of Rhodococcus rhodochrous J1 consists of beta- and alpha-subunits encoded by the nhlBA genes, respectively. An ORF, nhlE, just downstream of nhlBA, was found to be necessary for L-NHase activation. In contrast to the cobalt-containing L-NHase (holo-L-NHase containing Cys-SO(2)(-) and Cys-SO(-) metal ligands) derived from nhlBAE, the gene products derived from nhlBA were cobalt-free L-NHase (apo-L-NHase lacking oxidized cysteine residues). We discovered an L-NHase maturation mediator, NhlAE, consisting of NhlE and the cobalt- and oxidized cysteine-containing alpha-subunit of L-NHase. The incorporation of cobalt into L-NHase was shown to depend on the exchange of the nonmodified cobalt-free alpha-subunit of apo-L-NHase with the cobalt-containing cysteine-modified alpha-subunit of NhlAE. This is a posttranslational maturation process different from general mechanisms of metallocenter biosynthesis known so far: the unexpected behavior of a protein in a protein complex, which we named "self-subunit swapping."
Topics: Apoenzymes; Bacterial Proteins; Cobalt; Cysteine; Enzyme Activation; Holoenzymes; Hydro-Lyases; Metalloproteins; Protein Processing, Post-Translational; Protein Subunits; Rhodococcus
PubMed: 18809911
DOI: 10.1073/pnas.0803428105 -
Nucleic Acids Research Sep 2021Tpt1, an essential component of the fungal and plant tRNA splicing machinery, catalyzes transfer of an internal RNA 2'-PO4 to NAD+ yielding RNA 2'-OH and...
Tpt1, an essential component of the fungal and plant tRNA splicing machinery, catalyzes transfer of an internal RNA 2'-PO4 to NAD+ yielding RNA 2'-OH and ADP-ribose-1',2'-cyclic phosphate products. Here, we report NMR structures of the Tpt1 ortholog from the bacterium Runella slithyformis (RslTpt1), as apoenzyme and bound to NAD+. RslTpt1 consists of N- and C-terminal lobes with substantial inter-lobe dynamics in the free and NAD+-bound states. ITC measurements of RslTpt1 binding to NAD+ (KD ∼31 μM), ADP-ribose (∼96 μM) and ADP (∼123 μM) indicate that substrate affinity is determined primarily by the ADP moiety; no binding of NMN or nicotinamide is observed by ITC. NAD+-induced chemical shift perturbations (CSPs) localize exclusively to the RslTpt1 C-lobe. NADP+, which contains an adenylate 2'-PO4 (mimicking the substrate RNA 2'-PO4), binds with lower affinity (KD ∼1 mM) and elicits only N-lobe CSPs. The RslTpt1·NAD+ binary complex reveals C-lobe contacts to adenosine ribose hydroxyls (His99, Thr101), the adenine nucleobase (Asn105, Asp112, Gly113, Met117) and the nicotinamide riboside (Ser125, Gln126, Asn163, Val165), several of which are essential for RslTpt1 activity in vivo. Proximity of the NAD+ β-phosphate to ribose-C1″ suggests that it may stabilize an oxocarbenium transition-state during the first step of the Tpt1-catalyzed reaction.
Topics: Apoenzymes; Bacterial Proteins; Binding Sites; Cytophagaceae; Ligands; Models, Molecular; Mutagenesis; NAD; Nuclear Magnetic Resonance, Biomolecular; Nucleotides; Phosphotransferases; Protein Binding; Protein Conformation; RNA
PubMed: 33880546
DOI: 10.1093/nar/gkab241 -
Drug Metabolism Reviews Feb 2011Heme is vital to our aerobic universe. Heme cellular content is finely tuned through an exquisite control of synthesis and degradation. Heme deficiency is deleterious to... (Review)
Review
Heme is vital to our aerobic universe. Heme cellular content is finely tuned through an exquisite control of synthesis and degradation. Heme deficiency is deleterious to cells, whereas excess heme is toxic. Most of the cellular heme serves as the prosthetic moiety of functionally diverse hemoproteins, including cytochromes P450 (P450s). In the liver, P450s are its major consumers, with >50% of hepatic heme committed to their synthesis. Prosthetic heme is the sine qua non of P450 catalytic biotransformation of both endo- and xenobiotics. This well-recognized functional role notwithstanding, heme also regulates P450 protein synthesis, assembly, repair, and disposal. These less well-appreciated aspects are reviewed herein.
Topics: Animals; Apoenzymes; Cytochrome P-450 Enzyme System; Endoplasmic Reticulum; Enzyme Stability; Heme; Liver; Molecular Structure; Protein Serine-Threonine Kinases; Transcriptional Activation
PubMed: 20860521
DOI: 10.3109/03602532.2010.515222 -
The FEBS Journal Sep 2015[Fe]-hydrogenase (Hmd), an enzyme of the methanogenic energy metabolism, harbors an iron-guanylylpyridinol (FeGP) cofactor used for H2 cleavage. The generated hydride is...
UNLABELLED
[Fe]-hydrogenase (Hmd), an enzyme of the methanogenic energy metabolism, harbors an iron-guanylylpyridinol (FeGP) cofactor used for H2 cleavage. The generated hydride is transferred to methenyl-tetrahydromethanopterin (methenyl-H4MPT(+)). Most hydrogenotrophic methanogens contain the hmd-related genes hmdII and hmdIII. Their function is still elusive. We were able to reconstitute the HmdII holoenzyme of Methanocaldococcus jannaschii with recombinantly produced apoenzyme and the FeGP cofactor, which is a prerequisite for in vitro functional analysis. Infrared spectroscopic and X-ray structural data clearly indicated binding of the FeGP cofactor. Methylene-H4MPT binding was detectable in the significantly altered infrared spectra of the HmdII holoenzyme and in the HmdII apoenzyme-methylene-H4 MPT complex structure. The related binding mode of the FeGP cofactor and methenyl-H4MPT(+) compared with Hmd and their multiple contacts to the polypeptide highly suggest a biological role in HmdII. However, holo-HmdII did not catalyze the Hmd reaction, not even in a single turnover process, as demonstrated by kinetic measurements. The found inactivity can be rationalized by an increased contact area between the C- and N-terminal folding units in HmdII compared with in Hmd, which impairs the catalytically necessary open-to-close transition, and by an exchange of a crucial histidine to a tyrosine. Mainly based on the presented data, a function of HmdII as Hmd isoenzyme, H2 sensor, FeGP-cofactor storage protein and scaffold protein for FeGP-cofactor biosynthesis could be excluded. Inspired by the recently found binding of HmdII to aminoacyl-tRNA synthetases and tRNA, we tentatively consider HmdII as a regulatory protein for protein synthesis that senses the intracellular methylene-H4 MPT concentration.
DATABASE
Structural data are available in the Protein Data Bank under the accession numbers 4YT8; 4YT2; 4YT4 and 4YT5.
Topics: Apoenzymes; Archaeal Proteins; Binding Sites; Coenzymes; Crystallography, X-Ray; Escherichia coli; Gene Expression; Holoenzymes; Hydrogenase; Iron-Sulfur Proteins; Kinetics; Methanocaldococcus; Models, Molecular; Protein Binding; Protein Biosynthesis; Protein Folding; Protein Interaction Domains and Motifs; Protein Structure, Secondary; Pterins; Pyridines; Recombinant Proteins; Spectrophotometry, Infrared
PubMed: 26094576
DOI: 10.1111/febs.13351