A homozygous loss-of-function mutation p.(Arg316Gln) in the fat mass and obesity-associated (FTO) gene, which encodes for an iron and 2-oxoglutarate-dependent oxygenase, was previously identified in ...a large family in which nine affected individuals present with a lethal syndrome characterised by growth retardation and multiple malformations. To date, no other pathogenic mutation in FTO has been identified as a cause of multiple congenital malformations.
We investigated a 21-month-old girl who presented distinctive facial features, failure to thrive, global developmental delay, left ventricular cardiac hypertrophy, reduced vision and bilateral hearing loss. We performed targeted next-generation sequencing of 4813 clinically relevant genes in the patient and her parents.
We identified a novel FTO homozygous missense mutation (c.956C>T; p.(Ser319Phe)) in the affected individual. This mutation affects a highly conserved residue located in the same functional domain as the previously characterised mutation p.(Arg316Gln). Biochemical studies reveal that p.(Ser319Phe) FTO has reduced 2-oxoglutarate turnover and N-methyl-nucleoside demethylase activity.
Our findings are consistent with previous reports that homozygous mutations in FTO can lead to rare growth retardation and developmental delay syndrome, and further support the proposal that FTO plays an important role in early development of human central nervous and cardiovascular systems.
Cryogenic X-ray diffraction is a powerful tool for crystallographic studies on enzymes including oxygenases and oxidases. Amongst the benefits that cryo-conditions (usually employing a nitrogen ...cryo-stream at 100 K) enable, is data collection of dioxygen-sensitive samples. Although not strictly anaerobic, at low temperatures the vitreous ice conditions severely restrict O
2
diffusion into and/or through the protein crystal. Cryo-conditions limit chemical reactivity, including reactions that require significant conformational changes. By contrast, data collection at room temperature imposes fewer restrictions on diffusion and reactivity; room-temperature serial methods are thus becoming common at synchrotrons and XFELs. However, maintaining an anaerobic environment for dioxygen-dependent enzymes has not been explored for serial room-temperature data collection at synchrotron light sources. This work describes a methodology that employs an adaptation of the `sheet-on-sheet' sample mount, which is suitable for the low-dose room-temperature data collection of anaerobic samples at synchrotron light sources. The method is characterized by easy sample preparation in an anaerobic glovebox, gentle handling of crystals, low sample consumption and preservation of a localized anaerobic environment over the timescale of the experiment (<5 min). The utility of the method is highlighted by studies with three X-ray-radiation-sensitive Fe(II)-containing model enzymes: the 2-oxoglutarate-dependent L-arginine hydroxylase VioC and the DNA repair enzyme AlkB, as well as the oxidase isopenicillin N synthase (IPNS), which is involved in the biosynthesis of all penicillin and cephalosporin antibiotics.
Lysyl and prolyl hydroxylations are well-known post-translational modifications to animal and plant proteins with extracellular roles. More recent work has indicated that the hydroxylation of ...intracellular animal proteins may be common. JMJD6 catalyses the iron- and 2-oxoglutarate-dependent hydroxylation of lysyl residues in arginine–serine-rich domains of RNA-splicing-related proteins. We report crystallographic studies on the catalytic domain of JMJD6 in complex with Ni(II) substituting for Fe(II). Together with mutational studies, the structural data suggest how JMJD6 binds its lysyl residues such that it can catalyse C-5 hydroxylation rather than Nɛ-demethylation, as for analogous enzymes.
Target validation remains a challenge in drug discovery, which leads to a high attrition rate in the drug discovery process, particularly in Phase II clinical trials. Consequently, new approaches to ...enhance target validation are valuable tools to improve the drug discovery process. Here, we report the combination of site-directed mutagenesis and electrophilic fragments to enable the rapid identification of small molecules that selectively inhibit the mutant protein. Using the bromodomain-containing protein BRD4 as an example, we employed a structure-based approach to identify the L94C mutation in the first bromodomain of BRD4 BRD4(1) as having a minimal effect on BRD4(1) function. We then screened a focused, KAc mimic-containing fragment set and a diverse fragment library against the mutant and wild-type proteins and identified a series of fragments that showed high selectivity for the mutant protein. These compounds were elaborated to include an alkyne click tag to enable the attachment of a fluorescent dye. These clickable compounds were then assessed in HEK293T cells, transiently expressing BRD4(1)WT or BRD4(1)L94C, to determine their selectivity for BRD4(1)L94C over other possible cellular targets. One compound was identified that shows very high selectivity for BRD4(1)L94C over all other proteins. This work provides a proof-of-concept that the combination of site-directed mutagenesis and electrophilic fragments, in a mutate and conjugate approach, can enable rapid identification of small molecule inhibitors for an appropriately mutated protein of interest. This technology can be used to assess the cellular phenotype of inhibiting the protein of interest, and the electrophilic ligand provides a starting point for noncovalent ligand development.
2-Oxoglutarate (2OG)-dependent oxygenases have important roles in the regulation of gene expression via demethylation of N-methylated chromatin components and in the hydroxylation of transcription ...factors and splicing factor proteins. Recently, 2OG-dependent oxygenases that catalyse hydroxylation of transfer RNA and ribosomal proteins have been shown to be important in translation relating to cellular growth, TH17-cell differentiation and translational accuracy. The finding that ribosomal oxygenases (ROXs) occur in organisms ranging from prokaryotes to humans raises questions as to their structural and evolutionary relationships. In Escherichia coli, YcfD catalyses arginine hydroxylation in the ribosomal protein L16; in humans, MYC-induced nuclear antigen (MINA53; also known as MINA) and nucleolar protein 66 (NO66) catalyse histidine hydroxylation in the ribosomal proteins RPL27A and RPL8, respectively. The functional assignments of ROXs open therapeutic possibilities via either ROX inhibition or targeting of differentially modified ribosomes. Despite differences in the residue and protein selectivities of prokaryotic and eukaryotic ROXs, comparison of the crystal structures of E. coli YcfD and Rhodothermus marinus YcfD with those of human MINA53 and NO66 reveals highly conserved folds and novel dimerization modes defining a new structural subfamily of 2OG-dependent oxygenases. ROX structures with and without their substrates support their functional assignments as hydroxylases but not demethylases, and reveal how the subfamily has evolved to catalyse the hydroxylation of different residue side chains of ribosomal proteins. Comparison of ROX crystal structures with those of other JmjC-domain-containing hydroxylases, including the hypoxia-inducible factor asparaginyl hydroxylase FIH and histone N(ε)-methyl lysine demethylases, identifies branch points in 2OG-dependent oxygenase evolution and distinguishes between JmjC-containing hydroxylases and demethylases catalysing modifications of translational and transcriptional machinery. The structures reveal that new protein hydroxylation activities can evolve by changing the coordination position from which the iron-bound substrate-oxidizing species reacts. This coordination flexibility has probably contributed to the evolution of the wide range of reactions catalysed by oxygenases.
Carbapenems are important antibacterials and are both substrates and inhibitors of some β-lactamases. We report studies on the reaction of the unusual carbapenem biapenem, with the subclass B1 ...metallo-β-lactamases VIM-1 and VIM-2 and the class A serine-β-lactamase KPC-2. X-ray diffraction studies with VIM-2 crystals treated with biapenem reveal the opening of the β-lactam ring to form a mixture of the (2
)-imine and enamine complexed at the active site. NMR studies on the reactions of biapenem with VIM-1, VIM-2, and KPC-2 reveal the formation of hydrolysed enamine and (2
)- and (2
)-imine products. The combined results support the proposal that SBL/MBL-mediated carbapenem hydrolysis results in a mixture of tautomerizing enamine and (2
)- and (2
)-imine products, with the thermodynamically favoured (2
)-imine being the major observed species over a relatively long-time scale. The results suggest that prolonging the lifetimes of β-lactamase carbapenem complexes by optimising tautomerisation of the nascently formed enamine to the (2
)-imine and likely more stable (2
)-imine tautomer is of interest in developing improved carbapenems.
The β-lactam antibiotics represent the most successful drug class for treatment of bacterial infections. Resistance to them, importantly via production of β-lactamases, which collectively are able to ...hydrolyse all classes of β-lactams, threatens their continued widespread use. Bicyclic boronates show potential as broad spectrum inhibitors of the mechanistically distinct serine- (SBL) and metallo- (MBL) β-lactamase families.
Using biophysical methods, including crystallographic analysis, we have investigated the binding mode of bicyclic boronates to clinically important β-lactamases. Induction experiments and agar-based MIC screening against MDR-Enterobacteriaceae (n = 132) were used to evaluate induction properties and the in vitro efficacy of a bicyclic boronate in combination with meropenem.
Crystallographic analysis of a bicyclic boronate in complex with AmpC from Pseudomonas aeruginosa reveals it binds to form a tetrahedral boronate species. Microbiological studies on the clinical coverage (in combination with meropenem) and induction of β-lactamases by bicyclic boronates further support the promise of such compounds as broad spectrum β-lactamase inhibitors.
Together with reported studies on the structural basis of their inhibition of class A, B and D β-lactamases, biophysical studies, including crystallographic analysis, support the proposal that bicyclic boronates mimic tetrahedral intermediates common to SBL and MBL catalysis.
Bicyclic boronates are a new generation of broad spectrum inhibitors of both SBLs and MBLs.
•Bicyclic boronate structures reveal mechanism of the broad spectrum β-lactamase inhibition by this new drug class.•Bicyclic boronates do not manifest detectable β-lactamase induction in some bacteria.•Microbiological studies reveal promising clinical coverage for this class of β-lactamase inhibitor.
Lipolysis in adipocytes is regulated by phosphorylation of lipid droplet-associated proteins, including perilipin 1A and hormone-sensitive lipase (HSL). Perilipin 1A is potentially phosphorylated by ...cAMP(adenosine 3',5'-cyclic monophosphate)-dependent protein kinase (PKA) on several sites, including conserved C-terminal residues, serine 497 (PKA-site 5) and serine 522 (PKA-site 6). To characterize perilipin 1A phosphorylation, novel monoclonal antibodies were developed, which selectively recognize perilipin 1A phosphorylation at PKA-site 5 and PKA-site 6. Utilizing these novel antibodies, as well as antibodies selectively recognizing HSL phosphorylation at serine 563 or serine 660, we used high content analysis to examine the phosphorylation of perilipin 1A and HSL in adipocytes exposed to lipolytic agents. We found that perilipin PKA-site 5 and HSL-serine 660 were phosphorylated to a similar extent in response to forskolin (FSK) and L-γ-melanocyte stimulating hormone (L-γ-MSH). In contrast, perilipin PKA-site 6 and HSL-serine 563 were phosphorylated more slowly and L-γ-MSH was a stronger agonist for these sites compared to FSK. When a panel of lipolytic agents was tested, including multiple concentrations of isoproterenol, FSK, and L-γ-MSH, the pattern of results was virtually identical for perilipin PKA-site 5 and HSL-serine 660, whereas a distinct pattern was observed for perilipin PKA-site 6 and HSL-serine 563. Notably, perilipin PKA-site 5 and HSL-serine 660 feature two arginine residues upstream from the phospho-acceptor site, which confers high affinity for PKA, whereas perilipin PKA-site 6 and HSL-serine 563 feature only a single arginine. Thus, we suggest perilipin 1A and HSL are differentially phosphorylated in a similar manner at the initiation of lipolysis and arginine residues near the target serines may influence this process.
Select an isoform: Linking of cosubstrate and substrate binding sites enables highly selective inhibiton of isoforms of human histone lysine demethylases. The results should provide a basis for the ...development of potent and selective JmjC inhibitors, possibly suitable for clinical use.
Crystallographic analyses of the VIM-5 metallo-β-lactamase (MBL) with isoquinoline inhibitors reveal non zinc ion binding modes. Comparison with other MBL-inhibitor structures directed addition of a ...zinc-binding thiol enabling identification of potent B1 MBL inhibitors. The inhibitors potentiate meropenem activity against clinical isolates harboring MBLs.
PDF
