Natural supramolecular protein assemblies

Supramolecular protein assemblies are an emerging area within the chemical sciences, which combine the topological structures of the field of supramolecular chemistry and the state-of-the-art chemical biology approaches to unravel the formation and function of protein assemblies. Recent chemical and biological studies on natural multimeric protein structures, including fibers, rings, tubes, catenanes, knots, and cages, have shown that the quaternary structures of proteins are a prerequisite for their highly specific biological functions. In this review, we illustrate that a striking structural diversity of protein assemblies is present in nature. Furthermore, we describe structure–function relationship studies for selected classes of protein architectures, and we highlight the techniques that enable the characterisation of supramolecular protein structures

Substrate selectivity and inhibition of histidine JmjC hydroxylases MINA53 and NO66.

Non-haem Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases catalyse oxidation of multiple proteins in organisms ranging from bacteria to humans. We describe studies on the substrate selectivity and inhibition of the human ribosomal oxygenases (ROX) MINA53 and NO66, members of the JmjC 2OG oxygenase subfamily, which catalyse C-3 hydroxylation of histidine residues in Rpl27a and Rpl8, respectively. Assays with natural and unnatural histidine analogues incorporated into Rpl peptides provide evidence that MINA53 and NO66 have narrow substrate selectivities compared to some other human JmjC hydroxylases, including factor inhibiting HIF and JMJD6. Notably, the results of inhibition assays with Rpl peptides containing histidine analogues with acyclic side chains, including Asn, Gln and homoGln, suggest the activities of MINA53/NO66, and by implication related 2OG dependent protein hydroxylases/demethylases, might be regulated in vivo by competition with non-oxidised proteins/peptides. The inhibition results also provide avenues for development of inhibitors selective for MINA53 and NO66

Selective Inhibitors of the JMJD2 Histone Demethylases: Combined Nondenaturing Mass Spectrometric Screening and Crystallographic Approaches†

Ferrous ion and 2-oxoglutarate (2OG) oxygenases catalyze the demethylation of N(epsilon)-methylated lysine residues in histones. Here we report studies on the inhibition of the JMJD2 subfamily of histone demethylases, employing binding analyses by nondenaturing mass spectrometry (MS), dynamic combinatorial chemistry coupled to MS, turnover assays, and crystallography. The results of initial binding and inhibition assays directed the production and analysis of a set of N-oxalyl-d-tyrosine derivatives to explore the extent of a subpocket at the JMJD2 active site. Some of the inhibitors were shown to be selective for JMJD2 over the hypoxia-inducible factor prolyl hydroxylase PHD2. A crystal structure of JMJD2A in complex with one of the potent inhibitors was obtained; modeling other inhibitors based on this structure predicts interactions that enable improved inhibition for some compounds

Mechanism of biomolecular recognition of trimethyllysine by the fluorinated aromatic cage of KDM5A PHD3 finger

The understanding of biomolecular recognition of posttranslationally modified histone proteins is centrally important to the histone code hypothesis. Despite extensive binding and structural studies on the readout of histones, the molecular language by which posttranslational modifications on histone proteins are read remains poorly understood. Here we report physical-organic chemistry studies on the recognition of the positively charged trimethyllysine by the electron-rich aromatic cage containing PHD3 finger of KDM5A. The aromatic character of two tryptophan residues that solely constitute the aromatic cage of KDM5A was fine-tuned by the incorporation of fluorine substituents. Our thermodynamic analyses reveal that the wild-type and fluorinated KDM5A PHD3 fingers associate equally well with trimethyllysine. This work demonstrates that the biomolecular recognition of trimethyllysine by fluorinated aromatic cages is associated with weaker cation-π interactions that are compensated by the energetically more favourable trimethyllysine-mediated release of high-energy water molecules that occupy the aromatic cage

2-Oxoglutarate oxygenases are inhibited by a range of transition metals.

2-Oxoglutarate oxygenases are inhibited by a range of transition metals, as exemplified by studies on human histone demethylases and prolyl hydroxylase domain 2 (PHD2 or EGLN1). The biological effects associated with 2-oxoglutarate oxygenase inhibition may result from inhibition of more than one enzyme and by mechanisms in addition to simple competition with the Fe(ii) cofactor

Use of mass spectrometry to probe the nucleophilicity of cysteinyl residues of prolyl hydroxylase domain 2.

Prolyl hydroxylase domain 2 (PHD2) plays an important role in hypoxic sensing in humans. Here we report studies on the reactivity of cysteinyl residues of the catalytic domain of PHD2 using an approach in which nondenaturing electrospray ionization-mass spectrometry (ESI-MS) analyses were combined with the use of a thiol library and residue substitution. Among the seven cysteinyl residues of the PHD2 catalytic domain, Cys201 was found to be predominantly modified by thiols or N-ethylmaleimide. Selective modification of Cys201 was further demonstrated with methanethiosulfonate, a spin-labeled probe. The modified PHD2 will be useful in electron paramagnetic resonance studies on PHD2. The results demonstrate the use of a combined library/residue substitution/ESI-MS approach for analyzing residue reactivity

2-Oxoglutarate analogue inhibitors of prolyl hydroxylase domain 2.

Analogues of the 2-oxoglutarate cosubstrate of the human oxygen sensing enzyme prolyl hydroxylase domain 2 (PHD2) with variations in the potential iron-chelating group were screened as inhibitors and for binding (using non-denaturing electrospray ionization mass spectrometry) to PHD2

Recognition of dimethylarginine analogues by tandem Tudor domain protein Spindlin1

Epigenetic readout of the combinatorial posttranslational modification comprised of trimethyllysine and asymmetric dimethylarginine (H3K4me3R8me2a) takes place via biomolecular recognition of tandem Tudor-domain-containing protein Spindlin1. Through comparative thermodynamic data and molecular dynamics simulations, we sought to explore the binding scope of asymmetric dimethylarginine mimics by Spindlin1. Herein, we provide evidence that the biomolecular recognition of H3K4me2R8me2a is not significantly affected when R8me2a is replaced by dimethylarginine analogues, implying that the binding of K4me3 provides the major binding contribution. High-energy water molecules inside both aromatic cages of the ligand binding sites contribute to the reader–histone association upon displacement by histone peptide, with the K4me3 hydration site being lower in free energy due to a flip of Trp151

Carboxymethylproline synthase catalysed syntheses of functionalised N-heterocycles.

The utility of wild-type and variant carboxymethylproline synthases for biocatalysis was demonstrated by preparing functionalised 5-, 6- and 7-membered N-heterocycles from amino acid aldehydes and (alkylated) malonyl-coenzyme A derivatives; the N-heterocycles produced were converted to the corresponding bicyclic beta-lactams by a carbapenem synthetase