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1.
Appl Magn Reson ; 52(8): 995-1015, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34720439

RESUMO

In the study of biological structures, pulse dipolar spectroscopy (PDS) is used to elucidate spin-spin distances at nanometre-scale by measuring dipole-dipole interactions between paramagnetic centres. The PDS methods of Double Electron Electron Resonance (DEER) and Relaxation Induced Dipolar Modulation Enhancement (RIDME) are employed, and their results compared, for the measurement of the dipolar coupling between nitroxide spin labels and copper-II (Cu(II)) paramagnetic centres within the copper amine oxidase from Arthrobacter globiformis (AGAO). The distance distribution results obtained indicate that two distinct distances can be measured, with the longer of these at c.a. 5 nm. Conditions for optimising the RIDME experiment such that it may outperform DEER for these long distances are discussed. Modelling methods are used to show that the distances obtained after data analysis are consistent with the structure of AGAO. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00723-021-01321-6.

2.
Nat Commun ; 12(1): 4045, 2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-34193876

RESUMO

RAS mutations are the most common oncogenic drivers across human cancers, but there remains a paucity of clinically-validated pharmacological inhibitors of RAS, as druggable pockets have proven difficult to identify. Here, we identify two RAS-binding Affimer proteins, K3 and K6, that inhibit nucleotide exchange and downstream signaling pathways with distinct isoform and mutant profiles. Affimer K6 binds in the SI/SII pocket, whilst Affimer K3 is a non-covalent inhibitor of the SII region that reveals a conformer of wild-type RAS with a large, druggable SII/α3 pocket. Competitive NanoBRET between the RAS-binding Affimers and known RAS binding small-molecules demonstrates the potential to use Affimers as tools to identify pharmacophores. This work highlights the potential of using biologics with small interface surfaces to select unseen, druggable conformations in conjunction with pharmacophore identification for hard-to-drug proteins.


Assuntos
Produtos Biológicos/farmacologia , Técnicas de Visualização da Superfície Celular/métodos , Descoberta de Drogas/métodos , Neoplasias/tratamento farmacológico , Proteínas ras/antagonistas & inibidores , Sítio Alostérico , Produtos Biológicos/química , Humanos , Neoplasias/química , Neoplasias/enzimologia , Transdução de Sinais , Proteínas ras/metabolismo
3.
Int J Mol Sci ; 22(13)2021 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-34203139

RESUMO

Fibrinogen is one of the key molecular players in haemostasis. Thrombin-mediated release of fibrinopeptides from fibrinogen converts this soluble protein into a network of fibrin fibres that form a building block for blood clots. Thrombin-activated factor XIII further crosslinks the fibrin fibres and incorporates antifibrinolytic proteins into the network, thus stabilising the clot. The conversion of fibrinogen to fibrin also exposes binding sites for fibrinolytic proteins to limit clot formation and avoid unwanted extension of the fibrin fibres. Altered clot structure and/or incorporation of antifibrinolytic proteins into fibrin networks disturbs the delicate equilibrium between clot formation and lysis, resulting in either unstable clots (predisposing to bleeding events) or persistent clots that are resistant to lysis (increasing risk of thrombosis). In this review, we discuss the factors responsible for alterations in fibrin(ogen) that can modulate clot stability, in turn predisposing to abnormal haemostasis. We also explore the mechanistic pathways that may allow the use of fibrinogen as a potential therapeutic target to treat vascular thrombosis or bleeding disorders. Better understanding of fibrinogen function will help to devise future effective and safe therapies to modulate thrombosis and bleeding risk, while maintaining the fine balance between clot formation and lysis.


Assuntos
Fator XIIIa/metabolismo , Fibrina/metabolismo , Fibrinogênio/metabolismo , Trombose/metabolismo , Animais , Fator XIIIa/genética , Fibrina/genética , Fibrinogênio/genética , Fibrinólise/genética , Fibrinólise/fisiologia , Humanos , Trombose/genética
4.
Biochemistry ; 57(36): 5301-5314, 2018 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-30110143

RESUMO

Copper amine oxidases (CuAOs) are metalloenzymes that reduce molecular oxygen to hydrogen peroxide during catalytic turnover of primary amines. In addition to Cu2+ in the active site, two peripheral calcium sites, ∼32 Šfrom the active site, have roles in Escherichia coli amine oxidase (ECAO). The buried Ca2+ (Asp533, Leu534, Asp535, Asp678, and Ala679) is essential for full-length protein production, while the surface Ca2+ (Glu573, Tyr667, Asp670, and Glu672) modulates biogenesis of the 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor. The E573Q mutation at the surface site prevents calcium binding and TPQ biogenesis. However, TPQ biogenesis can be restored by a suppressor mutation (I342F) in the proposed oxygen delivery channel to the active site. While supporting TPQ biogenesis (∼60% WTECAO TPQ), I342F/E573Q has almost no amine oxidase activity (∼4.6% WTECAO activity). To understand how these long-range mutations have major effects on TPQ biogenesis and catalysis, we employed ultraviolet-visible spectroscopy, steady-state kinetics, inhibition assays, and X-ray crystallography. We show that the surface metal site controls the equilibrium (disproportionation) of the Cu2+-substrate reduced TPQ (TPQAMQ) Cu+-TPQ semiquinone (TPQSQ) couple. Removal of the calcium ion from this site by chelation or mutagenesis shifts the equilibrium to Cu2+-TPQAMQ or destabilizes Cu+-TPQSQ. Crystal structure analysis shows that TPQ biogenesis is stalled at deprotonation in the Cu2+-tyrosinate state. Our findings support WTECAO using the inner sphere electron transfer mechanism for oxygen reduction during catalysis, and while a Cu+-tyrosyl radical intermediate is not essential for TPQ biogenesis, it is required for efficient biogenesis.


Assuntos
Amina Oxidase (contendo Cobre)/química , Amina Oxidase (contendo Cobre)/metabolismo , Cobre/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Espécies Reativas de Oxigênio/metabolismo , Amina Oxidase (contendo Cobre)/genética , Sítios de Ligação , Catálise , Domínio Catalítico , Cristalografia por Raios X , Proteínas de Escherichia coli/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Conformação Proteica , Espécies Reativas de Oxigênio/química
5.
Chembiochem ; 16(4): 559-64, 2015 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-25607656

RESUMO

For some homodimeric copper amine oxidases (CuAO), there is suggestive evidence of differential activity at the two active sites implying potential cooperativity between the two monomers. To examine this phenomenon for the Arthrobacter globiformis CuAO (AGAO), we purified a heterodimeric form of the enzyme for comparison with the homodimer. The heterodimer comprises an active wild-type monomer and an inactive monomer in which an active-site tyrosine is mutated to phenylalanine (Y382F). This mutation prevents the formation of the trihydroxyphenylalanine quinone (TPQ) cofactor. A pETDuet vector and a dual fusion tag strategy was used to purify heterodimers (WT/Y382F) from homodimers. Purity was confirmed by western blot and native PAGE analyses. Spectral and kinetic studies support the view that whether there are one or two functional monomers in the dimer, the properties of each functional monomer are the same, thus indicating no communication between the active sites in this bacterial enzyme.


Assuntos
Amina Oxidase (contendo Cobre)/química , Arthrobacter/enzimologia , Amina Oxidase (contendo Cobre)/metabolismo , Arthrobacter/química , Cinética , Modelos Moleculares , Conformação Proteica , Multimerização Proteica
6.
J Neural Transm (Vienna) ; 118(7): 1043-53, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21547391

RESUMO

Copper amine oxidases are important for the metabolism of a range of biogenic amines. Here, we focus on substrate specificity in the E. coli copper amine oxidase (ECAO) and specifically the role of Tyr 381. This residue, and its equivalent, in other copper amine oxidases has been referred to as a "gating" residue able to move position depending upon the presence or absence of amine substrate. The position of this residue suggests a role in substrate selectivity. We have compared the properties of two variant forms of ECAO, Y381F and Y381A, with wild-type enzyme by steady-state kinetics of oxidation of a number of amine substrates, modes of inhibitor interactions and X-ray structure determination. Y381F displays a similar catalytic efficiency to wild type against the preferred substrate ß-phenylethylamine. In both cases oxidation of the alternative aromatic amine substrate benzylamine is relatively poor, although Y381F represents an efficient benzylamine oxidase. By contrast, Y381A performed poorly against both aromatic substrates predominantly due to an increased K (M) which we propose is due to the lack of an aromatic residue to orient substrate towards the TPQ and active site base. These results are supported by different behaviour of Y381A to inhibition with 2-hydrazinopyridine. We also report on methylamine turnover by the three enzymes. We propose that Y381, together with another residue Y387, may be considered of critical importance for the substrate selectivity of ECAO, through stacking or hydrophobic interactions with substrate.


Assuntos
Amina Oxidase (contendo Cobre)/química , Amina Oxidase (contendo Cobre)/fisiologia , Escherichia coli/enzimologia , Tirosina/química , Tirosina/fisiologia , Amina Oxidase (contendo Cobre)/genética , Sequência de Aminoácidos , Domínio Catalítico/genética , Escherichia coli/genética , Interações Hidrofóbicas e Hidrofílicas , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/fisiologia , Metilaminas/química , Piridonas/química , Especificidade por Substrato/genética , Tirosina/genética
7.
Biochemistry ; 49(6): 1268-80, 2010 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-20052994

RESUMO

To investigate the role of the active site copper in Escherichia coli copper amine oxidase (ECAO), we initiated a metal-substitution study. Copper reconstitution of ECAO (Cu-ECAO) restored only approximately 12% wild-type activity as measured by k(cat(amine)). Treatment with EDTA, to remove exogenous divalent metals, increased Cu-ECAO activity but reduced the activity of wild-type ECAO. Subsequent addition of calcium restored wild-type ECAO and further enhanced Cu-ECAO activities. Cobalt-reconstituted ECAO (Co-ECAO) showed lower but significant activity. These initial results are consistent with a direct electron transfer from TPQ to oxygen stabilized by the metal. If a Cu(I)-TPQ semiquinone mechanism operates, then an alternative outer-sphere electron transfer must also exist to account for the catalytic activity of Co-ECAO. The positive effect of calcium on ECAO activity led us to investigate the peripheral calcium binding sites of ECAO. Crystallographic analysis of wild-type ECAO structures, determined in the presence and absence of EDTA, confirmed that calcium is the normal ligand of these peripheral sites. The more solvent exposed calcium can be easily displaced by mono- and divalent cations with no effect on activity, whereas removal of the more buried calcium ion with EDTA resulted in a 60-90% reduction in ECAO activity and the presence of a lag phase, which could be overcome under oxygen saturation or by reoccupying the buried site with various divalent cations. Our studies indicate that binding of metal ions in the peripheral sites, while not essential, is important for maximal enzymatic activity in the mature enzyme.


Assuntos
Amina Oxidase (contendo Cobre)/química , Proteínas de Escherichia coli/química , Metais Pesados/química , Amina Oxidase (contendo Cobre)/antagonistas & inibidores , Amina Oxidase (contendo Cobre)/metabolismo , Sequência de Aminoácidos , Cálcio/química , Cálcio/metabolismo , Domínio Catalítico/genética , Cobre/química , Cristalografia por Raios X , Di-Hidroxifenilalanina/análogos & derivados , Di-Hidroxifenilalanina/química , Ácido Edético/química , Ativação Enzimática/genética , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/metabolismo , Humanos , Metais Alcalinoterrosos/química , Metais Alcalinoterrosos/metabolismo , Metais Pesados/metabolismo , Dados de Sequência Molecular , Oxirredução , Ligação Proteica/genética
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