Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 58
Filtrar
1.
Nucleic Acids Res ; 50(8): 4732-4754, 2022 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-35420136

RESUMO

SUMOylation is critical for numerous cellular signalling pathways, including the maintenance of genome integrity via the repair of DNA double-strand breaks (DSBs). If misrepaired, DSBs can lead to cancer, neurodegeneration, immunodeficiency and premature ageing. Using systematic human proteome microarray screening combined with widely applicable carbene footprinting, genetic code expansion and high-resolution structural profiling, we define two non-conventional and topology-selective SUMO2-binding regions on XRCC4, a DNA repair protein important for DSB repair by non-homologous end-joining (NHEJ). Mechanistically, the interaction of SUMO2 and XRCC4 is incompatible with XRCC4 binding to three other proteins important for NHEJ-mediated DSB repair. These findings are consistent with SUMO2 forming a redundant NHEJ layer with the potential to regulate different NHEJ complexes at distinct levels including, but not limited to, XRCC4 interactions with XLF, LIG4 and IFFO1. Regulation of NHEJ is not only relevant for carcinogenesis, but also for the design of precision anti-cancer medicines and the optimisation of CRISPR/Cas9-based gene editing. In addition to providing molecular insights into NHEJ, this work uncovers a conserved SUMO-binding module and provides a rich resource on direct SUMO binders exploitable towards uncovering SUMOylation pathways in a wide array of cellular processes.


Assuntos
Reparo do DNA por Junção de Extremidades , Reparo do DNA , Quebras de DNA de Cadeia Dupla , Enzimas Reparadoras do DNA/metabolismo , Humanos , Análise em Microsséries , Ligação Proteica , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina , Sumoilação
2.
J Biol Chem ; 298(4): 101771, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35218772

RESUMO

The ubiquitous UbiD family of reversible decarboxylases is implicated in a wide range of microbial processes and depends on the prenylated flavin mononucleotide cofactor for catalysis. However, only a handful of UbiD family members have been characterized in detail, and comparison between these has suggested considerable variability in enzyme dynamics and mechanism linked to substrate specificity. In this study, we provide structural and biochemical insights into the indole-3-carboxylic acid decarboxylase, representing an UbiD enzyme activity distinct from those previously studied. Structural insights from crystal structure determination combined with small-angle X-ray scattering measurements reveal that the enzyme likely undergoes an open-closed transition as a consequence of domain motion, an event that is likely coupled to catalysis. We also demonstrate that the indole-3-carboxylic acid decarboxylase can be coupled with carboxylic acid reductase to produce indole-3-carboxyaldehyde from indole + CO2 under ambient conditions. These insights provide further evidence for a common mode of action in the widespread UbiD enzyme family.


Assuntos
Carboxiliases , Modelos Moleculares , Carboxiliases/química , Carboxiliases/metabolismo , Mononucleotídeo de Flavina/metabolismo , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Especificidade por Substrato
3.
J Biol Chem ; 298(5): 101903, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35398092

RESUMO

The sugars streptose and dihydrohydroxystreptose (DHHS) are unique to the bacteria Streptomyces griseus and Coxiella burnetii, respectively. Streptose forms the central moiety of the antibiotic streptomycin, while DHHS is found in the O-antigen of the zoonotic pathogen C. burnetii. Biosynthesis of these sugars has been proposed to follow a similar path to that of TDP-rhamnose, catalyzed by the enzymes RmlA, RmlB, RmlC, and RmlD, but the exact mechanism is unclear. Streptose and DHHS biosynthesis unusually requires a ring contraction step that could be performed by orthologs of RmlC or RmlD. Genome sequencing of S. griseus and C. burnetii has identified StrM and CBU1838 proteins as RmlC orthologs in these respective species. Here, we demonstrate that both enzymes can perform the RmlC 3'',5'' double epimerization activity necessary to support TDP-rhamnose biosynthesis in vivo. This is consistent with the ring contraction step being performed on a double epimerized substrate. We further demonstrate that proton exchange is faster at the 3''-position than the 5''-position, in contrast to a previously studied ortholog. We additionally solved the crystal structures of CBU1838 and StrM in complex with TDP and show that they form an active site highly similar to those of the previously characterized enzymes RmlC, EvaD, and ChmJ. These results support the hypothesis that streptose and DHHS are biosynthesized using the TDP pathway and that an RmlD paralog most likely performs ring contraction following double epimerization. This work will support the elucidation of the full pathways for biosynthesis of these unique sugars.


Assuntos
Antígenos de Bactérias/biossíntese , Carboidratos Epimerases , Coxiella burnetii/enzimologia , Streptomyces griseus/enzimologia , Carboidratos Epimerases/genética , Açúcares de Nucleosídeo Difosfato/biossíntese , Nucleotídeos de Timina/biossíntese
4.
J Am Chem Soc ; 144(47): 21648-21657, 2022 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-36379007

RESUMO

Understanding and controlling peptide foldamer conformation in phospholipid bilayers is a key step toward their use as molecular information relays in membranes. To this end, a new 19F "reporter" tag has been developed and attached to dynamic peptide foldamers. The (R)-1-(trifluoromethyl)ethylamido ((R)-TFEA) reporter was attached to the C-terminus of α-amino-iso-butyric acid (Aib) foldamers. Crystallography confirmed that the foldamers adopted 310 helical conformations. Variable temperature (VT) NMR spectroscopy in organic solvents showed that the (R)-TFEA reporter had an intrinsic preference for P helicity, but the overall screw-sense was dominated by a chiral "controller" at the N-terminus. The 19F NMR chemical shift of the CF3 resonance was correlated with the ability of different N-terminal groups to induce either an M or a P helix in solution. In bilayers, a similar correlation was found. Solution 19F NMR spectroscopy on small unilamellar vesicle (SUV) suspensions containing the same family of (R)-TFEA-labeled foldamers showed broadened but resolvable 19F resonances, with each chemical shift mirroring their relative positions in organic solvents. These studies showed that foldamer conformational preferences are the same in phospholipid bilayers as in organic solvents and also revealed that phospholipid chirality has little influence on conformation.


Assuntos
Aminoácidos , Peptídeos , Modelos Moleculares , Espectroscopia de Ressonância Magnética , Peptídeos/química , Aminoácidos/química , Fosfolipídeos/química , Solventes
5.
J Biol Chem ; 293(24): 9301-9310, 2018 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-29695509

RESUMO

Myelocytomatosis proto-oncogene transcription factor (Myc) is an intrinsically disordered protein with critical roles in cellular homeostasis and neoplastic transformation. It is tightly regulated in the cell, with Myc phosphorylation playing a major role. In addition to the well-described tandem phosphorylation of Thr-52 and Ser-62 in the Myc transactivation domain linked to its degradation, P21 (RAC1)-activated kinase 2 (PAK2)-mediated phosphorylation of serine and threonine residues in the C-terminal basic helix-loop-helix leucine zipper (bHLH-LZ) region regulates Myc transcriptional activity. Here we report that PAK2 preferentially phosphorylates Myc twice, at Thr-358 and Ser-373, with only a minor fraction being modified at the previously identified Thr-400 site. For transcriptional activity, Myc binds E-box DNA elements, requiring its heterodimerization with Myc-associated factor X (Max) via the bHLH-LZ regions. Using isothermal calorimetry (ITC), we found that Myc phosphorylation destabilizes this ternary protein-DNA complex by decreasing Myc's affinity for Max by 2 orders of magnitude, suggesting a major effect of phosphorylation on this complex. Phosphomimetic substitutions revealed that Ser-373 dominates the effect on Myc-Max heterodimerization. Moreover, a T400D substitution disrupted Myc's affinity for Max. ITC, NMR, and CD analyses of several Myc variants suggested that the effect of phosphorylation on the Myc-Max interaction is caused by secondary structure disruption during heterodimerization rather than by a change in the structurally disordered state of Myc or by phosphorylation-induced electrostatic repulsion in the heterodimer. Our findings provide critical insights into the effects of PAK2-catalyzed phosphorylation of Myc on its interactions with Max and DNA.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , DNA/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Sequência de Aminoácidos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/química , Humanos , Modelos Moleculares , Fosforilação , Ligação Proteica , Conformação Proteica , Conformação Proteica em alfa-Hélice , Mapas de Interação de Proteínas , Estabilidade Proteica , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-myc/química
6.
J Biol Chem ; 293(7): 2272-2287, 2018 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-29259125

RESUMO

The UbiD family of reversible decarboxylases act on aromatic, heteroaromatic, and unsaturated aliphatic acids and utilize a prenylated flavin mononucleotide (prFMN) as cofactor, bound adjacent to a conserved Glu-Arg-Glu/Asp ionic network in the enzyme's active site. It is proposed that UbiD activation requires oxidative maturation of the cofactor, for which two distinct isomers, prFMNketimine and prFMNiminium, have been observed. It also has been suggested that only the prFMNiminium form is relevant to catalysis, which requires transient cycloaddition between substrate and cofactor. Using Aspergillus niger Fdc1 as a model system, we reveal that isomerization of prFMNiminium to prFMNketimine is a light-dependent process that is largely independent of the Glu277-Arg173-Glu282 network and accompanied by irreversible loss of activity. On the other hand, efficient catalysis was highly dependent on an intact Glu-Arg-Glu network, as only Glu → Asp substitutions retain activity. Surprisingly, oxidative maturation to form the prFMNiminium species is severely affected only for the R173A variant. In summary, the unusual irreversible isomerization of prFMN is light-dependent and probably proceeds via high-energy intermediates but is independent of the Glu-Arg-Glu network. Our results from mutagenesis, crystallographic, spectroscopic, and kinetic experiments indicate a clear role for the Glu-Arg-Glu network in both catalysis and oxidative maturation.


Assuntos
Aspergillus niger/enzimologia , Carboxiliases/química , Carboxiliases/metabolismo , Mononucleotídeo de Flavina/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Aspergillus niger/química , Aspergillus niger/genética , Sítios de Ligação , Carboxiliases/genética , Catálise , Domínio Catalítico , Sequência Conservada , Mononucleotídeo de Flavina/química , Proteínas Fúngicas/genética , Isomerismo , Cinética , Oxirredução
7.
Proc Natl Acad Sci U S A ; 111(34): 12384-9, 2014 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-25104750

RESUMO

ß-Phosphoglucomutase (ßPGM) catalyzes isomerization of ß-D-glucose 1-phosphate (ßG1P) into D-glucose 6-phosphate (G6P) via sequential phosphoryl transfer steps using a ß-D-glucose 1,6-bisphosphate (ßG16BP) intermediate. Synthetic fluoromethylenephosphonate and methylenephosphonate analogs of ßG1P deliver novel step 1 transition state analog (TSA) complexes for ßPGM, incorporating trifluoromagnesate and tetrafluoroaluminate surrogates of the phosphoryl group. Within an invariant protein conformation, the ß-D-glucopyranose ring in the ßG1P TSA complexes (step 1) is flipped over and shifted relative to the G6P TSA complexes (step 2). Its equatorial hydroxyl groups are hydrogen-bonded directly to the enzyme rather than indirectly via water molecules as in step 2. The (C)O-P bond orientation for binding the phosphate in the inert phosphate site differs by ∼ 30° between steps 1 and 2. By contrast, the orientations for the axial O-Mg-O alignment for the TSA of the phosphoryl group in the catalytic site differ by only ∼ 5°, and the atoms representing the five phosphorus-bonded oxygens in the two transition states (TSs) are virtually superimposable. The conformation of ßG16BP in step 1 does not fit into the same invariant active site for step 2 by simple positional interchange of the phosphates: the TS alignment is achieved by conformational change of the hexose rather than the protein.


Assuntos
Hexoses/química , Hexoses/metabolismo , Organofosfonatos/química , Organofosfonatos/metabolismo , Fosfoglucomutase/química , Fosfoglucomutase/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Catálise , Cristalografia por Raios X , Flúor/química , Glucose-6-Fosfato/química , Glucose-6-Fosfato/metabolismo , Glucofosfatos/química , Glucofosfatos/metabolismo , Isomerismo , Cinética , Lactococcus lactis/enzimologia , Magnésio/química , Modelos Moleculares , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Eletricidade Estática , Termodinâmica
8.
Angew Chem Int Ed Engl ; 56(33): 9732-9735, 2017 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-28498638

RESUMO

We report X-ray crystallographic and 19 F NMR studies of the G-protein RhoA complexed with MgF3- , GDP, and RhoGAP, which has the mutation Arg85'Ala. When combined with DFT calculations, these data permit the identification of changes in transition state (TS) properties. The X-ray data show how Tyr34 maintains solvent exclusion and the core H-bond network in the active site by relocating to replace the missing Arg85' sidechain. The 19 F NMR data show deshielding effects that indicate the main function of Arg85' is electronic polarization of the transferring phosphoryl group, primarily mediated by H-bonding to O3G and thence to PG . DFT calculations identify electron-density redistribution and pinpoint why the TS for guanosine 5'-triphosphate (GTP) hydrolysis is higher in energy when RhoA is complexed with RhoGAPArg85'Ala relative to wild-type (WT) RhoGAP. This study demonstrates that 19 F NMR measurements, in combination with X-ray crystallography and DFT calculations, can reliably dissect the response of small GTPases to site-specific modifications.


Assuntos
Teoria da Densidade Funcional , GTP Fosfo-Hidrolases/genética , Cristalografia por Raios X , Flúor/química , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Mutação
9.
J Biomol NMR ; 62(1): 43-52, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25737243

RESUMO

Spectral resolution in proton NMR spectroscopy is reduced by the splitting of resonances into multiplets due to the effect of homonuclear scalar couplings. Although these effects are often hidden in protein NMR spectroscopy by low digital resolution and routine apodization, behind the scenes homonuclear scalar couplings increase spectral overcrowding. The possibilities for biomolecular NMR offered by new pure shift NMR methods are illustrated here. Both resolution and sensitivity are improved, without any increase in experiment time. In these experiments, free induction decays are collected in short bursts of data acquisition, with durations short on the timescale of J-evolution, interspersed with suitable refocusing elements. The net effect is real-time (t 2) broadband homodecoupling, suppressing the multiplet structure caused by proton-proton interactions. The key feature of the refocusing elements is that they discriminate between the resonances of active (observed) and passive (coupling partner) spins. This can be achieved either by using band-selective refocusing or by the BIRD element, in both cases accompanied by a nonselective 180° proton pulse. The latter method selects the active spins based on their one-bond heteronuclear J-coupling to (15)N, while the former selects a region of the (1)H spectrum. Several novel pure shift experiments are presented, and the improvements in resolution and sensitivity they provide are evaluated for representative samples: the N-terminal domain of PGK; ubiquitin; and two mutants of the small antifungal protein PAF. These new experiments, delivering improved sensitivity and resolution, have the potential to replace the current standard HSQC experiments.


Assuntos
Proteínas Fúngicas/química , Ressonância Magnética Nuclear Biomolecular/métodos , Penicillium chrysogenum/metabolismo , Ubiquitina/química , Antifúngicos/química , Mutação , Isótopos de Nitrogênio/química , Fosfoglicerato Quinase/química , Dobramento de Proteína , Prótons , Sensibilidade e Especificidade
10.
Biomacromolecules ; 16(3): 951-61, 2015 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-25645947

RESUMO

Proteoglycan spatiotemporal organization underpins extracellular matrix biology, but atomic scale glimpses of this microarchitecture are obscured by glycosaminoglycan size and complexity. To overcome this, multimicrosecond aqueous simulations of chondroitin and dermatan sulfates were abstracted into a prior coarse-grained model, which was extended to heterogeneous glycosaminoglycans and small leucine-rich proteoglycans. Exploration of relationships between sequence and shape led to hypotheses that proteoglycan size is dependent on glycosaminoglycan unit composition but independent of sequence permutation. Uronic acid conformational equilibria were modulated by adjacent hexosamine sulfonation and iduronic acid increased glycosaminoglycan chain volume and rigidity, while glucuronic acid imparted chain plasticity. Consequently, block copolymeric glycosaminoglycans contained microarchitectures capable of multivalent binding to growth factors and collagen, with potential for interactional synergy at greater chain number. The described atomic scale views of proteoglycans and heterogeneous glycosaminoglycans provide structural routes to understanding their fundamental signaling and mechanical biological roles and development of new biomaterials.


Assuntos
alfa-Globulinas/química , Sulfatos de Condroitina/química , Decorina/química , Dermatan Sulfato/química , Animais , Configuração de Carboidratos , Cartilagem/química , Bovinos , Humanos , Intestinos/química , Simulação de Dinâmica Molecular , Conformação Proteica , Tubarões , Sus scrofa , Traqueia/química
11.
Proc Natl Acad Sci U S A ; 109(18): 6910-5, 2012 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-22505741

RESUMO

Experimental observations of fluoromagnesate and fluoroaluminate complexes of ß-phosphoglucomutase (ß-PGM) have demonstrated the importance of charge balance in transition-state stabilization for phosphoryl transfer enzymes. Here, direct observations of ground-state analog complexes of ß-PGM involving trifluoroberyllate establish that when the geometry and charge distribution closely match those of the substrate, the distribution of conformers in solution and in the crystal predominantly places the reacting centers in van der Waals proximity. Importantly, two variants are found, both of which satisfy the criteria for near attack conformers. In one variant, the aspartate general base for the reaction is remote from the nucleophile. The nucleophile remains protonated and forms a nonproductive hydrogen bond to the phosphate surrogate. In the other variant, the general base forms a hydrogen bond to the nucleophile that is now correctly orientated for the chemical transfer step. By contrast, in the absence of substrate, the solvent surrounding the phosphate surrogate is arranged to disfavor nucleophilic attack by water. Taken together, the trifluoroberyllate complexes of ß-PGM provide a picture of how the enzyme is able to organize itself for the chemical step in catalysis through the population of intermediates that respond to increasing proximity of the nucleophile. These experimental observations show how the enzyme is capable of stabilizing the reaction pathway toward the transition state and also of minimizing unproductive catalysis of aspartyl phosphate hydrolysis.


Assuntos
Fosfotransferases (Fosfomutases)/química , Fosfotransferases (Fosfomutases)/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Berílio/química , Cristalografia por Raios X , Fluoretos/química , Lactococcus lactis/enzimologia , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Eletricidade Estática , Termodinâmica
12.
Structure ; 32(10): 1834-1846.e3, 2024 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-39106858

RESUMO

Enzymes facilitating the transfer of phosphate groups constitute the most extensive protein families across all kingdoms of life. They make up approximately 10% of the proteins found in the human genome. Understanding the mechanisms by which enzymes catalyze these reactions is essential in characterizing the processes they regulate. Metal fluorides can be used as multifunctional tools to study these enzymes. These ionic species bear the same charge as phosphate and the transferring phosphoryl group and, in addition, allow the enzyme to be trapped in catalytically important states with spectroscopically sensitive atoms interacting directly with active site residues. The ionic nature of these phosphate surrogates also allows their removal and replacement with other analogs. Here, we describe the best practices to obtain these complexes, their use in NMR, X-ray crystallography, cryo-EM, and SAXS and describe a new metal fluoride, scandium tetrafluoride, which has significant anomalous signal using soft X-rays.


Assuntos
Fluoretos , Fluoretos/química , Fluoretos/metabolismo , Cristalografia por Raios X , Microscopia Crioeletrônica , Modelos Moleculares , Domínio Catalítico , Humanos , Espalhamento a Baixo Ângulo , Difração de Raios X , Fosfotransferases/metabolismo , Fosfotransferases/química
13.
Commun Biol ; 7(1): 909, 2024 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-39068257

RESUMO

Metabolic regulation occurs through precise control of enzyme activity. Allomorphy is a post-translational fine control mechanism where the catalytic rate is governed by a conformational switch that shifts the enzyme population between forms with different activities. ß-Phosphoglucomutase (ßPGM) uses allomorphy in the catalysis of isomerisation of ß-glucose 1-phosphate to glucose 6-phosphate via ß-glucose 1,6-bisphosphate. Herein, we describe structural and biophysical approaches to reveal its allomorphic regulatory mechanism. Binding of the full allomorphic activator ß-glucose 1,6-bisphosphate stimulates enzyme closure, progressing through NAC I and NAC III conformers. Prior to phosphoryl transfer, loops positioned on the cap and core domains are brought into close proximity, modulating the environment of a key proline residue. Hence accelerated isomerisation, likely via a twisted anti/C4-endo transition state, leads to the rapid predominance of active cis-P ßPGM. In contrast, binding of the partial allomorphic activator fructose 1,6-bisphosphate arrests ßPGM at a NAC I conformation and phosphoryl transfer to both cis-P ßPGM and trans-P ßPGM occurs slowly. Thus, allomorphy allows a rapid response to changes in food supply while not otherwise impacting substantially on levels of important metabolites.


Assuntos
Domínio Catalítico , Fosfoglucomutase , Prolina , Fosfoglucomutase/metabolismo , Fosfoglucomutase/química , Fosfoglucomutase/genética , Prolina/metabolismo , Prolina/química , Isomerismo , Glucofosfatos/metabolismo , Conformação Proteica , Humanos , Catálise , Modelos Moleculares , Glucose-6-Fosfato/análogos & derivados
14.
Biomol NMR Assign ; 18(1): 33-44, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38472728

RESUMO

The backbone 1H, 13C and 15N resonance assignment of Ubiquitin Specific Protease 7 catalytic domain (residues 208-554) was performed in its complex with a small molecule ligand and in its apo form as a reference. The amide 1H-15N signal intensities were boosted by an amide hydrogen exchange protocol, where expressed 2H, 13C, 15N-labeled protein was unfolded and re-folded to ensure exchange of amide deuterons to protons. The resonance assignments were used to determine chemical shift perturbations on ligand binding, which are consistent with the binding site observed by crystallography.


Assuntos
Domínio Catalítico , Ressonância Magnética Nuclear Biomolecular , Humanos , Ligantes , Isótopos de Nitrogênio
15.
Nat Commun ; 15(1): 2740, 2024 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-38548733

RESUMO

Photoreceptor proteins utilise chromophores to sense light and trigger a biological response. The discovery that adenosylcobalamin (or coenzyme B12) can act as a light-sensing chromophore heralded a new field of B12-photobiology. Although microbial genome analysis indicates that photoactive B12-binding domains form part of more complex protein architectures, regulating a range of molecular-cellular functions in response to light, experimental evidence is lacking. Here we identify and characterise a sub-family of multi-centre photoreceptors, termed photocobilins, that use B12 and biliverdin (BV) to sense light across the visible spectrum. Crystal structures reveal close juxtaposition of the B12 and BV chromophores, an arrangement that facilitates optical coupling. Light-triggered conversion of the B12 affects quaternary structure, in turn leading to light-activation of associated enzyme domains. The apparent widespread nature of photocobilins implies involvement in light regulation of a wider array of biochemical processes, and thus expands the scope for B12 photobiology. Their characterisation provides inspiration for the design of broad-spectrum optogenetic tools and next generation bio-photocatalysts.


Assuntos
Pigmentos Biliares , Fotorreceptores Microbianos , Fotoquímica , Biliverdina , Proteínas de Bactérias/metabolismo , Fotorreceptores Microbianos/química , Luz
16.
Proc Natl Acad Sci U S A ; 107(10): 4555-60, 2010 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-20164409

RESUMO

Prior evidence supporting the direct observation of phosphorane intermediates in enzymatic phosphoryl transfer reactions was based on the interpretation of electron density corresponding to trigonal species bridging the donor and acceptor atoms. Close examination of the crystalline state of beta-phosphoglucomutase, the archetypal phosphorane intermediate-containing enzyme, reveals that the trigonal species is not PO-3 , but is MgF-3 (trifluoromagnesate). Although MgF-3 complexes are transition state analogues rather than phosphoryl group transfer reaction intermediates, the presence of fluorine nuclei in near-transition state conformations offers new opportunities to explore the nature of the interactions, in particular the independent measures of local electrostatic and hydrogen-bonding distributions using 19F NMR. Measurements on three beta-PGM-MgF-3 -sugar phosphate complexes show a remarkable relationship between NMR chemical shifts, primary isotope shifts, NOEs, cross hydrogen bond F...H-N scalar couplings, and the atomic positions determined from the high-resolution crystal structure of the beta-PGM-MgF--3 -G6P complex. The measurements provide independent validation of the structural and isoelectronic MgF--3 model of near-transition state conformations.


Assuntos
Fluoretos/química , Compostos de Magnésio/química , Fosfoglucomutase/química , Fosforanos/química , Domínio Catalítico , Cristalografia por Raios X , Fluoretos/metabolismo , Glucofosfatos/química , Glucofosfatos/metabolismo , Ligação de Hidrogênio , Compostos de Magnésio/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Fosfatos/química , Fosfatos/metabolismo , Fosfoglucomutase/metabolismo , Fosforanos/metabolismo , Ligação Proteica , Conformação Proteica
17.
Angew Chem Int Ed Engl ; 52(44): 11616-9, 2013 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-24014213

RESUMO

BIRD's eye view: Adding periodic BIRD J-refocusing (BIRD=bilinear rotation decoupling) to data acquisition in an HSQC experiment causes broadband homonuclear decoupling, giving a single signal for each proton chemical shift. This pure shift method improves both resolution and signal-to-noise ratio, without the need for special data processing.

18.
ACS Catal ; 12(5): 3149-3164, 2022 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-35692864

RESUMO

Understanding the factors that underpin the enormous catalytic proficiencies of enzymes is fundamental to catalysis and enzyme design. Enzymes are, in part, able to achieve high catalytic proficiencies by utilizing the binding energy derived from nonreacting portions of the substrate. In particular, enzymes with substrates containing a nonreacting phosphodianion group coordinated in a distal site have been suggested to exploit this binding energy primarily to facilitate a conformational change from an open inactive form to a closed active form, rather than to either induce ground state destabilization or stabilize the transition state. However, detailed structural evidence for the model is limited. Here, we use ß-phosphoglucomutase (ßPGM) to investigate the relationship between binding a phosphodianion group in a distal site, the adoption of a closed enzyme form, and catalytic proficiency. ßPGM catalyzes the isomerization of ß-glucose 1-phosphate to glucose 6-phosphate via phosphoryl transfer reactions in the proximal site, while coordinating a phosphodianion group of the substrate(s) in a distal site. ßPGM has one of the largest catalytic proficiencies measured and undergoes significant domain closure during its catalytic cycle. We find that side chain substitution at the distal site results in decreased substrate binding that destabilizes the closed active form but is not sufficient to preclude the adoption of a fully closed, near-transition state conformation. Furthermore, we reveal that binding of a phosphodianion group in the distal site stimulates domain closure even in the absence of a transferring phosphoryl group in the proximal site, explaining the previously reported ß-glucose 1-phosphate inhibition. Finally, our results support a trend whereby enzymes with high catalytic proficiencies involving phosphorylated substrates exhibit a greater requirement to stabilize the closed active form.

19.
J Am Chem Soc ; 133(11): 3989-94, 2011 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-21348513

RESUMO

The direct observation of a transition state analogue (TSA) complex for tyrosine phosphorylation by a signaling kinase has been achieved using (19)F NMR analysis of MEK6 in complex with tetrafluoroaluminate (AlF(4)(-)), ADP, and p38α MAP kinase (acceptor residue: Tyr182). Solvent-induced isotope shifts and chemical shifts for the AlF(4)(-) moiety indicate that two fluorine atoms are coordinated by the two catalytic magnesium ions of the kinase active site, while the two remaining fluorides are liganded by protein residues only. An equivalent, yet distinct, AlF(4)(-) complex involving the alternative acceptor residue in p38α (Thr180) is only observed when the Tyr182 is mutated to phenylalanine. The formation of octahedral AlF(4)(-) species for both acceptor residues, rather than the trigonal bipyramidal AlF(3)(0) previously identified in the only other metal fluoride complex with a protein kinase, shows the requirement of MEK6 for a TSA that is isoelectronic with the migrating phosphoryl group. This requirement has hitherto only been demonstrated for proteins having a single catalytic magnesium ion.


Assuntos
Proteínas Quinases/metabolismo , Compostos de Alumínio/farmacologia , Fluoretos/farmacologia , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Fosforilação , Conformação Proteica , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/química , Especificidade por Substrato
20.
Biomol NMR Assign ; 15(2): 389-395, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34173222

RESUMO

DNA double-strand breaks (DSBs) represent the most cytotoxic DNA lesions, as-if mis- or unrepaired-they can cause cell death or lead to genome instability, which in turn can cause cancer. DSBs are repaired by two major pathways termed homologous recombination and non-homologous end-joining (NHEJ). NHEJ is responsible for repairing the vast majority of DSBs arising in human cells. Defects in NHEJ factors are also associated with microcephaly, primordial dwarfism and immune deficiencies. One of the key proteins important for mediating NHEJ is XRCC4. XRCC4 is a dimer, with the dimer interface mediated by an extended coiled-coil. The N-terminal head domain forms a mixed alpha-beta globular structure. Numerous factors interact with the C-terminus of the coiled-coil domain, which is also associated with significant self-association between XRCC4 dimers. A range of construct lengths of human XRCC4 were expressed and purified, and the 1-164 variant had the best NMR properties, as judged by consistent linewidths, and chemical shift dispersion. In this work we report the 1H, 15 N and 13C backbone resonance assignments of human XRCC4 in the solution form of the 1-164 construct. Assignments were obtained by heteronuclear multidimensional NMR spectroscopy. In total, 156 of 161 assignable residues of XRCC4 were assigned to resonances in the TROSY spectrum, with an additional 11 resonances assigned to His-Tag residues. Prediction of solution secondary structure from a chemical shift analysis using the TALOS + webserver is in good agreement with the published X-ray crystal structures of this protein.


Assuntos
Reparo do DNA por Junção de Extremidades
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA