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1.
Int J Mol Sci ; 25(5)2024 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-38473729

RESUMO

The toxicity of botulinum multi-domain neurotoxins (BoNTs) arises from a sequence of molecular events, in which the translocation of the catalytic domain through the membrane of a neurotransmitter vesicle plays a key role. A recent structural study of the translocation domain of BoNTs suggests that the interaction with the membrane is driven by the transition of an α helical switch towards a ß hairpin. Atomistic simulations in conjunction with the mesoscopic Twister model are used to investigate the consequences of this proposition for the toxin-membrane interaction. The conformational mobilities of the domain, as well as the effect of the membrane, implicitly examined by comparing water and water-ethanol solvents, lead to the conclusion that the transition of the switch modifies the internal dynamics and the effect of membrane hydrophobicity on the whole protein. The central two α helices, helix 1 and helix 2, forming two coiled-coil motifs, are analyzed using the Twister model, in which the initial deformation of the membrane by the protein is caused by the presence of local torques arising from asymmetric positions of hydrophobic residues. Different torque distributions are observed depending on the switch conformations and permit an origin for the mechanism opening the membrane to be proposed.


Assuntos
Toxinas Botulínicas , Humanos , Domínios Proteicos , Domínio Catalítico , Vesícula , Translocação Genética , Água
2.
Int J Mol Sci ; 25(5)2024 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-38473811

RESUMO

Alzheimer's disease (AD) is the leading cause of dementia and is characterized by a presence of amyloid plaques, composed mostly of the amyloid-ß (Aß) peptides, in the brains of AD patients. The peptides are generated from the amyloid precursor protein (APP), which undergoes a sequence of cleavages, referred as trimming, performed by γ-secretase. Here, we investigated conformational changes in a series of ß-amyloid substrates (from less and more amyloidogenic pathways) in the active site of presenilin-1, the catalytic subunit of γ-secretase. The substrates are trimmed every three residues, finally leading to Aß40 and Aß42, which are the major components of amyloid plaques. To study conformational changes, we employed all-atom molecular dynamics simulations, while for unfolding, we used steered molecular dynamics simulations in an implicit membrane-water environment to accelerate changes. We have found substantial differences in the flexibility of extended C-terminal parts between more and less amyloidogenic pathway substrates. We also propose that the positively charged residues of presenilin-1 may facilitate the stretching and unfolding of substrates. The calculated forces and work/energy of pulling were exceptionally high for Aß40, indicating why trimming of this substrate is so infrequent.


Assuntos
Doença de Alzheimer , Secretases da Proteína Precursora do Amiloide , Humanos , Secretases da Proteína Precursora do Amiloide/metabolismo , Presenilina-1/metabolismo , Domínio Catalítico , Placa Amiloide , Peptídeos beta-Amiloides/metabolismo , Doença de Alzheimer/metabolismo
3.
Bioprocess Biosyst Eng ; 47(3): 313-323, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38438572

RESUMO

Molecular docking is an important computational analysis widely used to predict the interaction of enzymes with several starting materials for developing new valuable products from several starting materials, including oils and fats. In the present study, molecular docking was used as an efficient in silico screening tool to select biocatalysts with the highest catalytic performance in butyl esters production in a solvent-free system, an eco-friendly approach, via direct esterification of free fatty acids from Licuri oil with butanol. For such purpose, three commercial lipase preparations were used to perform molecular docking studies such as Burkholderia cepacia (BCL), Porcine pancreatic (PPL), and Candida rugosa (CRL). Concurrently, the results obtained in BCL and CRL are the most efficient in the esterification process due to their higher preference for catalyzing the esterification of lauric acid, the main fatty acid found in the licuri oil composition. Meanwhile, PPL was the least efficient because it preferentially interacts with minor fatty acids. Molecular docking with the experimental results indicated the better performance in the synthesis of esters was BCL. In conclusion, experimental results analysis shows higher enzymatic productivity in esterification reactions of 1294.83 µmol/h.mg, while the CRL and PPL demonstrated the lowest performance (189.87 µmol / h.mg and 23.96 µmol / h.mg, respectively). Thus, molecular docking and experimental results indicate that BCL is a more efficient lipase to produce fatty acids and esters from licuri oil with a high content of lauric acid. In addition, this study also demonstrates the application of molecular docking as an important tool for lipase screening to achieve more sustainable production of butyl esters with a view synthesis of biolubricants.


Assuntos
Ácidos Graxos , Lipase , Animais , Suínos , Lipase/química , Simulação de Acoplamento Molecular , Domínio Catalítico , Ácidos Graxos/química , Esterificação , Ésteres , Ácidos Láuricos , Enzimas Imobilizadas/metabolismo
4.
J Phys Chem B ; 128(10): 2228-2235, 2024 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-38441478

RESUMO

Raman optical activity (ROA) is a chiral sensitive technique to measure the difference in Raman scattering intensity between right and left circularly polarized light. The method has been applied to the study of biological molecules such as proteins, and it is now recognized as a powerful tool for investigating biomolecular structures. We have expanded the capability of this chiroptical technique to colored molecules, such as photoreceptor proteins, by using a near-infrared excitation. A photoreceptor protein contains a light-absorbing chromophore as an active site, and the precise determination of its structure is vital for comprehending the protein's function at the atomic level. In a photoreceptor protein, the protein environment can distort an achiral chromophore into a chiral conformation. ROA spectroscopy offers detailed structural information about the chromophore under physiological conditions. Here we explore recent progress in near-infrared ROA spectroscopy and its application to biological systems.


Assuntos
Proteínas , Análise Espectral Raman , Rotação Ocular , Domínio Catalítico , Proteínas/química , Análise Espectral Raman/métodos
5.
Int J Med Sci ; 21(4): 714-724, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38464839

RESUMO

Sepsis-induced cardiomyopathy (SIC) represents a severe complication of systemic infection, characterized by significant cardiac dysfunction. This study examines the role of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and Inverted Formin 2 (INF2) in the pathogenesis of SIC, focusing on their impact on mitochondrial homeostasis and dynamics. Our research demonstrates that silencing DNA-PKcs alleviates lipopolysaccharide (LPS)-induced cardiomyocyte death and dysfunction. Using HL-1 cardiomyocytes treated with LPS, we observed that DNA-PKcs knockdown notably reverses LPS-induced cytotoxicity, indicating a protective role against cellular damage. This effect is further substantiated by the reduction in caspase-3 and caspase-9 activation, key markers of apoptosis, upon DNA-PKcs knockdown. Besides, our data further reveal that DNA-PKcs knockdown attenuates LPS-induced mitochondrial dysfunction, evidenced by improved ATP production, enhanced activities of mitochondrial respiratory complexes, and preserved mitochondrial membrane potential. Moreover, DNA-PKcs deletion counteracts LPS-induced shifts towards mitochondrial fission, indicating its regulatory influence on mitochondrial dynamics. Conclusively, our research elucidates the intricate interplay between DNA-PKcs and INF2 in the modulation of mitochondrial function and dynamics during sepsis-induced cardiomyopathy. These findings offer new insights into the molecular mechanisms underpinning SIC and suggest potential therapeutic targets for mitigating mitochondrial dysfunction in this critical condition.


Assuntos
Cardiomiopatias , Doenças Mitocondriais , Sepse , Humanos , Proteína Quinase Ativada por DNA/metabolismo , Dinâmica Mitocondrial , Lipopolissacarídeos/toxicidade , Lipopolissacarídeos/metabolismo , Domínio Catalítico , Cardiomiopatias/genética , Miócitos Cardíacos , Sepse/complicações , Sepse/genética , Doenças Mitocondriais/patologia , DNA/efeitos adversos , DNA/metabolismo
6.
PLoS One ; 19(3): e0300035, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38457483

RESUMO

The development of effective drugs targeting the K-Ras oncogene product is a significant focus in anticancer drug development. Despite the lack of successful Ras signaling inhibitors, recent research has identified PDEδ, a KRAS transporter, as a potential target for inhibiting the oncogenic KRAS signaling pathway. This study aims to investigate the interactions between eight K-Ras inhibitors (deltarazine, deltaflexin 1 and 2, and its analogues) and PDEδ to understand their binding modes. The research will utilize computational techniques such as density functional theory (DFT) and molecular electrostatic surface potential (MESP), molecular docking, binding site analyses, molecular dynamic (MD) simulations, electronic structure computations, and predictions of the binding free energy. Molecular dynamic simulations (MD) will be used to predict the binding conformations and pharmacophoric features in the active site of PDEδ for the examined structures. The binding free energies determined using the MMPB(GB)SA method will be compared with the observed potency values of the tested compounds. This computational approach aims to enhance understanding of the PDEδ selective mechanism, which could contribute to the development of novel selective inhibitors for K-Ras signaling.


Assuntos
Simulação de Dinâmica Molecular , Proteínas Proto-Oncogênicas p21(ras) , Simulação de Acoplamento Molecular , Proteínas Proto-Oncogênicas p21(ras)/genética , Sítios de Ligação , Domínio Catalítico
7.
Protein Sci ; 33(4): e4934, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38501460

RESUMO

AlphaFold protein structure database (AlphaFold DB) archives a vast number of predicted models. We conducted systematic data mining against AlphaFold DB and discovered an uncharacterized P-loop NTPase family. The structure of the protein family was surprisingly novel, showing an atypical topology for P-loop NTPases, noticeable twofold symmetry, and two pairs of independent putative active sites. Our findings show that structural data mining is a powerful approach to identifying undiscovered protein families.


Assuntos
Nucleosídeo-Trifosfatase , Proteínas , Nucleosídeo-Trifosfatase/química , Nucleosídeo-Trifosfatase/metabolismo , Proteínas/química , Domínio Catalítico , Proteínas AAA/metabolismo
8.
Protein Sci ; 33(4): e4920, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38501449

RESUMO

L-Asparaginases (ASNases) catalyze the hydrolysis of L-Asn to L-Asp and ammonia. Members of the ASNase family are used as drugs in the treatment of leukemia, as well as in the food industry. The protomers of bacterial ASNases typically contain 300-400 amino acids (typical class 1 ASNases). In contrast, the chain of ASNase from Rhodospirillum rubrum, reported here and referred to as RrA, consists of only 172 amino acid residues. RrA is homologous to the N-terminal domain of typical bacterial class 1 ASNases and exhibits millimolar affinity for L-Asn. In this study, we demonstrate that RrA belongs to a unique family of cytoplasmic, short-chain ASNases (scASNases). These proteins occupy a distinct region in the sequence space, separate from the regions typically assigned to class 1 ASNases. The scASNases are present in approximately 7% of eubacterial species, spanning diverse bacterial lineages. They seem to be significantly enriched in species that encode for more than one class 1 ASNase. Here, we report biochemical, biophysical, and structural properties of RrA, a member of scASNases family. Crystal structures of the wild-type RrA, both with and without bound L-Asp, as well as structures of several RrA mutants, reveal topologically unique tetramers. Moreover, the active site of one protomer is complemented by two residues (Tyr21 and Asn26) from another protomer. Upon closer inspection, these findings clearly outline scASNases as a stand-alone subfamily of ASNases that can catalyze the hydrolysis of L-Asn to L-Asp despite the lack of the C-terminal domain that is present in all ASNases described structurally to date.


Assuntos
Asparaginase , Rhodospirillum rubrum , Asparaginase/química , Rhodospirillum rubrum/genética , Rhodospirillum rubrum/metabolismo , Subunidades Proteicas , Ácido Aspártico , Domínio Catalítico
9.
Eur J Med Chem ; 268: 116282, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38430853

RESUMO

The Son of Sevenless 1 (SOS1) guanine nucleotide exchange factor, prevalent across eukaryotic species, plays a pivotal role in facilitating the attachment of RAS protein to GTP, thereby regulating the activation of intracellular RAS proteins. This regulation is part of a feedback mechanism involving SOS1, which allows both activators and inhibitors of SOS1 to exert control over downstream signaling pathways, demonstrating potential anti-tumor effects. Predominantly, small molecule modulators that target SOS1 focus on a hydrophobic pocket within the CDC25 protein domain. The effectiveness of these modulators largely depends on their ability to interact with specific amino acids, notably Phe890 and Tyr884. This interaction is crucial for influencing the protein-protein interaction (PPI) between RAS and the catalytic domain of SOS1. Currently, most small molecule modulators targeting SOS1 are in the preclinical research phase, with a few advancing to clinical trials. This progression raises safety concerns, making the assurance of drug safety a primary consideration alongside the enhancement of efficacy in the development of SOS1 modulators. This review encapsulates recent advancements in the chemical categorization of SOS1 inhibitors and activators. It delves into the evolution of small molecule modulation targeting SOS1 and offers perspectives on the design of future generations of selective SOS1 small molecule modulators.


Assuntos
Núcleo Familiar , Transdução de Sinais , Descoberta de Drogas , Domínio Catalítico
10.
Proc Natl Acad Sci U S A ; 121(12): e2313513121, 2024 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-38483989

RESUMO

Cooperative interactions between amino acids are critical for protein function. A genetic reflection of cooperativity is epistasis, which is when a change in the amino acid at one position changes the sequence requirements at another position. To assess epistasis within an enzyme active site, we utilized CTX-M ß-lactamase as a model system. CTX-M hydrolyzes ß-lactam antibiotics to provide antibiotic resistance, allowing a simple functional selection for rapid sorting of modified enzymes. We created all pairwise mutations across 17 active site positions in the ß-lactamase enzyme and quantitated the function of variants against two ß-lactam antibiotics using next-generation sequencing. Context-dependent sequence requirements were determined by comparing the antibiotic resistance function of double mutations across the CTX-M active site to their predicted function based on the constituent single mutations, revealing both positive epistasis (synergistic interactions) and negative epistasis (antagonistic interactions) between amino acid substitutions. The resulting trends demonstrate that positive epistasis is present throughout the active site, that epistasis between residues is mediated through substrate interactions, and that residues more tolerant to substitutions serve as generic compensators which are responsible for many cases of positive epistasis. Additionally, we show that a key catalytic residue (Glu166) is amenable to compensatory mutations, and we characterize one such double mutant (E166Y/N170G) that acts by an altered catalytic mechanism. These findings shed light on the unique biochemical factors that drive epistasis within an enzyme active site and will inform enzyme engineering efforts by bridging the gap between amino acid sequence and catalytic function.


Assuntos
Escherichia coli , beta-Lactamases , Escherichia coli/genética , Domínio Catalítico/genética , Mutação , Substituição de Aminoácidos , beta-Lactamases/química
11.
Epigenetics Chromatin ; 17(1): 5, 2024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-38429855

RESUMO

Protein and nucleic acid methylation are important biochemical modifications. In addition to their well-established roles in gene regulation, they also regulate cell signaling, metabolism, and translation. Despite this high biological relevance, little is known about the general regulation of methyltransferase function. Methyltransferases are divided into superfamilies based on structural similarities and further classified into smaller families based on sequence/domain/target similarity. While members within superfamilies differ in substrate specificity, their structurally similar active sites indicate a potential for shared modes of regulation. Growing evidence from one superfamily suggests a common regulatory mode may be through heterooligomerization with other family members. Here, we describe examples of methyltransferase regulation through intrafamily heterooligomerization and discuss how this can be exploited for therapeutic use.


Assuntos
Metiltransferases , Proteínas , Humanos , Metiltransferases/metabolismo , Sequência de Aminoácidos , Metilação , Proteínas/metabolismo , Domínio Catalítico
12.
Plant Mol Biol ; 114(2): 22, 2024 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-38443687

RESUMO

The dynamic interaction of RNA-binding proteins (RBPs) with their target RNAs contributes to the diversity of ribonucleoprotein (RNP) complexes that are involved in a myriad of biological processes. Identifying the RNP components at high resolution and defining their interactions are key to understanding their regulation and function. Expressing fusions between an RBP of interest and an RNA editing enzyme can result in nucleobase changes in target RNAs, representing a recent addition to experimental approaches for profiling RBP/RNA interactions. Here, we have used the MS2 protein/RNA interaction to test four RNA editing proteins for their suitability to detect target RNAs of RBPs in planta. We have established a transient test system for fast and simple quantification of editing events and identified the hyperactive version of the catalytic domain of an adenosine deaminase (hADARcd) as the most suitable editing enzyme. Examining fusions between homologs of polypyrimidine tract binding proteins (PTBs) from Arabidopsis thaliana and hADARcd allowed determining target RNAs with high sensitivity and specificity. Moreover, almost complete editing of a splicing intermediate provided insight into the order of splicing reactions and PTB dependency of this particular splicing event. Addition of sequences for nuclear localisation of the fusion protein increased the editing efficiency, highlighting this approach's potential to identify RBP targets in a compartment-specific manner. Our studies have established the editing-based analysis of interactions between RBPs and their RNA targets in a fast and straightforward assay, offering a new system to study the intricate composition and functions of plant RNPs in vivo.


Assuntos
Arabidopsis , Splicing de RNA , Splicing de RNA/genética , Arabidopsis/genética , Domínio Catalítico , Éxons , RNA
14.
Protein Sci ; 33(4): e4935, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38501462

RESUMO

Flavin-dependent monooxygenases (FMOs) constitute a diverse enzyme family that catalyzes crucial hydroxylation, epoxidation, and Baeyer-Villiger reactions across various metabolic pathways in all domains of life. Due to the intricate nature of this enzyme family's mechanisms, some aspects of their functioning remain unknown. Here, we present the results of molecular dynamics computations, supplemented by a bioinformatics analysis, that clarify the early stages of their catalytic cycle. We have elucidated the intricate binding mechanism of NADPH and L-Orn to a class B monooxygenase, the ornithine hydroxylase from Aspergillus $$ Aspergillus $$ fumigatus $$ fumigatus $$ known as SidA. Our investigation involved a comprehensive characterization of the conformational changes associated with the FAD (Flavin Adenine Dinucleotide) cofactor, transitioning from the out to the in position. Furthermore, we explored the rotational dynamics of the nicotinamide ring of NADPH, shedding light on its role in facilitating FAD reduction, supported by experimental evidence. Finally, we also analyzed the extent of conservation of two Tyr-loops that play critical roles in the process.


Assuntos
Flavina-Adenina Dinucleotídeo , Oxigenases de Função Mista , Oxigenases de Função Mista/química , NADP/química , Oxirredução , Domínio Catalítico , Flavina-Adenina Dinucleotídeo/química
15.
Sci Rep ; 14(1): 3026, 2024 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-38321125

RESUMO

[NiFe]-hydrogenases have a bimetallic NiFe(CN)2CO cofactor in their large, catalytic subunit. The 136 Da Fe(CN)2CO group of this cofactor is preassembled on a distinct HypC-HypD scaffold complex, but the intracellular source of the iron ion is unresolved. Native mass spectrometric analysis of HypCD complexes defined the [4Fe-4S] cluster associated with HypD and identified + 26 to 28 Da and + 136 Da modifications specifically associated with HypC. A HypCC2A variant without the essential conserved N-terminal cysteine residue dissociated from its complex with native HypD lacked all modifications. Native HypC dissociated from HypCD complexes isolated from Escherichia coli strains deleted for the iscS or iscU genes, encoding core components of the Isc iron-sulfur cluster biogenesis machinery, specifically lacked the + 136 Da modification, but this was retained on HypC from suf mutants. The presence or absence of the + 136 Da modification on the HypCD complex correlated with the hydrogenase enzyme activity profiles of the respective mutant strains. Notably, the [4Fe-4S] cluster on HypD was identified in all HypCD complexes analyzed. These results suggest that the iron of the Fe(CN)2CO group on HypCD derives from the Isc machinery, while either the Isc or the Suf machinery can deliver the [4Fe-4S] cluster to HypD.


Assuntos
Proteínas de Escherichia coli , Hidrogenase , Proteínas Ferro-Enxofre , Escherichia coli/genética , Ferro/metabolismo , Proteínas de Escherichia coli/metabolismo , Hidrogenase/metabolismo , Domínio Catalítico , Cisteína/química
16.
Emerg Top Life Sci ; 8(1): 57-60, 2024 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-38323345

RESUMO

Through homeostatic processes, bacterial cells maintain intracytoplasmic metal ions at concentrations which enable the 'correct' metal to be inserted into an enzyme, thereby ensuring function. However, fluctuations in intracytoplasmic metal ion concentrations mean that under different conditions certain enzymes may contain different metals at their active site. This perspective describes examples of such cases and suggests that metalloproteome plasticity may contribute to the dynamic adaptation of pathogens to stresses in the host environment.


Assuntos
Bactérias , Metais , Íons , Homeostase , Domínio Catalítico
17.
J Comput Aided Mol Des ; 38(1): 8, 2024 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-38324213

RESUMO

The Janus kinases (JAK) are crucial targets in drug development for several diseases. However, accounting for the impact of possible structural rearrangements on the binding of different kinase inhibitors is complicated by the extensive conformational variability of their catalytic kinase domain (KD). The dynamic KD contains mainly four prominent mobile structural motifs: the phosphate-binding loop (P-loop), the αC-helix within the N-lobe, the Asp-Phe-Gly (DFG) motif, and the activation loop (A-loop) within the C-lobe. These distinct structural orientations imply a complex signal transmission path for regulating the A-loop's flexibility and conformational preference for optimal JAK function. Nevertheless, the precise dynamical features of the JAK induced by different types of inhibitors still remain elusive. We performed comparative, microsecond-long, Gaussian accelerated molecular dynamics simulations in triplicate of three phosphorylated JAK2 systems: the KD alone, type-I ATP-competitive inhibitor (CI) bound KD in the catalytically active DFG-in conformation, and the type-II inhibitor (AI) bound KD in the catalytically inactive DFG-out conformation. Our results indicate significant conformational variations observed in the A-loop and αC helix motions upon inhibitor binding. Our studies also reveal that the DFG-out inactive conformation is characterized by the closed A-loop rearrangement, open catalytic cleft of N and C-lobe, the outward movement of the αC helix, and open P-loop states. Moreover, the outward positioning of the αC helix impacts the hallmark salt bridge formation between Lys882 and Glu898 in an inactive conformation. Finally, we compared their ligand binding poses and free energy by the MM/PBSA approach. The free energy calculations suggested that the AI's binding affinity is higher than CI against JAK2 due to an increased favorable contribution from the total non-polar interactions and the involvement of the αC helix. Overall, our study provides the structural and energetic insights crucial for developing more promising type I/II JAK2 inhibitors for treating JAK-related diseases.


Assuntos
Janus Quinase 2 , Simulação de Dinâmica Molecular , Domínio Catalítico , Desenvolvimento de Medicamentos
18.
J Am Chem Soc ; 146(6): 3710-3720, 2024 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-38308759

RESUMO

1/2H and 13C hyperfine coupling constants to 5'-deoxyadenosyl (5'-dAdo•) radical trapped within the active site of the radical S-adenosyl-l-methionine (SAM) enzyme, pyruvate formate lyase-activating enzyme (PFL-AE), both in the absence of substrate and the presence of a reactive peptide-model of the PFL substrate, are completely characteristic of a classical organic free radical whose unpaired electron is localized in the 2pπ orbital of the sp2 C5'-carbon (J. Am. Chem. Soc. 2019, 141, 12139-12146). However, prior electron-nuclear double resonance (ENDOR) measurements had indicated that this 5'-dAdo• free radical is never truly "free": tight van der Waals contact with its target partners and active-site residues guide it in carrying out the exquisitely precise, regioselective reactions that are hallmarks of RS enzymes. Here, our understanding of how the active site chaperones 5'-dAdo• is extended through the finding that this apparently unexceptional organic free radical has an anomalous g-tensor and exhibits significant 57Fe, 13C, 15N, and 2H hyperfine couplings to the adjacent, isotopically labeled, methionine-bound [4Fe-4S]2+ cluster cogenerated with 5'-dAdo• during homolytic cleavage of cluster-bound SAM. The origin of the 57Fe couplings through nonbonded radical-cluster contact is illuminated by a formal exchange-coupling model and broken symmetry-density functional theory computations. Incorporation of ENDOR-derived distances from C5'(dAdo•) to labeled-methionine as structural constraints yields a model for active-site positioning of 5'-dAdo• with a short, nonbonded C5'-Fe distance (∼3 Å). This distance involves substantial motion of 5'-dAdo• toward the unique Fe of the [4Fe-4S]2+ cluster upon S-C(5') bond-cleavage, plausibly an initial step toward formation of the Fe-C5' bond of the organometallic complex, Ω, the central intermediate in catalysis by radical-SAM enzymes.


Assuntos
Proteínas Ferro-Enxofre , S-Adenosilmetionina , S-Adenosilmetionina/metabolismo , Metionina , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Domínio Catalítico , Racemetionina , Radicais Livres/química , Proteínas Ferro-Enxofre/química
19.
Sci Adv ; 10(8): eadl1258, 2024 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-38381834

RESUMO

Adrenal Cushing's syndrome is a disease of cortisol hypersecretion often caused by mutations in protein kinase A catalytic subunit (PKAc). Using a personalized medicine screening platform, we discovered a Cushing's driver mutation, PKAc-W196G, in ~20% of patient samples analyzed. Proximity proteomics and photokinetic imaging reveal that PKAcW196G is unexpectedly distinct from other described Cushing's variants, exhibiting retained association with type I regulatory subunits (RI) and their corresponding A kinase anchoring proteins (AKAPs). Molecular dynamics simulations predict that substitution of tryptophan-196 with glycine creates a 653-cubic angstrom cleft between the catalytic core of PKAcW196G and type II regulatory subunits (RII), but only a 395-cubic angstrom cleft with RI. Endocrine measurements show that overexpression of RIα or redistribution of PKAcW196G via AKAP recruitment counteracts stress hormone overproduction. We conclude that a W196G mutation in the kinase catalytic core skews R subunit selectivity and biases AKAP association to drive Cushing's syndrome.


Assuntos
Síndrome de Cushing , Humanos , Síndrome de Cushing/genética , Proteínas de Ancoragem à Quinase A/genética , Proteínas de Ancoragem à Quinase A/metabolismo , Transdução de Sinais , Domínio Catalítico , Viés
20.
J Chem Theory Comput ; 20(5): 1783-1795, 2024 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-38410913

RESUMO

Enzyme design faces challenges related to the implementation of the basic principles that govern the catalytic activity in natural enzymes. In this work, we revisit basic electrostatic concepts that have been shown to explain the origin of enzymatic efficiency like preorganization and reorganization. Using magnitudes such as the electrostatic potential and the electric field generated by the protein, we explain how these concepts work in different enzymes and how they can be used to rationalize the consequences of point mutations. We also discuss examples of protein design in which electrostatic effects have been implemented. For the near future, molecular simulations, coupled with the use of machine learning methods, can be used to implement electrostatics as a guiding principle for enzyme designs.


Assuntos
Proteínas , Eletricidade Estática , Domínio Catalítico
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