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1.
Blood ; 2024 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-39447073

RESUMO

The structure of human coagulation factor XIII (FXIII), a heterotetrameric plasma pro-transglutaminase that covalently crosslinks pre-formed fibrin polymers, remains elusive until today. The heterotetrameric complex is composed of two catalytic FXIII-A and two protective FXIII-B subunits. Structural etiology underlying FXIII deficiency has so far been derived from crystallographic structures, all of which are currently available for the FXIII-A2 homodimer only. Here, we present the cryo-electron microscopy structure of a native, human plasma-derived FXIII-A2B2 complex at 2.4 Å resolution. The structure provides detailed information on FXIII subunit interacting interfaces as the two subunits interact strongly in plasma. The native FXIII-A2B2 complex reveals a pseudo-symmetric heterotetramer of two FXIII-B monomers intercalating with a symmetric FXIII-A2 dimer forming a "crown-like" assembly. The symmetry axes of the A2 and B2 homodimers are twisted relative to each other such that Sushi domain 1 interacts with the catalytic core of the A subunit and Sushi domain 2 with the symmetry related A' subunit and vice versa. We also report four novel mutations in the F13A1 gene encoding the FXIII-A subunit from a cohort of patients with severe FXIII deficiency. Our structure reveals the etiological basis of homozygous and heterozygous pathogenic mutations and explains the conditional dominant negative effects of heterozygous mutations. This atomistic description of complex interfaces is consistent with previous biochemical data and shows a congruence between the structural biochemistry of the FXIII complex and the clinical features of FXIII deficiency.

2.
Immunology ; 171(2): 181-197, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-37885279

RESUMO

Haemolytic disorders, such as sickle cell disease, are accompanied by the release of high amounts of labile heme into the intravascular compartment resulting in the induction of proinflammatory and prothrombotic complications in affected patients. In addition to the relevance of heme-regulated proteins from the complement and blood coagulation systems, activation of the TLR4 signalling pathway by heme was ascribed a crucial role in the progression of these pathological processes. Heme binding to the TLR4-MD2 complex has been proposed recently, however, essential mechanistic information of the processes at the molecular level, such as heme-binding kinetics, the heme-binding capacity and the respective heme-binding sites (HBMs) is still missing. We report the interaction of TLR4, MD2 and the TLR4-MD2 complex with heme and the consequences thereof by employing biochemical, spectroscopic, bioinformatic and physiologically relevant approaches. Heme binding occurs transiently through interaction with up to four HBMs in TLR4, two HBMs in MD2 and at least four HBMs in their complex. Functional studies highlight that mutations of individual HBMs in TLR4 preserve full receptor activation by heme, suggesting that heme interacts with TLR4 through different binding sites independently of MD2. Furthermore, we confirm and extend the major role of TLR4 for heme-mediated cytokine responses in human immune cells.


Assuntos
Transdução de Sinais , Receptor 4 Toll-Like , Humanos , Receptor 4 Toll-Like/metabolismo , Sítios de Ligação , Citocinas/metabolismo , Antígeno 96 de Linfócito/metabolismo , Lipopolissacarídeos
3.
Anal Chem ; 96(10): 4057-4066, 2024 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-38407829

RESUMO

Disulfide bridges in peptides and proteins play an essential role in maintaining their conformation, structural integrity, and consequently function. Despite ongoing efforts, it is still not possible to detect disulfide bonds and the connectivity of multiply bridged peptides directly through a simple and sufficiently validated protein sequencing or peptide mapping method. Partial or complete reduction and chemical cysteine modification are required as initial steps, followed by the application of a proper detection method. Edman degradation (ED) has been used for primary sequence determination but is largely neglected since the establishment of mass spectrometry (MS)-based protein sequencing. Here, we evaluated and thoroughly characterized the phenyl thiohydantoin (PTH) cysteine derivatives PTH-S-methyl cysteine and PTH-S-carbamidomethyl cysteine as bioanalytical standards for cysteine detection and quantification as well as for the elucidation of the disulfide connectivity in peptides by ED. Validation of the established derivatives was performed according to the guidelines of the International Committee of Harmonization on bioanalytical method validation, and their analytical properties were confirmed as reference standards. A series of model peptides was sequenced to test the usability of the PTH-Cys-derivatives as standards, whereas the native disulfide-bonded peptides CCAP-vil, µ-conotoxin KIIIA, and human insulin were used as case studies to determine their disulfide bond connectivity completely independent of MS analysis.


Assuntos
Cisteína , Dissulfetos , Humanos , Cisteína/química , Dissulfetos/química , Peptídeos/química , Proteínas , Sequência de Aminoácidos
4.
Biol Chem ; 405(5): 297-309, 2024 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-38353111

RESUMO

G proteins are interacting partners of G protein-coupled receptors (GPCRs) in eukaryotic cells. Upon G protein activation, the ability of the Gα subunit to exchange GDP for GTP determines the intracellular signal transduction. Although various studies have successfully shown that both Gαs and Gαi have an opposite effect on the intracellular cAMP production, with the latter being commonly described as "more active", the functional analysis of Gαs is a comparably more complicated matter. Additionally, the thorough investigation of the ubiquitously expressed variants of Gαs, Gαs(short) and Gαs(long), is still pending. Since the previous experimental evaluation of the activity and function of the Gαs isoforms is not consistent, the focus was laid on structural investigations to understand the GTPase activity. Herein, we examined recombinant human Gαs by applying an established methodological setup developed for Gαi characterization. The ability for GTP binding was evaluated with fluorescence and fluorescence anisotropy assays, whereas the intrinsic hydrolytic activity of the isoforms was determined by a GTPase assay. Among different nucleotide probes, BODIPY FL GTPγS exhibited the highest binding affinity towards the Gαs subunit. This work provides a deeper understanding of the Gαs subunit and provides novel information concerning the differences between the two protein variants.


Assuntos
Subunidades alfa Gs de Proteínas de Ligação ao GTP , Humanos , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Nucleotídeos de Guanina/metabolismo , Nucleotídeos de Guanina/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Guanosina Trifosfato/metabolismo
5.
J Pept Sci ; 30(6): e3565, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38232955

RESUMO

Bicyclic peptides are important chemical tools that can function, for example, as bioactive ligands switching on/off signaling pathways mediated by guanine nucleotide-binding proteins as bicycles are more broadly applicable. Despite their relevance in medicinal chemistry, the synthesis of such peptides is challenging, and the final yield is highly dependent on the chemical composition and physicochemical properties of the scaffold. We recently discovered novel, state-specific peptide modulators targeting the Gαi protein, namely, GPM-2/GPM-3, by screening a one-bead-two-compound combinatorial library. A more detailed analysis, including sequence alignments and computer-assisted conformational studies based on the hit compounds, revealed the new peptide 10 as a potential macrobicyclic Gαi ligand sharing high sequence similarity to the known Gαi modulators. The Gαs protein was included in this study for comparison and to unravel the criteria for the specificity of modulator binding to Gαi versus Gαs. This work provides in-depth computer-assisted experimental studies for the analysis of novel macrobicyclic, library-derived Gαi protein ligands. The sequence and structural comparison of 10 with the lead compounds GPM-2 and GPM-3 reveals the importance of the size and amino acid composition of one ring of the bicyclic system and suggests features enhancing the binding affinity of the peptides to the Gαi protein.


Assuntos
Desenho de Fármacos , Peptídeos Cíclicos , Peptídeos Cíclicos/química , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/farmacologia , Ligantes , Sequência de Aminoácidos , Humanos , Modelos Moleculares , Ligação Proteica
6.
Anal Chem ; 94(41): 14410-14418, 2022 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-36206384

RESUMO

Gα proteins as part of heterotrimeric G proteins are molecular switches essential for G protein-coupled receptor- mediated intracellular signaling. The role of the Gα subunits has been examined for decades with various guanine nucleotides to elucidate the activation mechanism and Gα protein-dependent signal transduction. Several approaches describe fluorescent ligands mimicking the GTP function, yet lack the efficient estimation of the proteins' GTP binding activity and the fraction of active protein. Herein, we report the development of a reliable fluorescence anisotropy-based method to determine the affinity of ligands at the GTP-binding site and to quantify the fraction of active Gαi1 protein. An advanced bacterial expression protocol was applied to produce active human Gαi1 protein, whose GTP binding capability was determined with novel fluorescently labeled guanine nucleotides acting as high-affinity Gαi1 binders compared to the commonly used BODIPY FL GTPγS. This study thus contributes a new method for future investigations of the characterization of Gαi and other Gα protein subunits, exploring their corresponding signal transduction systems and potential for biomedical applications.


Assuntos
Nucleotídeos de Guanina , Proteínas Heterotriméricas de Ligação ao GTP , Polarização de Fluorescência , Nucleotídeos de Guanina/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Ligantes , Ligação Proteica , Subunidades Proteicas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo
7.
Biol Chem ; 403(11-12): 1055-1066, 2022 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-36043538

RESUMO

Under hemolytic conditions, hemoglobin and subsequently heme are rapidly released, leading to the toxic effects characterizing diseases such as ß-thalassemia and sickle cell disease. Herein, we provide evidence that human hemoglobin can bind heme in a transient fashion via surface-exposed sequence motifs. Following the synthesis of potential heme-binding motifs (HBMs) as peptides, their heme-binding capacity was investigated by UV-vis spectroscopy and ranked according to their binding affinity. Heme binding to human hemoglobin was subsequently studied by UV-vis and surface plasmon resonance (SPR) spectroscopy, revealing a heme-binding affinity in the sub- to micromolar range and a stoichiometry that clearly exceeds a 1:1 ratio. In silico molecular docking and simulation studies confirmed heme binding to the respective motifs in the ß-chain of hemoglobin. Finally, the peroxidase-like activity of hemoglobin and the hemoglobin-heme complex was monitored, which indicated a much higher activity (>1800%) than other heme-peptide/protein complexes reported so far. The present study provides novel insights into the nature of intact hemoglobin concerning its transient interaction with heme, which suggests for the first time potential heme-scavenging properties of the protein at concomitant disassembly and, consequently, a potentiation of hemolysis and related processes.


Assuntos
Heme , Hemoglobinas , Humanos , Simulação de Acoplamento Molecular , Hemoglobinas/química , Hemoglobinas/metabolismo , Heme/química , Hemólise , Análise Espectral , Ligação Proteica
8.
Chembiochem ; 22(5): 861-864, 2021 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-33103835

RESUMO

Janus kinase 2 (JAK2) is the most important signal-transducing tyrosine kinase in erythropoietic precursor cells. Its malfunction drives several myeloproliferative disorders. Heme is a small metal-ion-carrying molecule that is incorporated into hemoglobin in erythroid precursor cells to transport oxygen. In addition, heme is a signaling molecule and regulator of various biochemical processes. Here, we show that heme exposure leads to hyperphosphorylation of JAK2 in a myeloid cancer cell line. Two peptides identified in JAK2 are heme-regulatory motifs and show low-micromolar affinities for heme. These peptides map to the kinase domain of JAK2, which is essential for downstream signaling. We suggest these motifs to be the interaction sites of heme with JAK2, which drive the heme-induced hyperphosphorylation. The results presented herein could facilitate the development of heme-related pharmacological tools to combat myeloproliferative disorders.


Assuntos
Heme/química , Heme/metabolismo , Janus Quinase 2/química , Janus Quinase 2/metabolismo , Leucemia Mieloide/patologia , Tirosina/química , Humanos , Leucemia Mieloide/metabolismo , Fosforilação , Conformação Proteica , Transdução de Sinais , Células Tumorais Cultivadas , Tirosina/metabolismo
9.
Biol Chem ; 402(6): 675-691, 2021 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-33581700

RESUMO

In hemolytic disorders, erythrocyte lysis results in massive release of hemoglobin and, subsequently, toxic heme. Hemopexin is the major protective factor against heme toxicity in human blood and currently considered for therapeutic use. It has been widely accepted that hemopexin binds heme with extraordinarily high affinity of <1 pM in a 1:1 ratio. However, several lines of evidence point to a higher stoichiometry and lower affinity than determined 50 years ago. Here, we re-analyzed these data. SPR and UV/Vis spectroscopy were used to monitor the interaction of heme with the human protein. The heme-binding sites of hemopexin were characterized using hemopexin-derived peptide models and competitive displacement assays. We obtained a KD value of 0.32 ± 0.04 nM and the ratio for the interaction was determined to be 1:1 at low heme concentrations and at least 2:1 (heme:hemopexin) at high concentrations. We were able to identify two yet unknown potential heme-binding sites on hemopexin. Furthermore, molecular modelling with a newly created homology model of human hemopexin suggested a possible recruiting mechanism by which heme could consecutively bind several histidine residues on its way into the binding pocket. Our findings have direct implications for the potential administration of hemopexin in hemolytic disorders.


Assuntos
Heme/química , Hemopexina/química , Humanos , Modelos Moleculares , Espectrofotometria Ultravioleta , Ressonância de Plasmônio de Superfície
10.
Int J Mol Sci ; 22(2)2021 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-33477282

RESUMO

The saliva of blood-sucking leeches contains a plethora of anticoagulant substances. One of these compounds derived from Haementeria ghilianii, the 66mer three-disulfide-bonded peptide tridegin, specifically inhibits the blood coagulation factor FXIIIa. Tridegin represents a potential tool for antithrombotic and thrombolytic therapy. We recently synthesized two-disulfide-bonded tridegin variants, which retained their inhibitory potential. For further lead optimization, however, structure information is required. We thus analyzed the structure of a two-disulfide-bonded tridegin isomer by solution 2D NMR spectroscopy in a combinatory approach with subsequent MD simulations. The isomer was studied using two fragments, i.e., the disulfide-bonded N-terminal (Lys1-Cys37) and the flexible C-terminal part (Arg38-Glu66), which allowed for a simplified, label-free NMR-structure elucidation of the 66mer peptide. The structural information was subsequently used in molecular modeling and docking studies to provide insights into the structure-activity relationships. The present study will prospectively support the development of anticoagulant-therapy-relevant compounds targeting FXIIIa.


Assuntos
Fator XIIIa/antagonistas & inibidores , Espectroscopia de Ressonância Magnética/métodos , Proteínas e Peptídeos Salivares/farmacologia , Sequência de Aminoácidos , Animais , Dissulfetos/química , Fator XIIIa/metabolismo , Fibrinolíticos/farmacologia , Humanos , Isomerismo , Sanguessugas/metabolismo , Imageamento por Ressonância Magnética/métodos , Modelos Moleculares , Simulação de Dinâmica Molecular , Proteínas e Peptídeos Salivares/química , Proteínas e Peptídeos Salivares/metabolismo , Relação Estrutura-Atividade
11.
BMC Bioinformatics ; 21(1): 124, 2020 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-32216745

RESUMO

BACKGROUND: The notion of heme as a regulator of many physiological processes via transient binding to proteins is one that is recently being acknowledged. The broad spectrum of the effects of heme makes it important to identify further heme-regulated proteins to understand physiological and pathological processes. Moreover, several proteins were shown to be functionally regulated by interaction with heme, yet, for some of them the heme-binding site(s) remain unknown. The presented application HeMoQuest enables identification and qualitative evaluation of such heme-binding motifs from protein sequences. RESULTS: We present HeMoQuest, an online interface (http://bit.ly/hemoquest) to algorithms that provide the user with two distinct qualitative benefits. First, our implementation rapidly detects transient heme binding to nonapeptide motifs from protein sequences provided as input. Additionally, the potential of each predicted motif to bind heme is qualitatively gauged by assigning binding affinities predicted by an ensemble learning implementation, trained on experimentally determined binding affinity data. Extensive testing of our implementation on both existing and new manually curated datasets reveal that our method produces an unprecedented level of accuracy (92%) in identifying those residues assigned "heme binding" in all of the datasets used. Next, the machine learning implementation for the prediction and qualitative assignment of binding affinities to the predicted motifs achieved 71% accuracy on our data. CONCLUSIONS: Heme plays a crucial role as a regulatory molecule exerting functional consequences via transient binding to surfaces of target proteins. HeMoQuest is designed to address this imperative need for a computational approach that enables rapid detection of heme-binding motifs from protein datasets. While most existing implementations attempt to predict sites of permanent heme binding, this application is to the best of our knowledge, the first of its kind to address the significance of predicting transient heme binding to proteins.


Assuntos
Motivos de Aminoácidos , Heme/metabolismo , Software , Algoritmos , Sítios de Ligação , Internet , Aprendizado de Máquina , Ligação Proteica , Análise de Sequência de Proteína
12.
Anal Chem ; 92(16): 10920-10924, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32806900

RESUMO

Disulfide bonds within cysteine-rich peptides are important for their stability and biological function. In this respect, the correct disulfide connectivity plays a decisive role. The differentiation of individual disulfide-bonded isomers by traditional high-performance liquid chromatography (HPLC) and mass spectrometry (MS) is limited due to the similarity in physicochemical properties of the isomers sharing the same amino acid sequence. By using trapped ion mobility spectrometry-mass spectrometry (TIMS-MS), several 2- and 3-disulfide-bonded isomers of the µ-conotoxin PIIIA were investigated for their distinguishability by collision cross section (CCS) values and their characteristic mobilogram traces. The isomers could be differentiated by TIMS-MS and also identified in mixing experiments. Thus, TIMS-MS provides a highly valuable and enriching addition to standard HPLC and MS analysis of conformational isomers of disulfide-rich peptides and proteins.


Assuntos
Conotoxinas/análise , Dissulfetos/análise , Sequência de Aminoácidos , Cromatografia Líquida , Conotoxinas/química , Dissulfetos/química , Espectrometria de Mobilidade Iônica , Isomerismo , Espectrometria de Massas/métodos
13.
Anal Chem ; 92(14): 9429-9440, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32490668

RESUMO

Many research institutions, clinical diagnostic laboratories, and blood banks are desperately searching for a possibility to identify and quantify heme in different physiological and pathological settings as well as various research applications. The reasons for this are the toxicity of the heme and the fact that it acts as a hemolytic and pro-inflammatory molecule. Heme only exerts these severe and undesired effects when it is not incorporated in hemoproteins. Upon release from the hemoproteins, it enters a biologically available state (labile heme), in which it is loosely associated with proteins, lipids, nucleic acids, or other molecules. While the current methods and procedures for quantitative determination of heme have been used for many years in different settings, their value is limited by the challenging chemical properties of heme. A major cause of inadequate quantification is the separation of labile and permanently bound heme and its high aggregation potential. Thus, none of the current methods are utilized as a generally applicable, standardized approach. The aim of this Feature is to describe and summarize the most common and frequently used chemical, analytical, and biochemical methods for the quantitative determination of heme. Based on this overview, the most promising approaches for future solutions to heme quantification are highlighted.


Assuntos
Cromatografia/métodos , Eletroforese Capilar/métodos , Ensaios Enzimáticos/métodos , Heme/química , Hemeproteínas/química , Humanos , Estrutura Molecular , Análise Espectral
14.
Anal Biochem ; 605: 113708, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32335064

RESUMO

The blood coagulation factor XIII (FXIII) plays an essential role in the stabilization of fibrin clots. This factor, belonging to the class of transglutaminases, catalyzes the final step of secondary hemostasis, i.e. the crosslinking of fibrin polymers. These crosslinks protect the clots against premature fibrinolysis. Consequently, FXIII is an interesting target for the therapeutic treatment of cardiovascular diseases. In this context, inhibitors can influence FXIII in the activation process of the enzyme itself or in its catalytic activity. To date, there is no FXIII inhibitor in medical application, but several studies have been conducted in the past. These studies provided a better understanding of FXIII and identified new lead structures for FXIII inhibitors. Next to small molecule inhibitors, the most promising candidates for the development of clinically applicable FXIII inhibitors are the peptide inhibitors tridegin and transglutaminase-inhibiting Michael acceptors (TIMAs) due to their selectivity towards activated FXIII (FXIIIa). In this review, select FXIII inhibitors and their pharmacological potential are discussed.


Assuntos
Coagulação Sanguínea , Doenças Cardiovasculares/sangue , Inibidores Enzimáticos , Fator XIIIa/antagonistas & inibidores , Animais , Sítios de Ligação , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Fibrina/metabolismo , Humanos , Ligação Proteica , Proteínas e Peptídeos Salivares/química , Proteínas e Peptídeos Salivares/metabolismo
15.
Mar Drugs ; 17(7)2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31269696

RESUMO

Cyclic µ-conotoxin PIIIA, a potent blocker of skeletal muscle voltage-gated sodium channel NaV1.4, is a 22mer peptide stabilized by three disulfide bonds. Combining electrophysiological measurements with molecular docking and dynamic simulations based on NMR solution structures, we investigated the 15 possible 3-disulfide-bonded isomers of µ-PIIIA to relate their blocking activity at NaV1.4 to their disulfide connectivity. In addition, three µ-PIIIA mutants derived from the native disulfide isomer, in which one of the disulfide bonds was omitted (C4-16, C5-C21, C11-C22), were generated using a targeted protecting group strategy and tested using the aforementioned methods. The 3-disulfide-bonded isomers had a range of different conformational stabilities, with highly unstructured, flexible conformations with low or no channel-blocking activity, while more constrained molecules preserved 30% to 50% of the native isomer's activity. This emphasizes the importance and direct link between correct fold and function. The elimination of one disulfide bond resulted in a significant loss of blocking activity at NaV1.4, highlighting the importance of the 3-disulfide-bonded architecture for µ-PIIIA. µ-PIIIA bioactivity is governed by a subtle interplay between an optimally folded structure resulting from a specific disulfide connectivity and the electrostatic potential of the conformational ensemble.


Assuntos
Conotoxinas/farmacocinética , Canal de Sódio Disparado por Voltagem NAV1.4/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Conotoxinas/química , Dissulfetos/química , Isomerismo , Simulação de Acoplamento Molecular , Conformação Proteica , Eletricidade Estática , Relação Estrutura-Atividade , Bloqueadores do Canal de Sódio Disparado por Voltagem/química
16.
Anal Chem ; 90(5): 3321-3327, 2018 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-29397705

RESUMO

Peptides and proteins carrying high numbers of cysteines can adopt various 3D structures depending on their disulfide connectivities. The unambiguous verification of such conformational isomers with more than two disulfide bonds is extremely challenging, and experimental strategies for their unequivocal structural analysis are largely lacking. We synthesized all 15 possible isomers of the 22mer conopeptide µ-PIIIA and applied 2D NMR spectroscopy and MS/MS for the elucidation of its structure. This study provides intriguing insights in how the disulfide connectivity alters the global fold of a toxin. We also show that analysis procedures involving comprehensive combinations of conventional methods are required for the unambiguous assignment of disulfides in cysteine-rich peptides and proteins and that standard compounds are crucially needed for the structural analysis of such complex molecules.

18.
Biochim Biophys Acta Gen Subj ; 1862(9): 1964-1972, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29908817

RESUMO

BACKGROUND: Tight regulation of heme homeostasis is a critical mechanism in pathogenic bacteria since heme functions as iron source and prosthetic group, but is also toxic at elevated concentrations. Hemolysin-activating lysine-acyltransferase (HlyC) from Escherichia coli is crucial for maturation of hemolysin A, which lyses several mammalian cells including erythrocytes liberating large amounts of heme for bacterial uptake. A possible impact and functional consequences of the released heme on events employing bacterial HlyC have remained unexplored. METHODS: Heme binding to HlyC was investigated using UV/vis and SPR spectroscopy. Functional impact of heme association was examined using an in vitro hemolysis assay. The interaction was further studied by homology modeling, molecular docking and dynamics simulations. RESULTS: We identified HlyC as potential heme-binding protein possessing heme-regulatory motifs. Using wild-type protein and a double alanine mutant we demonstrated that heme binds to HlyC via histidine 151 (H151). We could show further that heme inhibits the enzymatic activity of wild-type HlyC. Computational studies illustrated potential interaction sites in addition to H151 confirming the results from spectroscopy indicating more than one heme-binding site. CONCLUSIONS: Taken together, our results reveal novel insights into heme-protein interactions and regulation of a component of the heme uptake system in one of the major causative agents of urinary tract infections in humans. GENERAL SIGNIFICANCE: This study points to a possible novel mechanism of regulation as present in many uropathogenic E. coli strains at an early stage of heme iron acquisition from erythrocytes for subsequent internalization by the bacterial heme-uptake machinery.


Assuntos
Aciltransferases/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Heme/metabolismo , Proteínas Hemolisinas/metabolismo , Lisina Acetiltransferases/metabolismo , Aciltransferases/química , Animais , Sítios de Ligação , Eritrócitos/metabolismo , Proteínas de Escherichia coli/química , Heme/química , Proteínas Hemolisinas/química , Hemólise , Lisina Acetiltransferases/química , Ovinos
19.
Biochim Biophys Acta ; 1860(6): 1343-53, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27015758

RESUMO

BACKGROUND: The occurrence of free organismal heme can either contribute to serious diseases or beneficially regulate important physiological processes. Research on transient binding to heme-regulatory motifs (HRMs) in proteins resulted in the discovery of numerous Cys-based, especially Cys-Pro (CP)-based motifs. However, the number of His- and Tyr-based protein representatives is comparatively low so far, which is in part caused by a lack of information regarding recognition and binding requirements. METHODS: To understand transient heme association with such motifs on the molecular level, we analyzed a set of 44 His- and Tyr-based peptides using UV-vis, resonance Raman, cw-EPR and 2D NMR spectroscopy. RESULTS: We observed similarities with Cys-based sequences with respect to their spectral behavior and complex geometries. However, significant differences regarding heme-binding affinities and sequence requirements were also found. Compared to Cys-based peptides and proteins all sequences investigated structurally display increased flexibility already in the free-state, which is also maintained upon heme association. The acquired knowledge allowed for identification and prediction of a His-based HRM in chloramphenicol acetyltransferase from Escherichia coli as potential heme-regulated protein. The enzyme's heme-interacting capability was studied, and revealed an inhibitory effect of heme on the protein activity with an IC50 value of 57.69±4.37 µM. CONCLUSIONS: It was found that heme inhibits a bacterial protein carrying a potential His-based HRM. This finding brings microbial proteins more into focus of regulation by free heme. GENERAL SIGNIFICANCE: Understanding transient binding and regulatory action of heme with bacterial proteins, being crucial for survival, might promote new strategies for the treatment of bacterial infections.


Assuntos
Cloranfenicol O-Acetiltransferase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Escherichia coli/enzimologia , Heme/farmacologia , Motivos de Aminoácidos , Cloranfenicol O-Acetiltransferase/química , Espectroscopia de Ressonância de Spin Eletrônica , Histidina , Espectroscopia de Ressonância Magnética , Análise Espectral Raman , Tirosina
20.
Biochem Biophys Res Commun ; 482(4): 1135-1140, 2017 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-27916464

RESUMO

The neurotoxic cone snail peptide µ-GIIIA specifically blocks skeletal muscle voltage-gated sodium (NaV1.4) channels. The related conopeptides µ-PIIIA and µ-SIIIA, however, exhibit a wider activity spectrum by also inhibiting the neuronal NaV channels NaV1.2 and NaV1.7. Here we demonstrate that those µ-conopeptides with a broader target range also antagonize select subtypes of voltage-gated potassium channels of the KV1 family: µ-PIIIA and µ-SIIIA inhibited KV1.1 and KV1.6 channels in the nanomolar range, while being inactive on subtypes KV1.2-1.5 and KV2.1. Construction and electrophysiological evaluation of chimeras between KV1.5 and KV1.6 revealed that these toxins block KV channels involving their pore regions; the subtype specificity is determined in part by the sequence close to the selectivity filter but predominantly by the so-called turret domain, i.e. the extracellular loop connecting the pore with transmembrane segment S5. Conopeptides µ-SIIIA and µ-PIIIA, thus, are not specific for NaV channels, and the known structure of some KV channel subtypes may provide access to structural insight into the molecular interaction between µ-conopeptides and their target channels.


Assuntos
Conotoxinas/química , Canal de Potássio Kv1.1/antagonistas & inibidores , Canal de Potássio Kv1.2/antagonistas & inibidores , Canal de Potássio Kv1.4/antagonistas & inibidores , Canal de Potássio Kv1.6/antagonistas & inibidores , Bloqueadores dos Canais de Potássio/química , Superfamília Shaker de Canais de Potássio/antagonistas & inibidores , Eletrofisiologia , Células HEK293 , Humanos , Neurônios/metabolismo , Peptídeos/química , Domínios Proteicos
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