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1.
J Biol Chem ; 298(10): 102472, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36089066

RESUMO

The membrane-bound complex II family of proteins is composed of enzymes that catalyze succinate and fumarate interconversion coupled with reduction or oxidation of quinones within the membrane domain. The majority of complex II enzymes are protein heterotetramers with the different subunits harboring a variety of redox centers. These redox centers are used to transfer electrons between the site of succinate-fumarate oxidation/reduction and the membrane domain harboring the quinone. A covalently bound FAD cofactor is present in the flavoprotein subunit, and the covalent flavin linkage is absolutely required to enable the enzyme to oxidize succinate. Assembly of the covalent flavin linkage in eukaryotic cells and many bacteria requires additional protein assembly factors. Here, we provide mechanistic details for how the assembly factors work to enhance covalent flavinylation. Both prokaryotic SdhE and mammalian SDHAF2 enhance FAD binding to their respective apoprotein of complex II. These assembly factors also increase the affinity for dicarboxylates to the apoprotein-noncovalent FAD complex and stabilize the preassembly complex. These findings are corroborated by previous investigations of the roles of SdhE in enhancing covalent flavinylation in both bacterial succinate dehydrogenase and fumarate reductase flavoprotein subunits and of SDHAF2 in performing the same function for the human mitochondrial succinate dehydrogenase flavoprotein. In conclusion, we provide further insight into assembly factor involvement in building complex II flavoprotein subunit active site required for succinate oxidation.


Assuntos
Flavoproteínas , Succinato Desidrogenase , Humanos , Succinato Desidrogenase/metabolismo , Flavoproteínas/química , Flavina-Adenina Dinucleotídeo/metabolismo , Flavinas/metabolismo , Ácido Succínico , Apoproteínas/metabolismo , Fumaratos
2.
Int J Mol Sci ; 23(15)2022 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-35955810

RESUMO

Arrestins were first discovered as suppressors of G protein-mediated signaling by G protein-coupled receptors. It was later demonstrated that arrestins also initiate several signaling branches, including mitogen-activated protein kinase cascades. Arrestin-3-dependent activation of the JNK family can be recapitulated with peptide fragments, which are monofunctional elements distilled from this multi-functional arrestin protein. Here, we use maltose-binding protein fusions of arrestin-3-derived peptides to identify arrestin elements that bind kinases of the ASK1-MKK4/7-JNK3 cascade and the shortest peptide facilitating JNK signaling. We identified a 16-residue arrestin-3-derived peptide expressed as a Venus fusion that leads to activation of JNK3α2 in cells. The strength of the binding to the kinases does not correlate with peptide activity. The ASK1-MKK4/7-JNK3 cascade has been implicated in neuronal apoptosis. While inhibitors of MAP kinases exist, short peptides are the first small molecule tools that can activate MAP kinases.


Assuntos
Arrestina , Proteína Quinase 10 Ativada por Mitógeno , Arrestina/metabolismo , Arrestinas/metabolismo , Proteína Quinase 10 Ativada por Mitógeno/metabolismo , Peptídeos/metabolismo , Peptídeos/farmacologia , Fosforilação/fisiologia , Ligação Proteica/fisiologia , beta-Arrestina 2/metabolismo , beta-Arrestinas/metabolismo
3.
Nat Commun ; 12(1): 4070, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34210959

RESUMO

Mucins are a large family of heavily O-glycosylated proteins that cover all mucosal surfaces and constitute the major macromolecules in most body fluids. Mucins are primarily defined by their variable tandem repeat (TR) domains that are densely decorated with different O-glycan structures in distinct patterns, and these arguably convey much of the informational content of mucins. Here, we develop a cell-based platform for the display and production of human TR O-glycodomains (~200 amino acids) with tunable structures and patterns of O-glycans using membrane-bound and secreted reporters expressed in glycoengineered HEK293 cells. Availability of defined mucin TR O-glycodomains advances experimental studies into the versatile role of mucins at the interface with pathogenic microorganisms and the microbiome, and sparks new strategies for molecular dissection of specific roles of adhesins, glycoside hydrolases, glycopeptidases, viruses and other interactions with mucin TRs as highlighted by examples.


Assuntos
Mucinas/metabolismo , Mucosa/metabolismo , Polissacarídeos/genética , Polissacarídeos/metabolismo , Engenharia Genética , Glicosilação , Células HEK293 , Humanos , Microbiota , Mucina-1/genética , Mucina-1/metabolismo
4.
Biochem J ; 477(19): 3695-3707, 2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-32910185

RESUMO

Infective endocarditis (IE) is a cardiovascular disease often caused by bacteria of the viridans group of streptococci, which includes Streptococcus gordonii and Streptococcus sanguinis. Previous research has found that serine-rich repeat (SRR) proteins on the S. gordonii bacterial surface play a critical role in pathogenesis by facilitating bacterial attachment to sialylated glycans displayed on human platelets. Despite their important role in disease progression, there are currently no anti-adhesive drugs available on the market. Here, we performed structure-based virtual screening using an ensemble docking approach followed by consensus scoring to identify novel small molecule effectors against the sialoglycan binding domain of the SRR adhesin protein Hsa from the S. gordonii strain DL1. The screening successfully predicted nine compounds which were able to displace the native ligand (sialyl-T antigen) in an in vitro assay and bind competitively to Hsa. Furthermore, hierarchical clustering based on the MACCS fingerprints showed that eight of these small molecules do not share a common scaffold with the native ligand. This study indicates that SRR family of adhesin proteins can be inhibited by diverse small molecules and thus prevent the interaction of the protein with the sialoglycans. This opens new avenues for discovering potential drugs against IE.


Assuntos
Adesinas Bacterianas/química , Antibacterianos/química , Hemaglutininas Virais/química , Streptococcus gordonii/química , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Hemaglutininas Virais/genética , Hemaglutininas Virais/metabolismo , Domínios Proteicos , Streptococcus gordonii/genética , Streptococcus gordonii/metabolismo
5.
ACS Chem Biol ; 14(6): 1183-1194, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31058487

RESUMO

Extracellular signal-regulated kinases (ERK1/2) are mitogen-activated protein kinases (MAPKs) that play a pro-tumorigenic role in numerous cancers. ERK1/2 possess two protein-docking sites that are distinct from the active site: the D-recruitment site (DRS) and the F-recruitment site. These docking sites facilitate substrate recognition, intracellular localization, signaling specificity, and protein complex assembly. Targeting these sites on ERK in a therapeutic context may overcome many problems associated with traditional ATP-competitive inhibitors. Here, we identified a new class of inhibitors that target the ERK DRS by screening a synthetic combinatorial library of more than 30 million compounds. The screen detects the competitive displacement of a fluorescent peptide from the DRS of ERK2. The top molecular scaffold from the screen was optimized for structure-activity relationship by positional scanning of different functional groups. This resulted in 10 compounds with similar binding affinities and a shared core structure consisting of a tertiary amine hub with three functionalized cyclic guanidino branches. Compound 2507-1 inhibited ERK2 from phosphorylating a DRS-targeting substrate and prevented the phosphorylation of ERK2 by a constitutively active MEK1 (MAPK/ERK kinase 1) mutant. Interaction between an analogue, 2507-8, and the ERK2 DRS was confirmed by nuclear magnetic resonance and X-ray crystallography. 2507-8 forms critical interactions at the common docking domain residue Asp319 via an arginine-like moiety that is shared by all 10 hits, suggesting a common binding mode. The structural and biochemical insights reported here provide the basis for developing new ERK inhibitors that are not ATP-competitive but instead function by disrupting critical protein-protein interactions.


Assuntos
Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Ativação Enzimática , Guanidina/farmacologia , Humanos , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/química , Ressonância Magnética Nuclear Biomolecular , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Especificidade por Substrato
6.
Trends Biochem Sci ; 43(6): 412-423, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29636212

RESUMO

Arrestins are a small family of proteins with four isoforms in humans. Remarkably, two arrestins regulate signaling from >800 G protein-coupled receptors (GPCRs) or nonreceptor activators by simultaneously binding an activator and one out of hundreds of other signaling proteins. When arrestins are bound to GPCRs or other activators, the affinity for these signaling partners changes. Thus, it is proposed that an activator alters arrestin's ability to transduce a signal. The comparison of all available arrestin structures identifies several common conformational rearrangements associated with activation. In particular, it identifies elements that are directly involved in binding to GPCRs or other activators, elements that likely engage distinct downstream effectors, and elements that likely link the activator-binding sites with the effector-binding sites.


Assuntos
Arrestina/química , Arrestina/metabolismo , Transdução de Sinais , Animais , Humanos , Modelos Moleculares
7.
Cell Signal ; 36: 98-107, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28461104

RESUMO

Non-visual arrestins interact with hundreds of different G protein-coupled receptors (GPCRs). Here we show that by introducing mutations into elements that directly bind receptors, the specificity of arrestin-3 can be altered. Several mutations in the two parts of the central "crest" of the arrestin molecule, middle-loop and C-loop, enhanced or reduced arrestin-3 interactions with several GPCRs in receptor subtype and functional state-specific manner. For example, the Lys139Ile substitution in the middle-loop dramatically enhanced the binding to inactive M2 muscarinic receptor, so that agonist activation of the M2 did not further increase arrestin-3 binding. Thus, the Lys139Ile mutation made arrestin-3 essentially an activation-independent binding partner of M2, whereas its interactions with other receptors, including the ß2-adrenergic receptor and the D1 and D2 dopamine receptors, retained normal activation dependence. In contrast, the Ala248Val mutation enhanced agonist-induced arrestin-3 binding to the ß2-adrenergic and D2 dopamine receptors, while reducing its interaction with the D1 dopamine receptor. These mutations represent the first example of altering arrestin specificity via enhancement of the arrestin-receptor interactions rather than selective reduction of the binding to certain subtypes.


Assuntos
Arrestinas/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Sequência de Aminoácidos , Animais , Arrestinas/química , Células COS , Bovinos , Chlorocebus aethiops , Sequência Conservada , Células HEK293 , Humanos , Lisina/metabolismo , Proteínas Mutantes/metabolismo , Mutação/genética , Ligação Proteica , Estrutura Secundária de Proteína , Rodopsina/metabolismo
8.
Glycobiology ; 26(11): 1222-1234, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27037304

RESUMO

Serine-rich repeat glycoproteins are adhesins expressed by commensal and pathogenic Gram-positive bacteria. A subset of these adhesins, expressed by oral streptococci, binds sialylated glycans decorating human salivary mucin MG2/MUC7, and platelet glycoprotein GPIb. Specific sialoglycan targets were previously identified for the ligand-binding regions (BRs) of GspB and Hsa, two serine-rich repeat glycoproteins expressed by Streptococcus gordonii While GspB selectively binds sialyl-T antigen, Hsa displays broader specificity. Here we examine the binding properties of four additional BRs from Streptococcus sanguinis or Streptococcus mitis and characterize the molecular determinants of ligand selectivity and affinity. Each BR has two domains that are essential for sialoglycan binding by GspB. One domain is structurally similar to the glycan-binding module of mammalian Siglecs (sialic acid-binding immunoglobulin-like lectins), including an arginine residue that is critical for glycan recognition, and that resides within a novel, conserved YTRY motif. Despite low sequence similarity to GspB, one of the BRs selectively binds sialyl-T antigen. Although the other three BRs are highly similar to Hsa, each displayed a unique ligand repertoire, including differential recognition of sialyl Lewis antigens and sulfated glycans. These differences in glycan selectivity were closely associated with differential binding to salivary and platelet glycoproteins. Specificity of sialoglycan adherence is likely an evolving trait that may influence the propensity of streptococci expressing Siglec-like adhesins to cause infective endocarditis.


Assuntos
Glicoproteínas/química , Polissacarídeos/análise , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/química , Ácidos Siálicos/análise , Streptococcus/química , Humanos , Ligantes
9.
Sci Rep ; 6: 21025, 2016 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-26868142

RESUMO

Three-kinase mitogen-activated protein kinase (MAPK) signaling cascades are present in virtually all eukaryotic cells. MAPK cascades are organized by scaffold proteins, which assemble cognate kinases into productive signaling complexes. Arrestin-3 facilitates JNK activation in cells, and a short 25-residue arrestin-3 peptide was identified as the critical JNK3-binding element. Here we demonstrate that this peptide also binds MKK4, MKK7, and ASK1, which are upstream JNK3-activating kinases. This peptide is sufficient to enhance JNK3 activity in cells. A homologous arrestin-2 peptide, which differs only in four positions, binds MKK4, but not MKK7 or JNK3, and is ineffective in cells at enhancing activation of JNK3. The arrestin-3 peptide is the smallest MAPK scaffold known. This peptide or its mimics can regulate MAPKs, affecting cellular decisions to live or die.


Assuntos
Ativadores de Enzimas , Proteína Quinase 10 Ativada por Mitógeno/metabolismo , Peptídeos , beta-Arrestina 1/química , beta-Arrestina 2/química , Animais , Células COS , Chlorocebus aethiops , Ativação Enzimática/efeitos dos fármacos , Ativadores de Enzimas/síntese química , Ativadores de Enzimas/química , Ativadores de Enzimas/farmacologia , Humanos , Proteína Quinase 10 Ativada por Mitógeno/genética , Peptídeos/síntese química , Peptídeos/química , Peptídeos/farmacologia
10.
J Biol Chem ; 291(6): 2904-16, 2016 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-26644464

RESUMO

Escherichia coli harbors two highly conserved homologs of the essential mitochondrial respiratory complex II (succinate:ubiquinone oxidoreductase). Aerobically the bacterium synthesizes succinate:quinone reductase as part of its respiratory chain, whereas under microaerophilic conditions, the quinol:fumarate reductase can be utilized. All complex II enzymes harbor a covalently bound FAD co-factor that is essential for their ability to oxidize succinate. In eukaryotes and many bacteria, assembly of the covalent flavin linkage is facilitated by a small protein assembly factor, termed SdhE in E. coli. How SdhE assists with formation of the covalent flavin bond and how it binds the flavoprotein subunit of complex II remain unknown. Using photo-cross-linking, we report the interaction site between the flavoprotein of complex II and the SdhE assembly factor. These data indicate that SdhE binds to the flavoprotein between two independently folded domains and that this binding mode likely influences the interdomain orientation. In so doing, SdhE likely orients amino acid residues near the dicarboxylate and FAD binding site, which facilitates formation of the covalent flavin linkage. These studies identify how the conserved SdhE assembly factor and its homologs participate in complex II maturation.


Assuntos
Complexo II de Transporte de Elétrons/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Complexo II de Transporte de Elétrons/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Flavina-Adenina Dinucleotídeo/genética
11.
Curr Protoc Pharmacol ; 67: 2.11.1-2.11.19, 2014 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-25446290

RESUMO

Purified arrestin proteins are necessary for biochemical, biophysical, and crystallographic studies of these versatile regulators of cell signaling. Described herein is a basic protocol for arrestin expression in E. coli and purification of the tag-free wild-type and mutant arrestins. The method includes ammonium sulfate precipitation of arrestins from cell lysates, followed by heparin-Sepharose chromatography. Depending on the arrestin type and/or mutations, the next step is Q-Sepharose or SP-Sepharose chromatography. In many cases the nonbinding column is used as a filter to bind contaminants without retaining arrestin. In some cases both chromatographic steps must be performed sequentially to achieve high purity. Purified arrestins can be concentrated up to 10 mg/ml, remain fully functional, and withstand several cycles of freezing and thawing, provided that overall salt concentration is maintained at or above physiological levels.


Assuntos
Arrestinas/biossíntese , Proteínas de Escherichia coli/biossíntese , Escherichia coli/metabolismo , Arrestinas/isolamento & purificação , Cromatografia em Agarose , Proteínas de Escherichia coli/isolamento & purificação
12.
J Biol Chem ; 289(35): 24475-87, 2014 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-25037222

RESUMO

G protein activation by G protein-coupled receptors is one of the critical steps for many cellular signal transduction pathways. Previously, we and other groups reported that the α5 helix in the G protein α subunit plays a major role during this activation process. However, the precise signaling pathway between the α5 helix and the guanosine diphosphate (GDP) binding pocket remains elusive. Here, using structural, biochemical, and computational techniques, we probed different residues around the α5 helix for their role in signaling. Our data showed that perturbing the Phe-336 residue disturbs hydrophobic interactions with the ß2-ß3 strands and α1 helix, leading to high basal nucleotide exchange. However, mutations in ß strands ß5 and ß6 do not perturb G protein activation. We have highlighted critical residues that leverage Phe-336 as a relay. Conformational changes are transmitted starting from Phe-336 via ß2-ß3/α1 to Switch I and the phosphate binding loop, decreasing the stability of the GDP binding pocket and triggering nucleotide release. When the α1 and α5 helices were cross-linked, inhibiting the receptor-mediated displacement of the C-terminal α5 helix, mutation of Phe-336 still leads to high basal exchange rates. This suggests that unlike receptor-mediated activation, helix 5 rotation and translocation are not necessary for GDP release from the α subunit. Rather, destabilization of the backdoor region of the Gα subunit is sufficient for triggering the activation process.


Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Guanosina Difosfato/metabolismo , Fenilalanina/metabolismo , Cristalografia por Raios X , Subunidades alfa de Proteínas de Ligação ao GTP/química , Modelos Moleculares , Fenilalanina/química , Conformação Proteica
13.
J Biol Chem ; 288(50): 35982-96, 2013 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-24165132

RESUMO

The serine-rich repeat glycoproteins of Gram-positive bacteria comprise a large family of cell wall proteins. Streptococcus agalactiae (group B streptococcus, GBS) expresses either Srr1 or Srr2 on its surface, depending on the strain. Srr1 has recently been shown to bind fibrinogen, and this interaction contributes to the pathogenesis of GBS meningitis. Although strains expressing Srr2 appear to be hypervirulent, no ligand for this adhesin has been described. We now demonstrate that Srr2 also binds human fibrinogen and that this interaction promotes GBS attachment to endothelial cells. Recombinant Srr1 and Srr2 bound fibrinogen in vitro, with affinities of KD = 2.1 × 10(-5) and 3.7 × 10(-6) M, respectively, as measured by surface plasmon resonance spectroscopy. The binding site for Srr1 and Srr2 was localized to tandem repeats 6-8 of the fibrinogen Aα chain. The structures of both the Srr1 and Srr2 binding regions were determined and, in combination with mutagenesis studies, suggest that both Srr1 and Srr2 interact with a segment of these repeats via a "dock, lock, and latch" mechanism. Moreover, properties of the latch region may account for the increased affinity between Srr2 and fibrinogen. Together, these studies identify how greater affinity of Srr2 for fibrinogen may contribute to the increased virulence associated with Srr2-expressing strains.


Assuntos
Proteínas de Bactérias/metabolismo , Fibrinogênio/metabolismo , Glicoproteínas/metabolismo , Streptococcus agalactiae/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Glicoproteínas/química , Glicoproteínas/genética , Humanos , Modelos Moleculares , Mutagênese , Ligação Proteica , Estrutura Terciária de Proteína , Especificidade por Substrato
14.
J Biol Chem ; 288(34): 24293-301, 2013 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-23836905

RESUMO

Respiratory processes often use quinone oxidoreduction to generate a transmembrane proton gradient, making the 2H(+)/2e(-) quinone chemistry important for ATP synthesis. There are a variety of quinones used as electron carriers between bioenergetic proteins, and some respiratory proteins can functionally interact with more than one quinone type. In the case of complex II homologs, which couple quinone chemistry to the interconversion of succinate and fumarate, the redox potentials of the biologically available ubiquinone and menaquinone aid in driving the chemical reaction in one direction. In the complex II homolog quinol:fumarate reductase, it has been demonstrated that menaquinol oxidation requires at least one proton shuttle, but many of the remaining mechanistic details of menaquinol oxidation are not fully understood, and little is known about ubiquinone reduction. In the current study, structural and computational studies suggest that the sequential removal of the two menaquinol protons may be accompanied by a rotation of the naphthoquinone ring to optimize the interaction with a second proton shuttling pathway. However, kinetic measurements of site-specific mutations of quinol:fumarate reductase variants show that ubiquinone reduction does not use the same pathway. Computational docking of ubiquinone followed by mutagenesis instead suggested redundant proton shuttles lining the ubiquinone-binding site or from direct transfer from solvent. These data show that the quinone-binding site provides an environment that allows multiple amino acid residues to participate in quinone oxidoreduction. This suggests that the quinone-binding site in complex II is inherently plastic and can robustly interact with different types of quinones.


Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Simulação de Acoplamento Molecular , Oxirredutases/química , Ubiquinona/química , Domínio Catalítico , Cinética , Relação Estrutura-Atividade
16.
Proc Natl Acad Sci U S A ; 110(3): 942-7, 2013 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-23277586

RESUMO

Solution NMR spectroscopy of labeled arrestin-1 was used to explore its interactions with dark-state phosphorylated rhodopsin (P-Rh), phosphorylated opsin (P-opsin), unphosphorylated light-activated rhodopsin (Rh*), and phosphorylated light-activated rhodopsin (P-Rh*). Distinct sets of arrestin-1 elements were seen to be engaged by Rh* and inactive P-Rh, which induced conformational changes that differed from those triggered by binding of P-Rh*. Although arrestin-1 affinity for Rh* was seen to be low (K(D) > 150 µM), its affinity for P-Rh (K(D) ~80 µM) was comparable to the concentration of active monomeric arrestin-1 in the outer segment, suggesting that P-Rh generated by high-gain phosphorylation is occupied by arrestin-1 under physiological conditions and will not signal upon photo-activation. Arrestin-1 was seen to bind P-Rh* and P-opsin with fairly high affinity (K(D) of~50 and 800 nM, respectively), implying that arrestin-1 dissociation is triggered only upon P-opsin regeneration with 11-cis-retinal, precluding noise generated by opsin activity. Based on their observed affinity for arrestin-1, P-opsin and inactive P-Rh very likely affect the physiological monomer-dimer-tetramer equilibrium of arrestin-1, and should therefore be taken into account when modeling photoreceptor function. The data also suggested that complex formation with either P-Rh* or P-opsin results in a global transition in the conformation of arrestin-1, possibly to a dynamic molten globule-like structure. We hypothesize that this transition contributes to the mechanism that triggers preferential interactions of several signaling proteins with receptor-activated arrestins.


Assuntos
Arrestina/química , Arrestina/metabolismo , Rodopsina/metabolismo , Arrestina/genética , Sítios de Ligação , Humanos , Cinética , Modelos Moleculares , Complexos Multiproteicos/química , Mutagênese Insercional , Ressonância Magnética Nuclear Biomolecular , Opsinas/química , Opsinas/metabolismo , Fosforilação , Processos Fotoquímicos , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rodopsina/química
17.
J Biol Chem ; 288(5): 3394-405, 2013 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-23250748

RESUMO

Arrestin-1 preferentially binds active phosphorylated rhodopsin. Previously, a mutant with enhanced binding to unphosphorylated active rhodopsin (Rh*) was shown to partially compensate for lack of rhodopsin phosphorylation in vivo. Here we showed that reengineering of the receptor binding surface of arrestin-1 further improves the binding to Rh* while preserving protein stability. In mammals, arrestin-1 readily self-associates at physiological concentrations. The biological role of this phenomenon can only be elucidated by replacing wild type arrestin-1 in living animals with a non-oligomerizing mutant retaining all other functions. We demonstrate that constitutively monomeric forms of arrestin-1 are sufficiently stable for in vivo expression. We also tested the idea that individual functions of arrestin-1 can be independently manipulated to generate mutants with the desired combinations of functional characteristics. Here we showed that this approach is feasible; stable forms of arrestin-1 with high Rh* binding can be generated with or without the ability to self-associate. These novel molecular tools open the possibility of testing of the biological role of arrestin-1 self-association and pave the way to elucidation of full potential of compensational approach to gene therapy of gain-of-function receptor mutations.


Assuntos
Arrestinas/metabolismo , Olho/metabolismo , Engenharia de Proteínas , Animais , Arrestinas/química , Arrestinas/genética , Células HEK293 , Humanos , Camundongos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação/genética , Fosfatos/metabolismo , Ligação Proteica , Estabilidade Proteica , Estrutura Terciária de Proteína , Rodopsina/metabolismo , Eletricidade Estática , Temperatura , beta-Arrestinas
18.
J Biol Chem ; 287(42): 35430-35438, 2012 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-22904323

RESUMO

Complex II couples oxidoreduction of succinate and fumarate at one active site with that of quinol/quinone at a second distinct active site over 40 Å away. This process links the Krebs cycle to oxidative phosphorylation and ATP synthesis. The pathogenic mutation or inhibition of human complex II or its assembly factors is often associated with neurodegeneration or tumor formation in tissues derived from the neural crest. This brief overview of complex II correlates the clinical presentations of a large number of symptom-associated alterations in human complex II activity and assembly with the biochemical manifestations of similar alterations in the complex II homologs from Escherichia coli. These analyses provide clues to the molecular basis for diseases associated with aberrant complex II function.


Assuntos
Trifosfato de Adenosina/biossíntese , Ciclo do Ácido Cítrico/fisiologia , Complexo II de Transporte de Elétrons/fisiologia , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Animais , Humanos , Fosforilação/fisiologia , Relação Estrutura-Atividade
19.
J Biol Chem ; 286(10): 8043-8054, 2011 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-21193409

RESUMO

Bacterial phosphopentomutases (PPMs) are alkaline phosphatase superfamily members that interconvert α-D-ribose 5-phosphate (ribose 5-phosphate) and α-D-ribose 1-phosphate (ribose 1-phosphate). We investigated the reaction mechanism of Bacillus cereus PPM using a combination of structural and biochemical studies. Four high resolution crystal structures of B. cereus PPM revealed the active site architecture, identified binding sites for the substrate ribose 5-phosphate and the activator α-D-glucose 1,6-bisphosphate (glucose 1,6-bisphosphate), and demonstrated that glucose 1,6-bisphosphate increased phosphorylation of the active site residue Thr-85. The phosphorylation of Thr-85 was confirmed by Western and mass spectroscopic analyses. Biochemical assays identified Mn(2+)-dependent enzyme turnover and demonstrated that glucose 1,6-bisphosphate treatment increases enzyme activity. These results suggest that protein phosphorylation activates the enzyme, which supports an intermolecular transferase mechanism. We confirmed intermolecular phosphoryl transfer using an isotope relay assay in which PPM reactions containing mixtures of ribose 5-[(18)O(3)]phosphate and [U-(13)C(5)]ribose 5-phosphate were analyzed by mass spectrometry. This intermolecular phosphoryl transfer is seemingly counter to what is anticipated from phosphomutases employing a general alkaline phosphatase reaction mechanism, which are reported to catalyze intramolecular phosphoryl transfer. However, the two mechanisms may be reconciled if substrate encounters the enzyme at a different point in the catalytic cycle.


Assuntos
Fosfatase Alcalina , Bacillus cereus/enzimologia , Proteínas de Bactérias/química , Glucose-6-Fosfato/análogos & derivados , Fosfotransferases/química , Ribosemonofosfatos/química , Proteínas de Bactérias/metabolismo , Catálise , Domínio Catalítico , Cristalografia por Raios X , Glucose-6-Fosfato/química , Glucose-6-Fosfato/metabolismo , Manganês/química , Manganês/metabolismo , Fosfotransferases/metabolismo , Ribosemonofosfatos/metabolismo
20.
J Biol Chem ; 286(4): 3047-56, 2011 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-21098488

RESUMO

Complex II superfamily members catalyze the kinetically difficult interconversion of succinate and fumarate. Due to the relative simplicity of complex II substrates and their similarity to other biologically abundant small molecules, substrate specificity presents a challenge in this system. In order to identify determinants for on-pathway catalysis, off-pathway catalysis, and enzyme inhibition, crystal structures of Escherichia coli menaquinol:fumarate reductase (QFR), a complex II superfamily member, were determined bound to the substrate, fumarate, and the inhibitors oxaloacetate, glutarate, and 3-nitropropionate. Optical difference spectroscopy and computational modeling support a model where QFR twists the dicarboxylate, activating it for catalysis. Orientation of the C2-C3 double bond of activated fumarate parallel to the C(4a)-N5 bond of FAD allows orbital overlap between the substrate and the cofactor, priming the substrate for nucleophilic attack. Off-pathway catalysis, such as the conversion of malate to oxaloacetate or the activation of the toxin 3-nitropropionate may occur when inhibitors bind with a similarly activated bond in the same position. Conversely, inhibitors that do not orient an activatable bond in this manner, such as glutarate and citrate, are excluded from catalysis and act as inhibitors of substrate binding. These results support a model where electronic interactions via geometric constraint and orbital steering underlie catalysis by QFR.


Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Modelos Químicos , Modelos Moleculares , Oxirredutases/química , Catálise , Complexo II de Transporte de Elétrons/química , Complexo II de Transporte de Elétrons/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Proteínas de Escherichia coli/metabolismo , Fumaratos/química , Fumaratos/metabolismo , Oxirredutases/metabolismo , Especificidade por Substrato/fisiologia
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