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1.
Mol Cell ; 81(10): 2123-2134.e5, 2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-33794146

RESUMO

A body of data supports the existence of core (α2-α5) dimers of BAK and BAX in the oligomeric, membrane-perturbing conformation of these essential apoptotic effector molecules. Molecular structures for these dimers have only been captured for truncated constructs encompassing the core domain alone. Here, we report a crystal structure of BAK α2-α8 dimers (i.e., minus its flexible N-terminal helix and membrane-anchoring C-terminal segment) that has been obtained through the activation of monomeric BAK with the detergent C12E8. Core dimers are evident, linked through the crystal by contacts via latch (α6-α8) domains. This crystal structure shows activated BAK dimers with the extended latch domain present. Our data provide direct evidence for the conformational change converting BAK from inert monomer to the functional dimer that destroys mitochondrial integrity. This dimer is the smallest functional unit for recombinant BAK or BAX described so far.


Assuntos
Detergentes/química , Multimerização Proteica , Proteína Killer-Antagonista Homóloga a bcl-2/química , Sequência de Aminoácidos , Animais , Lipossomos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Moleculares , Estrutura Secundária de Proteína , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo
2.
Cell ; 152(3): 519-31, 2013 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-23374347

RESUMO

In stressed cells, apoptosis ensues when Bcl-2 family members Bax or Bak oligomerize and permeabilize the mitochondrial outer membrane. Certain BH3-only relatives can directly activate them to mediate this pivotal, poorly understood step. To clarify the conformational changes that induce Bax oligomerization, we determined crystal structures of BaxΔC21 treated with detergents and BH3 peptides. The peptides bound the Bax canonical surface groove but, unlike their complexes with prosurvival relatives, dissociated Bax into two domains. The structures define the sequence signature of activator BH3 domains and reveal how they can activate Bax via its groove by favoring release of its BH3 domain. Furthermore, Bax helices α2-α5 alone adopted a symmetric homodimer structure, supporting the proposal that two Bax molecules insert their BH3 domain into each other's surface groove to nucleate oligomerization. A planar lipophilic surface on this homodimer may engage the membrane. Our results thus define critical Bax transitions toward apoptosis.


Assuntos
Apoptose , Cristalografia por Raios X , Proteína X Associada a bcl-2/química , Sequência de Aminoácidos , Animais , Citocromos c/metabolismo , Dimerização , Embrião de Mamíferos/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Lipossomos/metabolismo , Fígado/metabolismo , Camundongos , Mitocôndrias/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência , Proteína X Associada a bcl-2/metabolismo
3.
Mol Cell ; 68(4): 659-672.e9, 2017 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-29149594

RESUMO

Certain BH3-only proteins transiently bind and activate Bak and Bax, initiating their oligomerization and the permeabilization of the mitochondrial outer membrane, a pivotal step in the mitochondrial pathway to apoptosis. Here we describe the first crystal structures of an activator BH3 peptide bound to Bak and illustrate their use in the design of BH3 derivatives capable of inhibiting human Bak on mitochondria. These BH3 derivatives compete for the activation site at the canonical groove, are the first engineered inhibitors of Bak activation, and support the role of key conformational transitions associated with Bak activation.


Assuntos
Apoptose/efeitos dos fármacos , Proteína 11 Semelhante a Bcl-2 , Mitocôndrias , Peptídeos , Proteína Killer-Antagonista Homóloga a bcl-2 , Animais , Proteína 11 Semelhante a Bcl-2/química , Proteína 11 Semelhante a Bcl-2/farmacologia , Linhagem Celular Transformada , Humanos , Camundongos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Peptídeos/química , Peptídeos/farmacologia , Ligação Proteica , Relação Estrutura-Atividade , Proteína Killer-Antagonista Homóloga a bcl-2/química , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo
4.
Mol Biol Evol ; 40(10)2023 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-37797308

RESUMO

Lactate dehydrogenase (LDH, EC.1.1.127) is an important enzyme engaged in the anaerobic metabolism of cells, catalyzing the conversion of pyruvate to lactate and NADH to NAD+. LDH is a relevant enzyme to investigate structure-function relationships. The present work provides the missing link in our understanding of the evolution of LDHs. This allows to explain (i) the various evolutionary origins of LDHs in eukaryotic cells and their further diversification and (ii) subtle phenotypic modifications with respect to their regulation capacity. We identified a group of cyanobacterial LDHs displaying eukaryotic-like LDH sequence features. The biochemical and structural characterization of Cyanobacterium aponinum LDH, taken as representative, unexpectedly revealed that it displays homotropic and heterotropic activation, typical of an allosteric enzyme, whereas it harbors a long N-terminal extension, a structural feature considered responsible for the lack of allosteric capacity in eukaryotic LDHs. Its crystallographic structure was solved in 2 different configurations typical of the R-active and T-inactive states encountered in allosteric LDHs. Structural comparisons coupled with our evolutionary analyses helped to identify 2 amino acid positions that could have had a major role in the attenuation and extinction of the allosteric activation in eukaryotic LDHs rather than the presence of the N-terminal extension. We tested this hypothesis by site-directed mutagenesis. The resulting C. aponinum LDH mutants displayed reduced allosteric capacity mimicking those encountered in plants and human LDHs. This study provides a new evolutionary scenario of LDHs that unifies descriptions of regulatory properties with structural and mutational patterns of these important enzymes.


Assuntos
L-Lactato Desidrogenase , Lactato Desidrogenases , Humanos , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/química , L-Lactato Desidrogenase/metabolismo
5.
Mol Cell ; 55(6): 938-946, 2014 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-25175025

RESUMO

Apoptotic stimuli activate and oligomerize the proapoptotic proteins Bak and Bax, resulting in mitochondrial outer-membrane permeabilization and subsequent cell death. This activation can occur when certain BH3-only proteins interact directly with Bak and Bax. Recently published crystal structures reveal that Bax separates into core and latch domains in response to BH3 peptides. The distinguishing characteristics of BH3 peptides capable of directly activating Bax were also elucidated. Here we identify specific BH3 peptides capable of "unlatching" Bak and describe structural insights into Bak activation and oligomerization. Crystal structures and crosslinking experiments demonstrate that Bak undergoes a conformational change similar to that of Bax upon activation. A structure of the Bak core domain dimer provides a high-resolution image of this key intermediate in the pore-forming oligomer. Our results confirm an analogous mechanism for activation and dimerization of Bak and Bax in response to certain BH3 peptides.


Assuntos
Fragmentos de Peptídeos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/química , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/química , Animais , Cristalografia , Cisteína/metabolismo , Humanos , Camundongos , Mitocôndrias/metabolismo , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteína X Associada a bcl-2/metabolismo
6.
Immunity ; 36(4): 646-57, 2012 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-22483802

RESUMO

The immune system must distinguish viable cells from cells damaged by physical and infective processes. The damaged cell-recognition molecule Clec9A is expressed on the surface of the mouse and human dendritic cell subsets specialized for the uptake and processing of material from dead cells. Clec9A recognizes a conserved component within nucleated and nonnucleated cells, exposed when cell membranes are damaged. We have identified this Clec9A ligand as a filamentous form of actin in association with particular actin-binding domains of cytoskeletal proteins. We have determined the crystal structure of the human CLEC9A C-type lectin domain and propose a functional dimeric structure with conserved tryptophans in the ligand recognition site. Mutation of these residues ablated CLEC9A binding to damaged cells and to the isolated ligand complexes. We propose that Clec9A provides targeted recruitment of the adaptive immune system during infection and can also be utilized to enhance immune responses generated by vaccines.


Assuntos
Citoesqueleto de Actina/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Lectinas Tipo C/metabolismo , Receptores Imunológicos/metabolismo , Receptores Mitogênicos/metabolismo , Actinas/metabolismo , Imunidade Adaptativa , Animais , Sítios de Ligação , Linhagem Celular , Membrana Celular/metabolismo , Células Dendríticas/citologia , Feminino , Humanos , Lectinas Tipo C/química , Lectinas Tipo C/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Secundária de Proteína , Receptores Imunológicos/genética , Receptores Mitogênicos/química , Receptores Mitogênicos/genética , Espectrina/metabolismo
7.
Plant J ; 87(6): 641-53, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27232113

RESUMO

Growing pharmaceutical interest in benzylisoquinoline alkaloids (BIA) coupled with their chemical complexity make metabolic engineering of microbes to create alternative platforms of production an increasingly attractive proposition. However, precise knowledge of rate-limiting enzymes and negative feedback inhibition by end-products of BIA metabolism is of paramount importance for this emerging field of synthetic biology. In this work we report the structural characterization of (S)-norcoclaurine-6-O-methyltransferase (6OMT), a key rate-limiting step enzyme involved in the synthesis of reticuline, the final intermediate to be shared between the different end-products of BIA metabolism, such as morphine, papaverine, berberine and sanguinarine. Four different crystal structures of the enzyme from Thalictrum flavum (Tf 6OMT) were solved: the apoenzyme, the complex with S-adenosyl-l-homocysteine (SAH), the complexe with SAH and the substrate and the complex with SAH and a feedback inhibitor, sanguinarine. The Tf 6OMT structural study provides a molecular understanding of its substrate specificity, active site structure and reaction mechanism. This study also clarifies the inhibition of Tf 6OMT by previously suggested feedback inhibitors. It reveals its high and time-dependent sensitivity toward sanguinarine.


Assuntos
Metiltransferases/química , Metiltransferases/metabolismo , Thalictrum/enzimologia , Benzofenantridinas/metabolismo , Benzofenantridinas/farmacologia , Benzilisoquinolinas/metabolismo , Berberina/farmacologia , Sítios de Ligação , Cristalografia por Raios X , Inibidores Enzimáticos/farmacologia , Escherichia coli/genética , Isoquinolinas/metabolismo , Isoquinolinas/farmacologia , Metiltransferases/antagonistas & inibidores , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , Conformação Proteica , Multimerização Proteica , Thalictrum/metabolismo
8.
Arch Biochem Biophys ; 545: 33-43, 2014 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-24434006

RESUMO

Activation and oligomerisation of Bax, a key pro-apoptotic Bcl-2 family protein, are key steps in the mitochondrial pathway to apoptosis. The signals for apoptosis are conveyed by the distantly related BH3-only proteins, which use their short BH3 domain, an amphipathic α-helix, to interact with other Bcl-2 family members. Here we report an NMR study of interactions between BaxΔC and BH3 domain-containing peptides in the absence and presence of CHAPS, a zwitterionic detergent. We find for the first time that CHAPS interacts weakly with BaxΔC (fast exchange on the NMR chemical shift timescale), at concentrations below micelle formation and with an estimated Kd in the tens of mM. Direct and relatively strong-interactions (slow exchange on the NMR chemical shift timescale) were also observed for BaxΔC with BaxBH3 (estimated Kd of circa 150µM) or BimBH3 in the absence of CHAPS. The interaction with either peptide alone induced widespread chemical shift perturbations to BaxΔC in solution which implies that BaxΔC might have undergone significant conformation change upon binding the BH3 peptide. However, BaxΔC remained monomeric upon binding either CHAPS or a BH3 peptide alone, but the presence of both provoked it to form a dimer.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Ácidos Cólicos/metabolismo , Detergentes/metabolismo , Proteínas de Membrana/metabolismo , Fragmentos de Peptídeos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteína X Associada a bcl-2/metabolismo , Apoptose , Proteínas Reguladoras de Apoptose/química , Proteína 11 Semelhante a Bcl-2 , Humanos , Proteínas de Membrana/química , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/química , Conformação Proteica , Mapas de Interação de Proteínas , Multimerização Proteica , Proteínas Proto-Oncogênicas/química , Proteína X Associada a bcl-2/química
9.
J Biol Chem ; 286(29): 26061-70, 2011 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-21613226

RESUMO

4-Hydroxyphenylpyruvate dioxygenase (HPPD) catalyzes the conversion of 4-hydroxyphenylpyruvate (HPP) into homogentisate. HPPD is the molecular target of very effective synthetic herbicides. HPPD inhibitors may also be useful in treating life-threatening tyrosinemia type I and are currently in trials for treatment of Parkinson disease. The reaction mechanism of this key enzyme in both plants and animals has not yet been fully elucidated. In this study, using site-directed mutagenesis supported by quantum mechanical/molecular mechanical theoretical calculations, we investigated the role of catalytic residues potentially interacting with the substrate/intermediates. These results highlight the following: (i) the central role of Gln-272, Gln-286, and Gln-358 in HPP binding and the first nucleophilic attack; (ii) the important movement of the aromatic ring of HPP during the reaction, and (iii) the key role played by Asn-261 and Ser-246 in C1 hydroxylation and the final ortho-rearrangement steps (numbering according to the Arabidopsis HPPD crystal structure 1SQD). Furthermore, this study reveals that the last step of the catalytic reaction, the 1,2 shift of the acetate side chain, which was believed to be unique to the HPPD activity, is also catalyzed by a structurally unrelated enzyme.


Assuntos
4-Hidroxifenilpiruvato Dioxigenase/química , 4-Hidroxifenilpiruvato Dioxigenase/metabolismo , Biocatálise , Delftia acidovorans/enzimologia , 4-Hidroxifenilpiruvato Dioxigenase/genética , Domínio Catalítico , Sequência Conservada , Ácido Homogentísico/metabolismo , Hidroxilação , Transferases Intramoleculares/metabolismo , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Oxirredução
10.
Arch Biochem Biophys ; 519(2): 186-93, 2012 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-22079167

RESUMO

Based on recent X-ray structures and biochemical characterizations of aspartate kinases from different species, we show in this review how various organizations of a regulatory domain have contributed to the different mechanisms of control observed in aspartate kinases allowing simple to complex allosteric controls in branched pathways. The aim of this review is to show the relationships between domain organization, effector binding sites, mechanism of inhibition and regulatory function of an allosteric enzyme in a biosynthetic pathway.


Assuntos
Aspartato Quinase , Regulação Alostérica , Aspartato Quinase/química , Aspartato Quinase/metabolismo , Sítios de Ligação , Cinética , Estrutura Terciária de Proteína
11.
Cell Death Differ ; 29(9): 1757-1768, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35279694

RESUMO

Pro-apoptotic BAK and BAX are activated by BH3-only proteins to permeabilise the outer mitochondrial membrane. The antibody 7D10 also activates BAK on mitochondria and its epitope has previously been mapped to BAK residues in the loop connecting helices α1 and α2 of BAK. A crystal structure of the complex between the Fv fragment of 7D10 and the BAK mutant L100A suggests a possible mechanism of activation involving the α1-α2 loop residue M60. M60 mutants of BAK have reduced stability and elevated sensitivity to activation by BID, illustrating that M60, through its contacts with residues in helices α1, α5 and α6, is a linchpin stabilising the inert, monomeric structure of BAK. Our data demonstrate that BAK's α1-α2 loop is not a passive covalent connector between secondary structure elements, but a direct restraint on BAK's activation.


Assuntos
Apoptose , Proteína Killer-Antagonista Homóloga a bcl-2 , Anticorpos , Apoptose/fisiologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Membranas Mitocondriais/metabolismo , Estrutura Secundária de Proteína , Proteína Killer-Antagonista Homóloga a bcl-2/química , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/genética
12.
Inorg Chem ; 50(14): 6408-10, 2011 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-21671656

RESUMO

The coupling of electron and proton transfers is currently under intense scrutiny. This Communication reports a new kind of proton-coupled electron transfer within a homodinuclear first-row transition-metal complex. The triply-bridged complex [Fe(III)(µ-OPh)(µ(2)-mpdp)Fe(II)(NH(2)Bn)] (1; mpdp(2-) = m-phenylenedipropionate) bearing a terminal aminobenzyl ligand can be reversibly deprotonated to the anilinate complex 2 whose core [Fe(II)(µ-OPh)(µ(2)-mpdp)Fe(III)(NHBn)] features an inversion of the iron valences. This observation is supported by a combination of UV-visible, (1)H NMR, and Mössbauer spectroscopic studies.


Assuntos
Compostos Férricos/química , Compostos Ferrosos/química , Cristalografia por Raios X , Elétrons , Ligantes , Modelos Moleculares , Conformação Molecular , Prótons , Estereoisomerismo
13.
Structure ; 29(2): 114-124.e3, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-32966763

RESUMO

Bcl-2 proteins orchestrate the mitochondrial pathway of apoptosis, pivotal for cell death. Yet, the structural details of the conformational changes of pro- and antiapoptotic proteins and their interactions remain unclear. Pulse dipolar spectroscopy (double electron-electron resonance [DEER], also known as PELDOR) in combination with spin-labeled apoptotic Bcl-2 proteins unveils conformational changes and interactions of each protein player via detection of intra- and inter-protein distances. Here, we present the synthesis and characterization of pro-apoptotic BimBH3 peptides of different lengths carrying cysteines for labeling with nitroxide or gadolinium spin probes. We show by DEER that the length of the peptides modulates their homo-interactions in the absence of other Bcl-2 proteins and solve by X-ray crystallography the structure of a BimBH3 tetramer, revealing the molecular details of the inter-peptide interactions. Finally, we prove that using orthogonal labels and three-channel DEER we can disentangle the Bim-Bim, Bcl-xL-Bcl-xL, and Bim-Bcl-xL interactions in a simplified interactome.


Assuntos
Proteína 11 Semelhante a Bcl-2/química , Multimerização Proteica , Animais , Apoptose , Proteína 11 Semelhante a Bcl-2/metabolismo , Sítios de Ligação , Humanos , Camundongos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Ratos , Proteína bcl-X/química , Proteína bcl-X/metabolismo
14.
Cell Rep ; 27(2): 359-373.e6, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30970242

RESUMO

To elicit apoptosis, BAX metamorphoses from an inert cytosolic monomer into homo-oligomers that permeabilize the mitochondrial outer membrane (MOM). A long-standing puzzle is that BH3 domains apparently activate BAX by not only its canonical groove but also a proposed site involving helices α1 and α6. Our mutagenesis studies reveal that late steps like oligomerization require activation through the groove but probably not earlier steps like MOM association. Conversely, α1 or α6 obstruction and alanine mutagenesis scanning implicate these helices early in BAX activation. The α1 and α6 mutations lowered BH3 binding, altered the BAX conformation, and reduced its MOM translocation and integration; their exposure of the BAX α1-α2 loop allosterically sequestered its α9 membrane anchor in the groove. The crystal structure of an α6 mutant revealed additional allosteric effects. The results suggest that the α1 and α6 region drives MOM association and integration, whereas groove binding favors subsequent steps toward oligomerization.


Assuntos
Mitocôndrias Hepáticas/metabolismo , Membranas Mitocondriais/metabolismo , Mutação , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Fragmentos de Peptídeos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Alinhamento de Sequência
15.
Structure ; 26(10): 1346-1359.e5, 2018 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-30122452

RESUMO

BAX and BAK are essential mediators of intrinsic apoptosis that permeabilize the mitochondrial outer membrane. BAX activation requires its translocation from cytosol to mitochondria where conformational changes cause its oligomerization. To better understand the critical step of translocation, we examined its blockade by mutation near the C terminus (P168G) or by antibody binding near the N terminus. Similarities in the crystal structures of wild-type and BAX P168G but significant other differences suggest that cytosolic BAX exists as an ensemble of conformers, and that the distribution of conformers within the ensemble determines the different functions of wild-type and mutant proteins. We also describe the structure of BAX in complex with an antibody, 3C10, that inhibits cytosolic BAX by limiting exposure of the membrane-associating helix α9, as does the P168G mutation. Our data for both means of BAX inhibition argue for an allosteric model of BAX regulation that derives from properties of the ensemble of conformers.


Assuntos
Mutação , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/metabolismo , Regulação Alostérica , Animais , Anticorpos Monoclonais/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Citosol/metabolismo , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Humanos , Ictaluridae/metabolismo , Camundongos , Modelos Moleculares , Conformação Proteica , Proteína X Associada a bcl-2/genética
16.
Spectrochim Acta A Mol Biomol Spectrosc ; 64(2): 532-48, 2006 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-16386459

RESUMO

Experimental (IR and Raman) and theoretical (Kohn-Sham calculations) methods are used in a combined analysis aimed at refining the available structural data concerning the molecular guests in channels formed by stacked dibenzo-18-crown-6 (DB18C6) crown ether. The calculations are performed for a simplified model comprising isolated DB18C6 unit and its complexes with either H2O or H3O+ guests, which are the simplest model ingredients of a one-dimensional diluted acid chain, to get structural and energetic data concerning the formation of the complex and to assign the characteristic spectroscopic bands. The oxygen centers in the previously reported crystallographic structure are assigned to either H2O or protonated species.


Assuntos
Éteres de Coroa/química , Análise Espectral Raman , Água/química , Modelos Teóricos , Oniocompostos/química , Oxigênio/química , Espectroscopia de Infravermelho com Transformada de Fourier , Vibração
17.
Chem Commun (Camb) ; (36): 4548-50, 2005 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-16158110

RESUMO

For the first time in Ag coordination chemistry, two supramolecular isomers, a ring and a helix, are isolated from the same mother liquor as a result of concentration effects.

18.
J Mol Biol ; 399(2): 283-93, 2010 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-20398676

RESUMO

Aspartate kinases (AKs) can be divided in two subhomology divisions, AKalpha and AKbeta, depending on the presence of an extra sequence of about 60 amino acids, which is found only in the N-terminus of all AKalpha's. To date, the structures of AKalpha failed to provide a role for this additional N-terminal sequence. In this study, the structure of the AKbeta from the Cyanobacteria Synechocystis reveals that this supplementary sequence is linked to the dimerization mode of AKs. Its absence in AKbeta leads to the dimerization by the catalytic domain instead of involving the ACT domains [Pfam 01842; small regulatory domains initially found in AK, chorismate mutase and TyrA (prephenate dehydrogenase)] as observed in AKalpha. Thus, the structural analysis of the Synechocystis AKbeta revealed a dimer with a novel architecture. The four ACT domains of each monomer interact together and do not make any contact with those of the second monomer. The enzyme is inhibited synergistically by threonine and lysine with the binding of threonine first. The interaction between ACT1 and ACT4 or between ACT2 and ACT3 generates a threonine binding site and a lysine binding site at each interface, making a total of eight regulatory sites per dimer and allowing a fine-tuning of the AK activity by the end products, threonine and lysine.


Assuntos
Aspartato Quinase/química , Dimerização , Synechocystis/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Lisina/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Treonina/metabolismo
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