RESUMO
Cytochrome P450s display a remarkable range of conformations in parallel with activity toward a great diversity of substrates. This aspect of P450s now extends to include the dynamic behavior of the protein, as shown by recent crystal structures of Cyp51.
Assuntos
Sistema Enzimático do Citocromo P-450/química , Catálise , Sistema Enzimático do Citocromo P-450/metabolismo , Modelos Moleculares , Conformação ProteicaRESUMO
Sp18 is an 18 kDa protein that is released from abalone sperm during the acrosome reaction. It coats the acrosomal process where it is thought to mediate fusion between sperm and egg cell membranes. Sp18 is evolutionarily related to lysin, a 16 kDa abalone sperm protein that dissolves the vitelline envelope surrounding the egg. The two proteins were generated by gene duplication followed by rapid divergence by positive selection. Here, we present the crystal structure of green abalone sp18 resolved to 1.86 A. Sp18 is composed of a bundle of five alpha-helices with surface clusters of basic and hydrophobic residues, giving it a large dipole moment and making it extremely amphipathic. The large clusters of hydrophobic surface residues and domains of high positive electrostatic surface charge explain sp18's ability as a potent fusagen of liposomes. The overall fold of sp18 is similar to that of green abalone lysin; however, the surface features of the proteins are quite different, accounting for their different roles in fertilization. This is the first crystal structure of a protein implicated in sperm-egg fusion during animal fertilization.
Assuntos
Acrossomo/fisiologia , Fusão de Membrana/fisiologia , Proteínas/química , Interações Espermatozoide-Óvulo , Sequência de Aminoácidos , Animais , Cristalização , Cristalografia por Raios X , Dimerização , Concentração de Íons de Hidrogênio , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Moluscos , Mucoproteínas/química , Mucoproteínas/fisiologia , Dobramento de Proteína , Proteínas/fisiologia , Relação Estrutura-Atividade , Propriedades de SuperfícieRESUMO
Abalone sperm lysin is a non-enzymatic protein that creates a hole for sperm passage in the envelope surrounding the egg. Lysin exhibits species-specificity in making the hole and it evolves rapidly by positive selection. Our studies have focused on combining structural, biochemical, and evolutionary data to understand the mechanism of action and evolution of this remarkable protein. Currently, more is known about lysin than about any other protein involved in animal fertilization. We present an hypothesis to explain lysin's rapid evolution and the evolution of species-specific fertilization in this order of mollusks. We also propose a two-step model for lysin's action in which a dimer of lysin binds species-specifically to its glycoprotein receptor, and then monomerizes and binds the receptor in a non-species-specific manner. This experimental system yields data relevant to the general problem of molecular recognition between cell surfaces, and is also important to our thinking about how new species arise in the sea. BioEssays 23:95-103, 2001.
Assuntos
Evolução Molecular , Mucoproteínas/química , Espermatozoides/metabolismo , Acrossomo/metabolismo , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Cristalografia por Raios X , Dimerização , Fertilização , Masculino , Dados de Sequência Molecular , Mucoproteínas/metabolismo , Mucoproteínas/fisiologia , Reprodução/fisiologia , Seleção Genética , Especificidade da EspécieRESUMO
All naturally occurring ferredoxins that have Cys-X-X-Asp-X-X-Cys motifs contain [4Fe-4S](2+/+) clusters that can be easily and reversibly converted to [3Fe-4S](+/0) clusters. In contrast, ferredoxins with unmodified Cys-X-X-Cys-X-X-Cys motifs assemble [4Fe-4S](2+/+) clusters that cannot be easily interconverted with [3Fe-4S](+/0) clusters. In this study we changed the central cysteine of the Cys(39)-X-X-Cys(42)-X-X-Cys(45) of Azotobacter vinelandii FdI, which coordinates its [4Fe-4S](2+/+) cluster, into an aspartate. UV-visible, EPR, and CD spectroscopies, metal analysis, and x-ray crystallography show that, like native FdI, aerobically purified C42D FdI is a seven-iron protein retaining its [4Fe-4S](2+/+) cluster with monodentate aspartate ligation to one iron. Unlike known clusters of this type the reduced [4Fe-4S](+) cluster of C42D FdI exhibits only an S = 1/2 EPR with no higher spin signals detected. The cluster shows only a minor change in reduction potential relative to the native protein. All attempts to convert the cluster to a 3Fe cluster using conventional methods of oxygen or ferricyanide oxidation or thiol exchange were not successful. The cluster conversion was ultimately accomplished using a new electrochemical method. Hydrophobic and electrostatic interaction and the lack of Gly residues adjacent to the Asp ligand explain the remarkable stability of this cluster.
Assuntos
Azotobacter vinelandii/química , Ferredoxinas/química , Sequência de Aminoácidos , Ácido Aspártico/metabolismo , Dicroísmo Circular , Espectroscopia de Ressonância de Spin Eletrônica , Ferredoxinas/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Espectrofotometria UltravioletaRESUMO
dUTP pyrophosphatase (dUTPase) cleaves the alpha-beta phosphodiester of dUTP to form pyrophosphate and dUMP, preventing incorporation of uracil into DNA and providing the substrate for thymine synthesis. Seven crystal structures of feline immunodeficiency virus (FIV) dUTPase in three crystal forms have been determined, including complexes with substrate (dUTP), product (dUMP) or inhibitor (dUDP) bound. The native enzyme has been refined at 1.40 A resolution in a hexagonal crystal form and at 2.3 A resolution in an orthorhombic crystal form. In the dUDP complex in a cubic crystal form refined at 2.5 A resolution, the C-terminal conserved P-loop motif is fully ordered. The analysis defines the roles of five sequence motifs in interaction with uracil, deoxyribose and the alpha-, beta- and gamma-phosphates. The enzyme utilizes adaptive recognition to bind the alpha- and beta-phosphates. In particular, the alpha-beta phosphodiester adopts an unfavorable eclipsed conformation in the presence of the P-loop. This conformation may be relevant to the mechanism of alpha-beta phosphodiester bond cleavage.
Assuntos
Nucleotídeos de Desoxiuracil/química , Vírus da Imunodeficiência Felina/enzimologia , Pirofosfatases/química , Proteínas Virais/química , Animais , Gatos , Cristalização , Cristalografia por Raios X , Nucleotídeos de Desoxiuracil/metabolismo , Modelos Moleculares , Conformação Proteica , Estrutura Secundária de Proteína , Pirofosfatases/metabolismo , Proteínas Virais/metabolismoRESUMO
A crystal structure of a 108 nucleotide RNA-DNA complex containing a four-way junction was solved at 3.1 A resolution. The structure of the junction differs substantially from the "stacked-X" conformation observed previously, due to a 135 degrees rotation of the branches. Comparison of the two conformers provides insight into the factors contributing to the flexibility of four-way junctions. The stacked-X conformation maximizes base-stacking but causes unfavorable repulsion between phosphate groups, whereas the 135 degrees -rotated "crossed" conformation minimizes electrostatic clashes at the expense of reduced base-stacking. Despite the large rotation of the branches, both junction structures exhibit an antiparallel arrangement of the continuous strands and opposite polarity of the crossover strands.
Assuntos
Troca Genética/genética , DNA Catalítico , DNA/química , DNA/metabolismo , Conformação de Ácido Nucleico , RNA/química , RNA/metabolismo , Pareamento de Bases/genética , Sequência de Bases , Cristalografia por Raios X , DNA/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Isomerismo , Modelos Moleculares , Dados de Sequência Molecular , Ácidos Nucleicos Heteroduplexes/química , Ácidos Nucleicos Heteroduplexes/genética , Ácidos Nucleicos Heteroduplexes/metabolismo , Fosfatos/metabolismo , Maleabilidade , RNA/genética , Rotação , Eletricidade EstáticaRESUMO
The basis of the chemiosmotic theory is that energy from light or respiration is used to generate a trans-membrane proton gradient. This is largely achieved by membrane-spanning enzymes known as 'proton pumps. There is intense interest in experiments which reveal, at the molecular level, how protons are drawn through proteins. Here we report the mechanism, at atomic resolution, for a single long-range electron-coupled proton transfer. In Azotobacter vinelandii ferredoxin I, reduction of a buried iron-sulphur cluster draws in a solvent proton, whereas re-oxidation is 'gated' by proton release to the solvent. Studies of this 'proton-transferring module' by fast-scan protein film voltammetry, high-resolution crystallography, site-directed mutagenesis and molecular dynamics, reveal that proton transfer is exquisitely sensitive to the position and pK of a single amino acid. The proton is delivered through the protein matrix by rapid penetrative excursions of the side-chain carboxylate of a surface residue (Asp 15), whose pK shifts in response to the electrostatic charge on the iron-sulphur cluster. Our analysis defines the structural, dynamic and energetic requirements for proton courier groups in redox-driven proton-pumping enzymes.
Assuntos
Ferredoxinas/química , Prótons , Ácido Aspártico/química , Azotobacter vinelandii , Ferredoxinas/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Oxirredução , Bombas de Próton/químicaRESUMO
Abalone sperm lysin is a 16 kDa acrosomal protein used by sperm to create a hole in the egg vitelline envelope. Lysins from seven California abalone exhibit species-specificity in binding to their egg receptor, and range in sequence identity from 63 % to 90 %. The crystal structure of the sperm lysin dimer from Haliotis fulgens (green abalone) has been determined to 1.71 A by multiple isomorphous replacement. Comparisons with the structure of the lysin dimer from Haliotis rufescens (red abalone) reveal a similar overall fold and conservation of features contributing to lysin's amphipathic character. The two structures do, however, exhibit differences in surface residues and electrostatics. A large clustering of non-conserved surface residues around the waist and clefts of the dimer, and differences in charged residues around these regions, indicate areas of the molecule which may be involved in species-specific egg recognition.
Assuntos
Acrossomo/química , Moluscos/química , Mucoproteínas/química , Mucoproteínas/metabolismo , Óvulo/metabolismo , Proteínas/química , Proteínas/metabolismo , Receptores de Superfície Celular/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Sequência Conservada , Cristalização , Cristalografia por Raios X , Dimerização , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Dobramento de Proteína , Alinhamento de Sequência , Especificidade da Espécie , Eletricidade EstáticaRESUMO
Abalone sperm use lysin to make a hole in the egg's protective vitelline envelope (VE). When released from sperm, lysin first binds to the VE receptor for lysin (VERL) then dissolves the VE by a non-enzymatic mechanism. The structures of the monomeric and dimeric forms of Haliotis rufescens (red abalone) lysin, previously solved at 1.90 and 2.75 A, respectively, have now been refined to 1.35 and 2.07 A, respectively. The monomeric form of lysin was refined using previously obtained crystallization conditions, while the dimer was solved in a new crystal form with four molecules (two dimers) per asymmetric unit. These high-resolution structures reveal alternate residue conformations, enabling a thorough analysis of the conserved residues contributing to the amphipathic nature of lysin. The availability of five independent high-resolution copies of lysin permits comparisons leading to insights on the local flexibility of lysin and alternative conformations of the hypervariable N-terminus, thought to be involved in species-specific receptor recognition. The new analysis led to the discovery of the basic nature of a cleft formed upon dimerization and a patch of basic residues in the dimer interface. Identification of these features was not possible at lower resolution. In light of this new information, a model explaining the binding of sperm lysin to egg VERL and the subsequent dissolution of the egg VE is proposed.
Assuntos
Proteínas do Ovo/metabolismo , Moluscos/metabolismo , Mucoproteínas/química , Mucoproteínas/metabolismo , Receptores de Superfície Celular/metabolismo , Espermatozoides/metabolismo , Animais , Sítios de Ligação , Cristalografia por Raios X , Dimerização , Feminino , Masculino , Modelos Biológicos , Modelos Moleculares , Mucoproteínas/isolamento & purificação , Conformação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Eletricidade Estática , Membrana Vitelina/metabolismoRESUMO
The crystal structure of a 70-kilodalton ribonucleoprotein complex from the central domain of the Thermus thermophilus 30S ribosomal subunit was solved at 2.6 angstrom resolution. The complex consists of a 104-nucleotide RNA fragment composed of two three-helix junctions that lie at the end of a central helix, and the ribosomal proteins S15, S6, and S18. S15 binds the ribosomal RNA early in the assembly of the 30S ribosomal subunit, stabilizing a conformational reorganization of the two three-helix junctions that creates the RNA fold necessary for subsequent binding of S6 and S18. The structure of the complex demonstrates the central role of S15-induced reorganization of central domain RNA for the subsequent steps of ribosome assembly.
Assuntos
RNA Ribossômico/química , Ribonucleoproteínas/química , Proteínas Ribossômicas/química , Ribossomos/química , Thermus thermophilus/química , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Ribossômico/metabolismo , Ribonucleoproteínas/metabolismo , Proteína S6 Ribossômica , Proteínas Ribossômicas/metabolismo , Thermus thermophilus/ultraestruturaRESUMO
The nicotinamide nucleotide transhydrogenases (TH) of mitochondria and bacteria are membrane-intercalated proton pumps that transduce substrate binding energy and protonmotive force via protein conformational changes. In mitochondria, TH utilizes protonmotive force to promote direct hydride ion transfer from NADH to NADP, which are bound at the distinct extramembranous domains I and III, respectively. Domain II is the membrane-intercalated domain and contains the enzyme's proton channel. This paper describes the crystal structure of the NADP(H) binding domain III of bovine TH at 1.2 A resolution. The structure reveals that NADP is bound in a manner inverted from that previously observed for nucleotide binding folds. The non-classical binding mode exposes the NADP(H) nicotinamide ring for direct contact with NAD(H) in domain I, in accord with biochemical data. The surface of domain III surrounding the exposed nicotinamide is comprised of conserved residues presumed to form the interface with domain I during hydride ion transfer. Further, an adjacent region contains a number of acidic residues, forming a surface with negative electrostatic potential which may interact with extramembranous loops of domain II. Together, the distinctive surface features allow mechanistic considerations regarding the NADP(H)-promoted conformation changes that are involved in the interactions of domain III with domains I and II for hydride ion transfer and proton translocation.
Assuntos
Domínio Catalítico , NADP Trans-Hidrogenases/química , NADP Trans-Hidrogenases/metabolismo , NADP/metabolismo , NAD/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bovinos , Sequência Conservada , Cristalização , Cristalografia por Raios X , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Miocárdio/enzimologia , NAD/química , NADP/química , Dobramento de Proteína , Estrutura Secundária de Proteína , Prótons , Alinhamento de Sequência , Eletricidade Estática , Relação Estrutura-AtividadeRESUMO
The [4Fe-4S](2+/+) cluster of Azotobacter vinelandii ferredoxin I (FdI) has an unusually low reduction potential (E(0')) relative to other structurally similar ferredoxins. Previous attempts to raise that E(0') by modification of surface charged residues were unsuccessful. In this study mutants were designed to alter the E(0') by substitution of polar residues for nonpolar residues near the cluster and by modification of backbone amides. Three FdI variants, P21G, I40N, and I40Q, were purified and characterized, and electrochemical E(0') measurements show that all had altered E(0') relative to native FdI. For P21G FdI and I40Q FdI, the E(0') increased by +42 and +53 mV, respectively validating the importance of dipole orientation in control of E(0'). Protein Dipole Langevin Dipole calculations based on models for those variants accurately predicted the direction of the change in E(0') while overestimating the magnitude. For I40N FdI, initial calculations based on the model predicted a +168 mV change in E(0') while a -33 mV change was observed. The x-ray structure of that variant, which was determined to 2.8 A, revealed a number of changes in backbone and side chain dipole orientation and in solvent accessibility, that were not predicted by the model and that were likely to influence E(0'). Subsequent Protein Dipole Langevin Dipole calculations (using the actual I40N x-ray structures) did quite accurately predict the observed change in E(0').
Assuntos
Azotobacter vinelandii/metabolismo , Ferredoxinas/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte de Elétrons , Ferredoxinas/química , Ferredoxinas/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Eletricidade EstáticaRESUMO
One of the most difficult steps in the X-ray crystallography of nucleic acids is obtaining crystals that diffract to high resolution. The choice of the nucleotide sequence has proven to be more important in producing high-quality crystals than the composition of the crystallization solution. This manuscript describes a systematic procedure for identifying the optimal sizes of a multi-stranded nucleic acid complex which provide high-quality crystals. This approach was used to crystallize the in vitro evolved 10-23 DNA enzyme complexed with its RNA substrate. In less than two months, 81 different enzyme-substrate complexes were generated by combinatorial mixing and annealing of complementary oligonucleotides which differed in length, resulting in duplexes of varying length, with or without nucleotide overhangs. Each of these complexes was screened against a standard set of 48 crystallization conditions and evaluated for crystal formation. The screen resulted in over 40 crystal forms, the best of which diffracted to 2.8 A resolution when exposed to a synchrotron X-ray source.
Assuntos
Ácidos Nucleicos Heteroduplexes/química , Técnicas de Química Combinatória , Cristalização , DNA/química , Conformação de Ácido Nucleico , RNA/química , Difração de Raios XRESUMO
The refined structure of reduced Azotobacter vinelandii 7Fe ferredoxin FdI at 100 K and 1.4 A resolution is reported, permitting comparison of [3Fe-4S]+ and [3Fe-4S]0 clusters in the same protein at near atomic resolution. The reduced state of the [3Fe-4S]0 cluster is established by single-crystal EPR following data collection. Redundant structures are refined to establish the reproducibility and accuracy of the results for both oxidation states. The structure of the [4Fe-4S]2+ cluster in four independently determined FdI structures is the same within the range of derived standard uncertainties, providing an internal control on the experimental methods and the refinement results. The structures of the [3Fe-4S]+ and [3Fe-4S]0 clusters are also the same within experimental error, indicating that the protein may be enforcing an entatic state upon this cluster, facilitating electron-transfer reactions. The structure of the FdI [3Fe-4S]0 cluster allows direct comparison with the structure of a well-characterized [Fe3S4]0 synthetic analogue compound. The [3Fe-4S]0 cluster displays significant distortions with respect to the [Fe3S4]0 analogue, further suggesting that the observed [3Fe-4S]+/0 geometry in FdI may represent an entatic state. Comparison of oxidized and reduced FdI reveals conformational changes at the protein surface in response to reduction of the [3Fe-4S]+/0 cluster. The carboxyl group of Asp15 rotates approximately 90 degrees, Lys84, a residue hydrogen bonded to Asp15, adopts a single conformation, and additional H2O molecules become ordered. These structural changes imply a mechanism for H+ transfer to the [3Fe-4S]0 cluster in agreement with electrochemical and spectroscopic results.
Assuntos
Azotobacter vinelandii/química , Ferredoxinas/química , Ferro/química , Enxofre/química , Cristalização , Cristalografia por Raios X , Espectroscopia de Ressonância de Spin Eletrônica , Transporte de Elétrons , Ligação de Hidrogênio , Modelos Químicos , Modelos Moleculares , Oxirredução , Conformação Proteica , Prótons , Propriedades de SuperfícieRESUMO
CD38 is a type II transmembrane glycoprotein that is expressed by many cell types including lymphocytes. Signaling through CD38 on B lymphocytes can mediate B cell activation, proliferation, and cytokine secretion. Additionally, coligation of CD38 and the B cell Ag receptor can greatly augment B cell Ag receptor responses. Interestingly, the extracellular domain of CD38 catalyzes the conversion of NAD+ into nicotinamide, ADP-ribose (ADPR), and cyclic ADPR (cADPR). cADPR can induce intracellular calcium release in an inositol trisphosphate-independent manner and has been hypothesized to regulate CD38-mediated signaling. We demonstrate that replacement of the cytoplasmic tail and the transmembrane domains of CD38 did not impair CD38 signaling, coreceptor activity, or enzyme activity. In contrast, independent point mutations in the extracellular domain of CD38 dramatically impaired signal transduction. However, no correlation could be found between CD38-mediated signaling and the capacity of CD38 to catalyze an enzyme reaction and produce cADPR, ADPR, and/or nicotinamide. Instead, we propose that CD38 signaling and coreceptor activity in vitro are regulated by conformational changes induced in the extracellular domain upon ligand/substrate binding, rather than on actual turnover or generation of products.
Assuntos
Adenosina Difosfato Ribose/análogos & derivados , Adenosina Difosfato Ribose/fisiologia , Antígenos CD/fisiologia , Linfócitos B/fisiologia , Sequência de Aminoácidos , Animais , Antígenos CD/química , Antígeno CD48 , ADP-Ribose Cíclica , Camundongos , Dados de Sequência Molecular , NAD/metabolismo , Coelhos , Transdução de Sinais , TransfecçãoRESUMO
The structure of a large nucleic acid complex formed by the 10-23 DNA enzyme bound to an RNA substrate was determined by X-ray diffraction at 3.0 A resolution. The 82-nucleotide complex contains two strands of DNA and two strands of RNA that form five double-helical domains. The spatial arrangement of these helices is maintained by two four-way junctions that exhibit extensive base-stacking interactions and sharp turns of the phosphodiester backbone stabilized by metal ions coordinated to nucleotides at these junctions. Although it is unlikely that the structure corresponds to the catalytically active conformation of the enzyme, it represents a novel nucleic acid fold with implications for the Holliday junction structure.
Assuntos
DNA/química , Enzimas/química , RNA/química , Sequência de Bases , Catálise , DNA/metabolismo , Enzimas/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , RNA/metabolismo , Especificidade por Substrato , Difração de Raios XRESUMO
In Azotobacter vinelandii, deletion of the fdxA gene, which encodes ferredoxin I (FdI), leads to activation of the expression of the fpr gene, which encodes NADPH-ferredoxin reductase (FPR). In order to investigate the relationship of these two proteins further, the interactions of the two purified proteins have been examined. AvFdI forms a specific 1:1 cross-linked complex with AvFPR through ionic interactions formed between the Lys residues of FPR and Asp/Glu residues of FdI. The Lys in FPR has been identified as Lys258, a residue that forms a salt bridge with one of the phosphate oxygens of FAD in the absence of FdI. UV-Vis and circular dichroism data show that on binding FdI, the spectrum of the FPR flavin is hyperchromatic and red-shifted, confirming the interaction region close to the FAD. Cytochrome c reductase assays and electron paramagnetic resonance data show that electron transfer between the two proteins is pH-dependent and that the [3Fe-4S]+ cluster of FdI is specifically reduced by NADPH via FPR, suggesting that the [3Fe-4S] cluster is near FAD in the complex. To further investigate the FPR:FdI interaction, the electrostatic potentials for each protein were calculated. Strongly negative regions around the [3Fe-4S] cluster of FdI are electrostatically complementary with a strongly positive region overlaying the FAD of FPR, centered on Lys258. These proposed interactions of FdI with FPR are consistent with cross-linking, peptide mapping, spectroscopic, and electron transfer data and strongly support the suggestion that the two proteins are physiological redox partners.
Assuntos
Azotobacter vinelandii/metabolismo , Ferredoxina-NADP Redutase/metabolismo , Ferredoxinas/metabolismo , Sequência de Aminoácidos , Anabaena , Dicroísmo Circular , Espectroscopia de Ressonância de Spin Eletrônica , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Ácido Glutâmico/metabolismo , Lisina/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , NADP/metabolismo , Conformação Proteica , Pseudomonas aeruginosa , Eletricidade EstáticaRESUMO
The crystal structure of the S642A mutant of mitochondrial aconitase (mAc) with citrate bound has been determined at 1.8 A resolution and 100 K to capture this binding mode of substrates to the native enzyme. The 2.0 A resolution, 100 K crystal structure of the S642A mutant with isocitrate binding provides a control, showing that the Ser --> Ala replacement does not alter the binding of substrates in the active site. The aconitase mechanism requires that the intermediate product, cis-aconitate, flip over by 180 degrees about the C alpha-C beta double bond. Only one of these two alternative modes of binding, that of the isocitrate mode, has been previously visualized. Now, however, the structure revealing the citrate mode of binding provides direct support for the proposed enzyme mechanism.
Assuntos
Aconitato Hidratase/química , Ácido Cítrico/química , Isocitratos/química , Mitocôndrias/química , Aconitato Hidratase/genética , Cristalografia por Raios X , Escherichia coli/química , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Proteínas Recombinantes/químicaRESUMO
[4Fe-4S]2+/+ clusters that are ligated by Cys-X-X-Cys-X-X-Cys sequence motifs share the general feature of being hard to convert to [3Fe-4S]+/0 clusters, whereas those that contain a Cys-X-X-Asp-X-X-Cys motif undergo facile and reversible cluster interconversion. Little is known about the factors that control the in vivo assembly and conversion of these clusters. In this study we have designed and constructed a 3Fe to 4Fe cluster conversion variant of Azotobacter vinelandii ferredoxin I (FdI) in which the sequence that ligates the [3Fe-4S] cluster in native FdI was altered by converting a nearby residue, Thr-14, to Cys. Spectroscopic and electrochemical characterization shows that when purified in the presence of dithionite, T14C FdI is an O2-sensitive 8Fe protein. Both the new and the indigenous clusters have reduction potentials that are significantly shifted compared with those in native FdI, strongly suggesting a significantly altered environment around the clusters. Interestingly, whole cell EPR have revealed that T14C FdI exists as a 7Fe protein in vivo. This 7Fe form of T14C FdI is extremely similar to native FdI in its spectroscopic, electrochemical, and structural features. However, unlike native FdI which does not undergo facile cluster conversion, the 7Fe form T14C FdI quickly converts to the 8Fe form with a high efficiency under reducing conditions.
Assuntos
Azotobacter vinelandii/metabolismo , Ferredoxinas/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Dicroísmo Circular , Cristalografia por Raios X , Primers do DNA , Eletroquímica , Ferredoxinas/química , Ferredoxinas/genética , Proteínas Ferro-Enxofre/química , Dados de Sequência Molecular , Mutagênese , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
NADPH:ferredoxin reductase (AvFPR) is involved in the response to oxidative stress in Azotobacter vinelandii. The crystal structure of AvFPR has been determined at 2.0 A resolution. The polypeptide fold is homologous with six other oxidoreductases whose structures have been solved including Escherichia coli flavodoxin reductase (EcFldR) and spinach, and Anabaena ferredoxin:NADP+ reductases (FNR). AvFPR is overall most homologous to EcFldR. The structure is comprised of a N-terminal six-stranded antiparallel beta-barrel domain, which binds FAD, and a C-terminal five-stranded parallel beta-sheet domain, which binds NADPH/NADP+ and has a classical nucleotide binding fold. The two domains associate to form a deep cleft where the NADPH and FAD binding sites are juxtaposed. The structure displays sequence conserved motifs in the region surrounding the two dinucleotide binding sites, which are characteristic of the homologous enzymes. The folded over conformation of FAD in AvFPR is similar to that in EcFldR due to stacking of Phe255 on the adenine ring of FAD, but it differs from that in the FNR enzymes, which lack a homologous aromatic residue. The structure of AvFPR displays three unique features in the environment of the bound FAD. Two features may affect the rate of reduction of FAD: the absence of an aromatic residue stacked on the isoalloxazine ring in the NADPH binding site; and the interaction of a carbonyl group with N10 of the flavin. Both of these features are due to the substitution of a conserved C-terminal tyrosine residue with alanine (Ala254) in AvFPR. An additional unique feature may affect the interaction of AvFPR with its redox partner ferredoxin I (FdI). This is the extension of the C-terminus by three residues relative to EcFldR and by four residues relative to FNR. The C-terminal residue, Lys258, interacts with the AMP phosphate of FAD. Consequently, both phosphate groups are paired with a basic group due to the simultaneous interaction of the FMN phosphate with Arg51 in a conserved FAD binding motif. The fourth feature, common to homologous oxidoreductases, is a concentration of 10 basic residues on the face of the protein surrounding the active site, in addition to Arg51 and Lys258.