RESUMEN
In the cyanobacterium Thermosynechococcus elongatus BP-1, living in hot springs, the light environment directly regulates expression of genes that encode key components of the photosynthetic multi-subunit protein-pigment complex photosystem II (PSII). Light is not only essential as an energy source to power photosynthesis, but leads to formation of aggressive radicals which induce severe damage of protein subunits and organic cofactors. Photosynthetic organisms develop several protection mechanisms against this photo-damage, such as the differential expression of genes coding for the reaction center subunit D1 in PSlI. Testing the expression of the three different genes (psbAI, psbAII, psbAIII) coding for D1 in T. elongatus under culture conditions used for preparing the material used in crystallization of PSII showed that under these conditions only subunit PsbA1 is present. However, exposure to high-light intensity induced partial replacement of PsbA1 with PsbA3. Modeling of the variant amino acids of the three different D1 copies in the 3.0 A resolution crystal structure of PSII revealed that most of them are in the direct vicinity to redox-active cofactors of the electron transfer chain. Possible structural and mechanistic consequences for electron transfer are discussed.
Asunto(s)
Cianobacterias/química , Cianobacterias/genética , Complejo de Proteína del Fotosistema II/química , Secuencia de Aminoácidos , Grupo Citocromo b/genética , Grupo Citocromo c/genética , Bases de Datos Genéticas , Transporte de Electrón/fisiología , Genes Bacterianos , Modelos Moleculares , Datos de Secuencia Molecular , Complejo de Proteína del Fotosistema II/genética , Unión Proteica , Quinonas/química , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The crystal structure of cytokinin-specific binding protein (CSBP) containing four independent molecules with 4 x 155 = 620 residues in the asymmetric unit of the P6(4) unit cell has been solved by three-wavelength MAD using 1.8 angstroms resolution data recorded from a crystal derivatized with the dodecabromohexatantalum cation (Ta6Br12)2+. The diffraction data contained a very strong anomalous signal (allowing successful phasing even using peak SAD data alone) despite the fact that the five (Ta6Br12)2+ clusters found in the asymmetric unit have low occupancy (about 0.3). The derivative structure has been successfully refined to R = 0.158, providing interesting details on the geometry of the (Ta6Br12)2+ cluster, its interactions with the protein and on the backsoaking of a cytokinin ligand that was originally part of a CSBP-cytokinin complex in the native crystals used for (Ta6Br12)2+ derivatization. A simulation analysis of the phasing power of the (Ta6Br12)2+ ions at artificially imposed resolution limits shows that it is not possible to resolve the individual Ta atoms if the dmin limit of the data is higher than 2.9 angstroms. Additionally, for successful Ta identification the (Ta6Br12)2+ complex should be specifically bound and ordered. Good binding at the protein surface is facilitated by the presence of acidic groups, indicating higher pH buffer conditions to be preferable. In addition, the water channels in the crystal should be sufficiently wide (at least 11 angstroms) to allow free diffusion of the (Ta6Br12)2+ ions on soaking. A retrospective look at the initial molecular-replacement calculations provides interesting insights into how the peculiar packing mode and strong bias of the molecular-replacement-phased electron-density maps had hindered successful solution of the structure by this method.
Asunto(s)
Proteínas Portadoras/química , Citocininas/química , Proteínas de Plantas/química , Cristalografía por Rayos X/métodos , Fabaceae/química , Modelos Moleculares , Unión Proteica , Pliegue de Proteína , Estudios Retrospectivos , Difracción de Rayos XRESUMEN
The catabolite control protein A (CcpA) from Bacillus megaterium is a member of the bacterial repressor protein family GalR-LacI. CcpA functions as master transcriptional regulator of carbon catabolite repression/regulation in firmicutes. Here we present the crystal structure of full-length apo CcpA at 2.5 A resolution from B. megaterium. The structure reveals the location of the helix-turn-helix domain as well as the hinge region, which were not visible due to their high flexibility in earlier crystallographic studies on CcpA molecules. The structure of the apo CcpA homodimer in the present form is in contrast to other reported structures for CcpA.
Asunto(s)
Apoproteínas/química , Apoproteínas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Transcripción Genética/genética , Apoproteínas/genética , Bacillus megaterium/química , Bacillus megaterium/genética , Bacillus megaterium/metabolismo , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Proteínas de Unión al ADN/genética , Dimerización , Modelos Moleculares , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Proteínas Represoras/genéticaRESUMEN
The catalytic centre for light-induced water oxidation in photosystem II (PSII) is a multinuclear metal cluster containing four manganese and one calcium cations. Knowing the structure of this biological catalyst is of utmost importance for unravelling the mechanism of water oxidation in photosynthesis. In this review we describe the current state of the X-ray structure determination at 3.0 A resolution of the water oxidation complex (WOC) of PSII. The arrangement of metal cations in the cluster, their coordination and protein surroundings are discussed with regard to spectroscopic and mutagenesis studies. Limitations of the presently available structural data are pointed out and possible perspectives for the future are outlined, including the combination of X-ray diffraction and X-ray spectroscopy on single crystals.
Asunto(s)
Calcio/química , Manganeso/química , Complejo de Proteína del Fotosistema II/química , Calcio/metabolismo , Manganeso/metabolismo , Oxidación-Reducción , Complejo de Proteína del Fotosistema II/metabolismo , Agua/metabolismo , Difracción de Rayos XRESUMEN
Catabolite control protein A (CcpA) functions as master transcriptional regulator of carbon catabolism in Firmicutes. It belongs to the family of bacterial repressor/regulator proteins. Here, the crystal structure of the 76 kDa homodimeric CcpA protein from Lactococcus lactis subsp. lactis IL1403 is presented at 1.9 A resolution in the absence of cognate DNA. The phases were derived by molecular replacement and the structure was refined to crystallographic R and R(free) factors of 0.177 and 0.211, respectively. The presence of a sulfate molecule in the direct vicinity of a putative effector-binding site in the monomer allowed the derivation of a model for the possible binding of small organic effector molecules.
Asunto(s)
Proteínas Bacterianas/química , Proteínas de Unión al ADN/química , Lactococcus lactis/metabolismo , Proteínas Represoras/química , Transcripción Genética , Proteínas Bacterianas/metabolismo , Cristalización , Cristalografía por Rayos X , Proteínas de Unión al ADN/metabolismo , Lactococcus lactis/química , Conformación Proteica , Pliegue de Proteína , Proteínas Represoras/metabolismoRESUMEN
Photosystem II (PSII) is a homodimeric protein-cofactor complex embedded in the thylakoid membrane that catalyses light-driven charge separation accompanied by the oxidation of water during oxygenic photosynthesis. Biochemical analysis of the lipid content of PSII indicates a number of integral lipids, their composition being similar to the average lipid composition of the thylakoid membrane. The crystal structure of PSII at 3.0 A resolution allowed for the first time the assignment of 14 integral lipids within the protein scaffold, all of them being located at the interface of different protein subunits. The reaction centre subunits D1 and D2 are encircled by a belt of 11 lipids providing a flexible environment for the exchange of D1. Three lipids are located in the dimerization interface and mediate interactions between the PSII monomers. Several lipids are located close to the binding pocket of the mobile plastoquinone Q(B), forming part of a postulated diffusion pathway for plastoquinone. Furthermore two lipids were found, each ligating one antenna chlorophyll a. A detailed analysis of lipid-protein and lipid-cofactor interactions allows to derive some general principles of lipid binding pockets in PSII and to suggest possible functional properties of the various identified lipid molecules.
Asunto(s)
Metabolismo de los Lípidos , Lípidos/química , Complejo de Proteína del Fotosistema II/química , Complejo de Proteína del Fotosistema II/metabolismo , Proteínas/química , Cristalografía por Rayos X , Difusión , Dimerización , Modelos Biológicos , Modelos Químicos , Modelos Moleculares , Oxidación-Reducción , Fotosíntesis/fisiología , Plastoquinona/química , Plastoquinona/metabolismo , Unión Proteica , Proteínas/metabolismo , Tilacoides/química , Agua/metabolismoRESUMEN
The antenna proteins in photosystem II (PSII) not only promote energy transfer to the photosynthetic reaction center (RC) but provide also an efficient cation sink to re-reduce chlorophyll a if the electron transfer (ET) from the Mn-cluster is inhibited. Using the newest PSII dimer crystal structure (3.0 A resolution), in which 11 beta-carotene molecules (Car) and 14 lipids are visible in the PSII monomer, we calculated the redox potentials (Em) of one-electron oxidation for all Car (Em(Car)) by solving the Poisson-Boltzmann equation. In each PSII monomer, the D1 protein harbors a previously unlocated Car (CarD1) in van der Waals contact with the chlorin ring of ChlZ(D1). Each CarD1 in the PSII dimer complex is located in the interface between the D1 and CP47 subunits, together with another four Car of the other PSII monomer and several lipid molecules. The proximity of Car bridging between CarD1 and plastoquinone/Q(A) may imply a direct charge recombination of Car+Q(A)-. The calculated Em(CarD1) and Em(ChlZ(D1)) are, respectively, 83 and 126 mV higher than Em(CarD2) and Em(ChlZ(D2)), which could explain why CarD2+ and ChlZ(D2)+ are observed rather than the corresponding CarD1+ and ChlZ(D1)+.
Asunto(s)
Cianobacterias/química , Modelos Moleculares , Complejo de Proteína del Fotosistema II/química , beta Caroteno/química , Transporte de Electrón/fisiología , Modelos Químicos , Oxidación-Reducción , Complejo de Proteína del Fotosistema II/metabolismo , beta Caroteno/metabolismoRESUMEN
The oxidation of water to dioxygen is catalyzed within photosystem II (PSII) by a Mn(4)Ca cluster, the structure of which remains elusive. Polarized extended x-ray absorption fine structure (EXAFS) measurements on PSII single crystals constrain the Mn(4)Ca cluster geometry to a set of three similar high-resolution structures. Combining polarized EXAFS and x-ray diffraction data, the cluster was placed within PSII, taking into account the overall trend of the electron density of the metal site and the putative ligands. The structure of the cluster from the present study is unlike either the 3.0 or 3.5 angstrom-resolution x-ray structures or other previously proposed models.
Asunto(s)
Calcio/química , Cianobacterias/química , Manganeso/química , Oxígeno/química , Complejo de Proteína del Fotosistema II/química , Agua/química , Sitios de Unión , Cristalización , Cristalografía por Rayos X , Cianobacterias/metabolismo , Análisis de Fourier , Ligandos , Modelos Moleculares , Oxidación-Reducción , Oxígeno/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Análisis Espectral , Agua/metabolismo , Difracción de Rayos X , Rayos XRESUMEN
At the heart of photosynthetic reaction centers (RCs) are pairs of chlorophyll a (Chla), P700 in photosystem I (PSI) and P680 in photosystem II (PSII) of cyanobacteria, algae, or plants, and a pair of bacteriochlorophyll a (BChla), P870 in purple bacterial RCs (PbRCs). These pairs differ greatly in their redox potentials for one-electron oxidation, E(m). For P680, E(m) is 1,100-1,200 mV, but for P700 and P870, E(m) is only 500 mV. Calculations with the linearized Poisson-Boltzmann equation reproduce these measured E(m) differences successfully. Analyzing the origin for these differences, we found as major factors in PSII the unique Mn(4)Ca cluster (relative to PSI and PbRC), the position of P680 close to the luminal edge of transmembrane alpha-helix d (relative to PSI), local variations in the cd loop (relative to PbRC), and the intrinsically higher E(m) of Chla compared with BChla (relative to PbRC).
Asunto(s)
Bacterioclorofilas/química , Clorofila/química , Fotosíntesis , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Oxidación-Reducción , Estructura Secundaria de ProteínaRESUMEN
The crystal structure of photosystem II (PSII) at 3.0-A resolution suggests that titratable residues on the lumenal side of D1/D2 and PsbO form a polar channel, which might serve as a proton exit pathway associated with water oxidation on the Mn-cluster. With full account of protein environment, we calculated the pK(a) of these residues by solving the linearized Poisson-Boltzmann equation. Along the prospective proton channel, the calculated pK(a) of titratable residues (namely via D1-Asp61, D1-Glu65, D2-Glu312, D2-Lys317 D1-Asp59, D1-Arg64, PsbO-Arg152, and PsbO-Asp224) monotonically increase from the Mn-cluster to the lumenal bulk side. We suggest that these residues form the exit pathway guiding protons, which are released at the Mn-cluster as a product of water oxidation, in an exergonic process out of PSII. Upon the S2 to S3 transition, CP43-Arg357 showed a dramatic deprotonation of ca. one H(+), suggesting that this residue is coupled to the redox states of the Mn-cluster and the tyrosine Y(Z). The calculated pK(a) values of 4.2-4.4 for D2-Glu312 and those of approximately 8-10.9 for D1-Asp59 and D1-Arg64 are indicative of the experimentally determined pK(a) values for inhibition of S-state transitions. Upon removal of the atomic coordinates of PsbO, the pK(a) of these residues are dramatically affected, indicating a significant role of PsbO in tuning the pK(a) of those residues in the proton exit pathway.
Asunto(s)
Complejo de Proteína del Fotosistema II/química , Protones , Agua/química , Sitios de Unión , Cadmio/química , Oxidación-ReducciónRESUMEN
An interesting property of certain peptides presented by major histocompatibility complex (MHC) molecules is their acquisition of a dual binding mode within the peptide binding groove. Using x-ray crystallography at 1.4 A resolution, we show here that the glucagon receptor-derived self-peptide pGR ((412)RRRWHRWRL(420)) is presented by the disease-associated human MHC class I subtype HLA-B*2705 in a dual conformation as well, with the middle of the peptide bent toward the floor of the peptide binding groove of the molecule in both binding modes. The conformations of pGR are compared here with those of another self-peptide (pVIPR, RRKWRRWHL) that is also displayed in two binding modes by HLA-B*2705 antigens and with that of the viral peptide pLMP2 (RRRWRRLTV). Conserved structural features suggest that the N-terminal halves of the peptides are crucial in allowing cytotoxic T lymphocyte (CTL) cross-reactivity. In addition, an analysis of T cell receptors (TCRs) from pGR- or pVIPR-directed, HLA-B27-restricted CTL clones demonstrates that TCR from distinct clones but with comparable reactivity may share CDR3alpha but not CDR3beta regions. Therefore, the cross-reactivity of these CTLs depends on TCR-CDR3alpha, is modulated by TCR-CDR3beta sequences, and is ultimately a consequence of the conformational dimorphism that characterizes binding of the self-peptides to HLA-B*2705. These results lend support to the concept that conformational dimorphisms of MHC class I-bound peptides might be connected with the occurrence of self-reactive CTL.
Asunto(s)
Antígeno HLA-B27/química , Péptidos/química , Antígenos/química , Línea Celular Tumoral , Cromo/química , Cristalografía por Rayos X , Electrones , Femenino , Antígenos HLA-B/química , Antígeno HLA-B27/metabolismo , Antígenos de Histocompatibilidad Clase I/química , Humanos , Enlace de Hidrógeno , Leucocitos Mononucleares/citología , Complejo Mayor de Histocompatibilidad , Masculino , Modelos Moleculares , Imitación Molecular , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Glucagón/química , Espondilitis Anquilosante/inmunología , Espondilitis Anquilosante/metabolismo , Linfocitos T/metabolismo , Linfocitos T Citotóxicos/metabolismo , Proteínas de la Matriz Viral/químicaRESUMEN
Oxygenic photosynthesis in plants, algae and cyanobacteria is initiated at photosystem II, a homodimeric multisubunit protein-cofactor complex embedded in the thylakoid membrane. Photosystem II captures sunlight and powers the unique photo-induced oxidation of water to atmospheric oxygen. Crystallographic investigations of cyanobacterial photosystem II have provided several medium-resolution structures (3.8 to 3.2 A) that explain the general arrangement of the protein matrix and cofactors, but do not give a full picture of the complex. Here we describe the most complete cyanobacterial photosystem II structure obtained so far, showing locations of and interactions between 20 protein subunits and 77 cofactors per monomer. Assignment of 11 beta-carotenes yields insights into electron and energy transfer and photo-protection mechanisms in the reaction centre and antenna subunits. The high number of 14 integrally bound lipids reflects the structural and functional importance of these molecules for flexibility within and assembly of photosystem II. A lipophilic pathway is proposed for the diffusion of secondary plastoquinone that transfers redox equivalents from photosystem II to the photosynthetic chain. The structure provides information about the Mn4Ca cluster, where oxidation of water takes place. Our study uncovers near-atomic details necessary to understand the processes that convert light to chemical energy.
Asunto(s)
Cianobacterias/química , Complejo de Proteína del Fotosistema II/química , Complejo de Proteína del Fotosistema II/metabolismo , Cristalografía por Rayos X , Citoplasma/metabolismo , Difusión , Transporte de Electrón , Metales/química , Metales/metabolismo , Modelos Moleculares , Oxígeno/metabolismo , Plastoquinona/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/metabolismoRESUMEN
The content and type of cofactors harboured in the Photosystem II core complex (PS IIcc) of the cyanobacterium Thermosynechococcus elongatus has been determined by biochemical and spectroscopic methods. 17 +/- 1 chlorophyll a per pheophytin a and 0.25 beta-carotene per chlorophyll a have been found in re-dissolved crystals of dimeric PS IIcc. The X-ray crystal structure of PS IIcc from Thermosynechococcus elongatus at 3.2 A resolution clearly shows chlorophyll a molecules arranged in two layers close to the cytoplasmic and lumenal sides of the thylakoid membrane. Each of the cytoplasmic layers contains 9 chlorophyll a, whose positions and orientations are related by a local twofold rotation pseudo-C2 axis passing through the non-haem Fe2+. These chlorophyll a are arranged comparably to those in the antenna domains of PsaA and PsaB of cyanobacterial Photosystem I affirming an evolutionary relation. The chlorophyll a in the lumenal layer are less well conserved between Photosystems I and II and even between CP43 and CP47 with 4 chlorophyll a in the former and 7 in the latter.
Asunto(s)
Complejo de Proteína del Fotosistema II/química , Complejo de Proteína del Fotosistema II/metabolismo , Synechococcus/química , Cromatografía Líquida de Alta Presión , Cristalografía por Rayos X , Modelos Moleculares , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , TemperaturaRESUMEN
X-ray absorption spectroscopy was used to measure the damage caused by exposure to x-rays to the Mn(4)Ca active site in single crystals of photosystem II as a function of dose and energy of x-rays, temperature, and time. These studies reveal that the conditions used for structure determination by x-ray crystallography cause serious damage specifically to the metal-site structure. The x-ray absorption spectra show that the structure changes from one that is characteristic of a high-valent Mn(4)(III(2),IV(2)) oxo-bridged Mn(4)Ca cluster to that of Mn(II) in aqueous solution. This damage to the metal site occurs at a dose that is more than one order of magnitude lower than the dose that results in loss of diffractivity and is commonly considered safe for protein crystallography. These results establish quantitative x-ray dose parameters that are applicable to redox-active metalloproteins. This case study shows that a careful evaluation of the structural intactness of the active site(s) by spectroscopic techniques can validate structures derived from crystallography and that it can be a valuable complementary method before structure-function correlations of metalloproteins can be made on the basis of high-resolution x-ray crystal structures.
Asunto(s)
Calcio/química , Cianobacterias/química , Compuestos de Manganeso/química , Compuestos de Manganeso/efectos de la radiación , Metaloproteínas/química , Complejo de Proteína del Fotosistema II/química , Cristalografía , Relación Dosis-Respuesta en la Radiación , Análisis Espectral , Temperatura , Factores de TiempoRESUMEN
Photosystem II from thylakoid membranes of the thermophilic cyanobacterium Thermosynechococcus elongatus was solubilized with n-beta-dodecylmaltoside and purified using anion exchange chromatography. Molecular weight, pigment stoichiometry and subunit composition were assayed using various techniques. The holocomplex is dimeric with a molecular mass of 756 +/- 18 kDa and functionally fully active. Crystals obtained from these samples showed significantly improved quality leading to a 3D structure at 3.2 A resolution. Several loop regions of the principal protein subunits are now defined that were not interpretable at lower (3.8 A) resolution, thus resulting in a more complete model. The head groups of the cofactors of the electron transfer chain and of the antennae have been modeled, coordinating and hydrogen bonding amino acids identified and the nature of the binding pockets derived. The orientations of these cofactors resemble those of the reaction centre from anoxygenic purple bacteria. For the two plastoquinones, electron density was only found for the head group of QA and none for QB indicating low or even no occupancy of this site in the crystal structure. Both binding pockets and problems related to the QB site are discussed here and compared to the situation in the purple bacterial reaction centre.
Asunto(s)
Cianobacterias/química , Complejo de Proteína del Fotosistema II/química , Complejo de Proteína del Fotosistema II/metabolismo , Plastoquinona/química , Plastoquinona/metabolismo , Sitios de Unión , Modelos Moleculares , Unión Proteica , Conformación Proteica , Subunidades de Proteína , Tilacoides/químicaRESUMEN
Water oxidation generating atmospheric oxygen occurs in photosystem II (PSII), a large protein-pigment complex located in the thylakoid membrane. The recent crystal structures at 3.2 and 3.5 A resolutions provide novel details on amino acid side chains, especially in the D1/D2 subunits. We calculated the redox potentials for one-electron oxidation of the chlorophyll a (Chla) molecules in PSII, considering the protein environment in atomic detail. The calculated redox potentials for the dimer Chla (P(D1/D2)) and accessory Chla (Chl(D1/D2)) were 1.11-1.30 V relative to the normal hydrogen electrode at pH 7, which is high enough for water oxidation. The D1/D2 proteins and their cofactors contribute approximately 390 mV to the enormous upshift of 470 mV compared to the redox potential of monomeric Chla in dimethylformamide. The other subunits are responsible for the remaining 80 mV. The high redox potentials of the two accessory Chla Chl(D1/D2) suggests that they also participate in the charge separation process.
Asunto(s)
Clorofila/química , Clorofila/metabolismo , Complejo de Proteína del Fotosistema II/química , Complejo de Proteína del Fotosistema II/metabolismo , Clorofila A , Cristalografía por Rayos X , Cianobacterias , Dimerización , Oxidación-Reducción , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Electricidad EstáticaRESUMEN
Accessory chlorophylls (B(A/B)) in bacterial photosynthetic reaction center play a key role in charge-separation. Although light-exposed and dark-adapted bRC crystal structures are virtually identical, the calculated B(A) redox potentials for one-electron reduction differ. This can be traced back to different orientations of the B(A) ester-group. This tuning ability of chlorophyll redox potentials modulates the electron transfer from SP* to B(A).
Asunto(s)
Clorofila/metabolismo , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Cristalografía por Rayos X , Transporte de Electrón , Ésteres/metabolismo , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Conformación Proteica , Rhodobacter sphaeroides/metabolismoRESUMEN
The product of the human leukocyte antigen (HLA) gene HLA-B*2703 differs from that of the prototypical subtype HLA-B*2705 by a single amino acid at heavy-chain residue 59 that is involved in anchoring the peptide N-terminus within the A pocket of the molecule. Two B*2703-peptide complexes were crystallized using the hanging-drop vapour-diffusion method using PEG 8000 as a precipitant. The crystals belong to space group P2(1) (pVIPR peptide) or P2(1)2(1)2(1) (pLMP2 peptide). Data sets were collected to 1.55 A (B*2703-pVIPR) or 2.0 A (B*2703-pLMP2) resolution using synchrotron radiation. With B*2705-pVIPR as a search model, a clear molecular-replacement solution was found for both B*2703 complexes.