Biomedical subjects

I Fita

Publications and source records attributed to I Fita.

At least 19 recordsLinked to original sources

PubMed2004

Diversity of structures and properties among catalases.

More than 300 catalase sequences are now available, divided among monofunctional catalases (> 225), bifunctional catalase-peroxidases (> 50) and manganese-containing catalases (> 25). When combined with the recent appearance of crystal structures from at least two representatives from each of these groups (nine from the monofunctional catalases), valuable insights into the catalatic reaction mechanism in its various forms and into catalase evolution have been gained. The structures have revealed an unusually large number of modifications unique to catalases, a result of interacting with reactive oxygen species. Biochemical and physiological characterization of catalases from many different organisms has revealed a surprisingly wide range of catalatic efficiencies, despite similar sequences. Catalase gene expression in micro-organisms generally is controlled either by sensors of reactive oxygen species or by growth phase regulons, although the detailed mechanisms vary considerably.

Bacteria↗

PubMed2001

Theoretical study of the mechanisms of substrate recognition by catalase.

A variety of theoretical methods including classical molecular interaction potentials, classical molecular dynamics, and activated molecular dynamics have been used to analyze the substrate recognition mechanisms of peroxisomal catalase from Saccharomyces cerevisiae. Special attention is paid to the existence of channels connecting the heme group with the exterior of the protein. On the basis of these calculations a rationale is given for the unique catalytic properties of this enzyme, as well as for the change in enzyme efficiency related to key mutations. According to our calculations the water is expected to be a competitive inhibitor of the enzyme, blocking the access of hydrogen peroxide to the active site. The main channel is the preferred route for substrate access to the enzyme and shows a cooperative binding to hydrogen peroxide. However, the overall affinity of the main channel for H(2)O(2) is only slightly larger than that for H(2)O. Alternative channels connecting the heme group with the monomer interface and the NADP(H) binding site are detected. These secondary channels might be important for product release.

Catalase↗

PubMed2001

Structure of the C2 domain from novel protein kinase Cepsilon. A membrane binding model for Ca(2+)-independent C2 domains.

Protein kinase Cepsilon (PKCepsilon) is a member of the novel PKCs which are activated by acidic phospholipids, diacylglycerol and phorbol esters, but lack the calcium dependence of classical PKC isotypes. The crystal structures of the C2 domain of PKCepsilon, crystallized both in the absence and in the presence of the two acidic phospholipids, 1,2-dicaproyl-sn-phosphatidyl-l-serine (DCPS) and 1,2-dicaproyl-sn-phosphatidic acid (DCPA), have now been determined at 2.1, 1.7 and 2.8 A resolution, respectively. The central feature of the PKCepsilon-C2 domain structure is an eight-stranded, antiparallel, beta-sandwich with a type II topology similar to that of the C2 domains from phospholipase C and from novel PKCdelta. Despite the similar topology, important differences are found between the structures of C2 domains from PKCs delta and epsilon, suggesting they be considered as different PKC subclasses. Site-directed mutagenesis experiments and structural changes in the PKCepsilon-C2 domain from crystals with DCPS or DCPA indicate, though phospholipids were not visible in these structures, that loops joining strands beta1-beta2 and beta5-beta6 participate in the binding to anionic membranes. The different behavior in membrane-binding and activation between PKCepsilon and classical PKCs appears to originate in localized structural changes, which include a major reorganization of the region corresponding to the calcium binding pocket in classical PKCs. A mechanism is proposed for the interaction of the PKCepsilon-C2 domain with model membranes that retains basic features of the docking of C2 domains from classical, calcium-dependent, PKCs.

Amino Acid Sequence↗

PubMed2001

Substrate flow in catalases deduced from the crystal structures of active site variants of HPII from Escherichia coli.

The active site of heme catalases is buried deep inside a structurally highly conserved homotetramer. Channels leading to the active site have been identified as potential routes for substrate flow and product release, although evidence in support of this model is limited. To investigate further the role of protein structure and molecular channels in catalysis, the crystal structures of four active site variants of catalase HPII from Escherichia coli (His128Ala, His128Asn, Asn201Ala, and Asn201His) have been determined at approximately 2.0-A resolution. The solvent organization shows major rearrangements with respect to native HPII, not only in the vicinity of the replaced residues but also in the main molecular channel leading to the heme distal pocket. In the two inactive His128 variants, continuous chains of hydrogen bonded water molecules extend from the molecular surface to the heme distal pocket filling the main channel. The differences in continuity of solvent molecules between the native and variant structures illustrate how sensitive the solvent matrix is to subtle changes in structure. It is hypothesized that the slightly larger H(2)O(2) passing through the channel of the native enzyme will promote the formation of a continuous chain of solvent and peroxide. The structure of the His128Asn variant complexed with hydrogen peroxide has also been determined at 2.3-A resolution, revealing the existence of hydrogen peroxide binding sites both in the heme distal pocket and in the main channel. Unexpectedly, the largest changes in protein structure resulting from peroxide binding are clustered on the heme proximal side and mainly involve residues in only two subunits, leading to a departure from the 222-point group symmetry of the native enzyme. An active role for channels in the selective flow of substrates through the catalase molecule is proposed as an integral feature of the catalytic mechanism. The Asn201His variant of HPII was found to contain unoxidized heme b in combination with the proximal side His-Tyr bond suggesting that the mechanistic pathways of the two reactions can be uncoupled.

Alleles↗

PubMed2001

Crystallization and preliminary X-ray analysis of clade I catalases from Pseudomonas syringae and Listeria seeligeri.

Haem-containing catalases are homotetrameric molecules that degrade hydrogen peroxide. Phylogenetically, the haem-containing catalases can be grouped into three main lines or clades. The crystal structures of seven catalases have been determined, all from clades II and III. In order to obtain a structure of an enzyme from clade I, which includes all plant, algae and some bacterial enzymes, two bacterial catalases, CatF from Pseudomonas syringae and Kat from Listeria seeligeri, have been crystallized by the hanging-drop vapour-diffusion technique, using PEG and ammonium sulfate as precipitants, respectively. Crystals of P. syringae CatF, with a plate-like morphology, belong to the monoclinic space group P2(1), with unit-cell parameters a = 60.6, b = 153.9, c = 109.2 A, beta = 102.8 degrees. From these crystals a diffraction data set to 1.8 A resolution with 98% completeness was collected using synchrotron radiation. Crystals of L. seeligeri Kat, with a well developed bipyramidal morphology, belong to space group I222 (or I2(1)2(1)2(1)), with unit-cell parameters a = 74.4, b = 121.3, c = 368.5 A. These crystals diffracted beyond 2.2 A resolution when using synchrotron radiation, but presented anisotropic diffraction, with the weakest direction perpendicular to the long c axis.

Catalase↗

PubMed2000

Structural and biochemical features distinguish the foot-and-mouth disease virus leader proteinase from other papain-like enzymes.

The structures of the two leader protease (Lpro) variants of foot-and-mouth disease virus known to date were solved using crystals in which molecules were organized as molecular fibers. Such crystals diffract to a resolution of only approximately 3 A. This singular, pseudo-polymeric organization is present in a new Lpro crystal form showing a cubic packing. As molecular fiber formation appeared unrelated to crystallization conditions, we mutated the reactive cysteine 133 residue, which makes a disulfide bridge between adjacent monomers in the fibers, to serine. None of the intermolecular contacts found in the molecular fibers was present in crystals of this variant. Analysis of this Lpro structure, refined at 1.9 A resolution, enables a detailed definition of the active center of the enzyme, including the solvent organization. Assay of Lpro activity on a fluorescent hexapeptide substrate showed that Lpro, in contrast to papain, was highly sensitive to increases in the cation concentration and was active only across a narrow pH range. Examination of the Lpro structure revealed that three aspartate residues near the active site, not present in papain-like enzymes, are probably responsible for these properties.

Amino Acid Substitution↗

PubMed2000

Structure of human rhinovirus serotype 2 (HRV2).

Human rhinoviruses are classified into a major and a minor group based on their binding to ICAM-1 or to members of the LDL-receptor family, respectively. They can also be divided into groups A and B, according to their sensitivity towards a panel of antiviral compounds. The structure of human rhinovirus 2 (HRV2), which uses the LDL receptor for cell attachment and is included in antiviral group B, has been solved and refined at 2.6 A resolution by X-ray crystallography to gain information on the peculiarities of rhinoviruses, in particular from the minor receptor group. The main structural differences between HRV2 and other rhinoviruses, including the minor receptor group serotype HRV1A, are located at the internal protein shell surface and at the external antigenic sites. In the interior, the N termini of VP1 and VP4 form a three-stranded beta-sheet in an arrangement similar to that present in poliovirus, although myristate was not visible at the amino terminus of VP4 in the HRV2 structure. The betaE-betaF loop of VP2, a linear epitope within antigenic site B recognized by monoclonal antibody 8F5, adopts a conformation considerably different from that found in the complex of 8F5 with a synthetic peptide of the same sequence. This either points to considerable structural changes impinged on this loop upon antibody binding, or to the existence of more than one single conformation of the loop when the virus is in solution. The hydrophobic pocket of VP1 was found to be occupied by a pocket factor apparently identical with that present in the major receptor group virus HRV16. Electron density, consistent with the presence of a viral RNA fragment, is seen stacked against a conserved tryptophan residue.

Antibodies, Viral↗

PubMed2000

The 1.5 A resolution crystal structure of the carbamate kinase-like carbamoyl phosphate synthetase from the hyperthermophilic Archaeon pyrococcus furiosus, bound to ADP, confirms that this thermostable enzyme is a carbamate kinase, and provides insight into substrate binding and stability in carbamate kinases.

Carbamoyl phosphate (CP), an essential precursor of arginine and the pyrimidine bases, is synthesized by CP synthetase (CPS) in three steps. The last step, the phosphorylation of carbamate, is also catalyzed by carbamate kinase (CK), an enzyme used by microorganisms to produce ATP from ADP and CP. Although the recently determined structures of CPS and CK show no obvious mutual similarities, a CK-like CPS reported in hyperthermophilic archaea was postulated to be a missing link in the evolution of CP biosynthesis. The 1.5 A resolution structure of this enzyme from Pyrococcus furiosus shows both a subunit topology and a homodimeric molecular organization, with a 16-stranded open beta-sheet core surrounded by alpha-helices, similar to those in CK. However, the pyrococcal enzyme exhibits many solvent-accessible ion-pairs, an extensive, strongly hydrophobic, intersubunit surface, and presents a bound ADP molecule, which does not dissociate at 22 degrees C from the enzyme. The ADP nucleotide is sequestered in a ridge formed over the C-edge of the core sheet, at the bottom of a large cavity, with the purine ring enclosed in a pocket specific for adenine. Overall, the enzyme structure is ill-suited for catalyzing the characteristic three-step reaction of CPS and supports the view that the CK-like CPS is in fact a highly thermostable and very slow (at 37 degrees C) CK that, in the extreme environment of P. furiosus, may have the new function of making, rather than using, CP. The thermostability of the enzyme may result from the extension of the hydrophobic intersubunit contacts and from the large number of exposed ion-pairs, some of which form ion-pair networks across several secondary structure elements in each enzyme subunit. The structure provides the first information on substrate binding and catalysis in CKs, and suggests that the slow rate at 37 degrees C is possibly a consequence of slow product dissociation.

Adenosine Diphosphate↗

PubMed2000

The need for a shared database infrastructure: combining X-ray crystallography and electron microscopy.

Advances in structural biology are opening greater opportunities for understanding biological structures from the cellular to the atomic level. Particularly promising are the links that can be established between the information provided by electron microscopy and the atomic structures derived from X-ray crystallography and nuclear magnetic resonance spectroscopy. Combining such different kinds of structural data can result in novel biological information on the interaction of biomolecules in large supramolecular assemblies. As a consequence, the need to develop new databases in the field of structural biology that allow for an integrated access to data from all the experimental techniques is becoming critical. Pilot studies performed in recent years have already established a solid background as far as the basic information that an integrated macromolecular structure database should contain, as well as the basic principles for integration. These efforts started in the context of the BioImage project, and resulted in a first complete database prototype that provided a versatile platform for the linking of atomic models or X-ray diffraction data with electron microscopy information. Analysis of the requirements needed to combine data at different levels of resolution have resulted in sets of specifications that make possible the integration of all these different types in the context of a web environment. The case of a structural study linking electron microscopy and X-ray data, which is already contained within the BioImage data base and in the Protein Data Bank, is used here to illustrate the current approach, while a general discussion highlights the urgent need for integrated databases.

Crystallography, X-Ray↗

PubMed2000

A multiply substituted G-H loop from foot-and-mouth disease virus in complex with a neutralizing antibody: a role for water molecules.

The crystal structure of a 15 amino acid synthetic peptide, corresponding to the sequence of the major antigenic site A (G-H loop of VP1) from a multiple variant of foot-and-mouth disease virus (FMDV), has been determined at 2.3 A resolution. The variant peptide includes four amino acid substitutions in the loop relative to the previously studied peptide representing FMDV C-S8c1 and corresponds to the loop of a natural FMDV isolate of subtype C(1). The peptide was complexed with the Fab fragment of the neutralizing monoclonal antibody 4C4. The peptide adopts a compact fold with a nearly cyclic conformation and a disposition of the receptor-recognition motif Arg-Gly-Asp that is closely related to the previously determined structure for the viral loop, as part of the virion, and for unsubstituted synthetic peptide antigen bound to neutralizing antibodies. New structural findings include the observation that well-defined solvent molecules appear to play a major role in stabilizing the conformation of the peptide and its interactions with the antibody. Structural results are supported by molecular-dynamic simulations. The multiply substituted peptide developed compensatory mechanisms to bind the antibody with a conformation very similar to that of its unsubstituted counterpart. One water molecule, which for steric reasons could not occupy the same position in the unsubstituted antigen, establishes hydrogen bonds with three peptide amino acids. The constancy of the structure of an antigenic domain despite multiple amino acid substitutions has implications for vaccine design.

Amino Acid Substitution↗

PubMed1999

Ca(2+) bridges the C2 membrane-binding domain of protein kinase Calpha directly to phosphatidylserine.

The C2 domain acts as a membrane-targeting module in a diverse group of proteins including classical protein kinase Cs (PKCs), where it plays an essential role in activation via calcium-dependent interactions with phosphatidylserine. The three-dimensional structures of the Ca(2+)-bound forms of the PKCalpha-C2 domain both in the absence and presence of 1, 2-dicaproyl-sn-phosphatidyl-L-serine have now been determined by X-ray crystallography at 2.4 and 2.6 A resolution, respectively. In the structure of the C2 ternary complex, the glycerophosphoserine moiety of the phospholipid adopts a quasi-cyclic conformation, with the phosphoryl group directly coordinated to one of the Ca(2+) ions. Specific recognition of the phosphatidylserine is reinforced by additional hydrogen bonds and hydrophobic interactions with protein residues in the vicinity of the Ca(2+) binding region. The central feature of the PKCalpha-C2 domain structure is an eight-stranded, anti-parallel beta-barrel with a molecular topology and organization of the Ca(2+) binding region closely related to that found in PKCbeta-C2, although only two Ca(2+) ions have been located bound to the PKCalpha-C2 domain. The structural information provided by these results suggests a membrane binding mechanism of the PKCalpha-C2 domain in which calcium ions directly mediate the phosphatidylserine recognition while the calcium binding region 3 might penetrate into the phospholipid bilayer.

Amino Acid Sequence↗

PubMed1999

Mutants that alter the covalent structure of catalase hydroperoxidase II from Escherichia coli.

The three-dimensional structures of two HPII variants, V169C and H392Q, have been determined at resolutions of 1.8 and 2.1 A, respectively. The V169C variant contains a new type of covalent bond between the sulfur atom of Cys(169) and a carbon atom on the imidazole ring of the essential His(128). This variant enzyme has only residual catalytic activity and contains heme b. The chain of water molecules visible in the main channel may reflect the organization of the hydrogen peroxide substrates in the active enzyme. Two alternative mechanisms, involving either compound I or free radical intermediates, are presented to explain the formation of the Cys-His covalent bond. The H392Q and H392E variants exhibit 75 and 25% of native catalytic activity, respectively. The Gln(392) variant contains only heme b, whereas the Glu(392) variant contains a mixture of heme b and cis and trans isomers of heme d, suggesting of a role for this residue in heme conversion. Replacement of either Gln(419) and Ser(414), both of which interact with the heme, affected the cis:trans ratio of spirolactone heme d. Implications for the heme oxidation mechanism and the His-Tyr bond formation in HPII are considered.

Amino Acid Sequence↗

PubMed1999

The carbamoyl-phosphate synthetase of Pyrococcus furiosus is enzymologically and structurally a carbamate kinase.

The hyperthermophiles Pyrococcus furiosus and Pyrococcus abyssi make pyrimidines and arginine from carbamoyl phosphate (CP) synthesized by an enzyme that differs from other carbamoyl-phosphate synthetases and that resembles carbamate kinase (CK) in polypeptide mass, amino acid sequence, and oligomeric organization. This enzyme was reported to use ammonia, bicarbonate, and two ATP molecules as carbamoyl-phosphate synthetases to make CP and to exhibit bicarbonatedependent ATPase activity. We have reexamined these findings using the enzyme of P. furiosus expressed in Escherichia coli from the corresponding gene cloned in a plasmid. We show that the enzyme uses chemically made carbamate rather than ammonia and bicarbonate and catalyzes a reaction with the stoichiometry and equilibrium that are typical for CK. Furthermore, the enzyme catalyzes actively full reversion of the CK reaction and exhibits little bicarbonate-dependent ATPase. In addition, it cross-reacts with antibodies raised against CK from Enterococcus faecium, and its three-dimensional structure, judged by x-ray crystallography of enzyme crystals, is very similar to that of CK. Thus, the enzyme is, in all respects other than its function in vivo, a CK. Because in other organisms the function of CK is to make ATP from ADP and CP derived from arginine catabolism, this is the first example of using CK for making rather than using CP. The reasons for this use and the adaptation of the enzyme to this new function are discussed.

Adenosine Diphosphate↗

PubMed1999

Flexibility of the major antigenic loop of foot-and-mouth disease virus bound to a Fab fragment of a neutralising antibody: structure and neutralisation.

The interaction of foot-and-mouth disease virus (FMDV) serotype C (clone C-S8c1) with a strongly neutralising monoclonal antibody (MAb) 4C4 has been studied by combining data from cryoelectron microscopy and x-ray crystallography. The MAb 4C4 binds to the exposed flexible GH-loop of viral protein 1 (VP1), which appears to retain its flexibility, allowing movement of the bound Fab. This is in striking contrast to MAb SD6, which binds to the same GH-loop of VP1 but exhibits no movement of the bound Fab when observed under identical conditions. However, MAbs 4C4 and SD6 have very similar neutralisation characteristics. The known atomic structure of FMDV C-S8c1 and that of the 4C4 Fab cocrystallised with a synthetic peptide corresponding to the GH-loop of VP1 were fitted to the cryoelectron microscope density map. The best fit of the 4C4 Fab is compatible only with monovalent binding of the MAb in agreement with the neutralisation data on 4C4 MAbs, Fab2s, and Fabs. The position of the bound GH-loop is related to other known positions of this loop by a hinge rotation about the base of the loop. The 4C4 Fab appears to interact almost exclusively with the G-H loop of VP1, making no other contacts with the viral capsid.

Animals↗

PubMed1999

Structure of catalase-A from Saccharomyces cerevisiae.

The structure of the peroxisomal catalase A from the budding yeast Saccharomyces cerevisiae, with 515 residues per subunit, has been determined and refined to 2.4 A resolution. The crystallographic agreement factors R and Rfree are 15.4% and 19.8%, respectively. A tetramer with accurate 222-molecular symmetry is located in the asymmetric unit of the crystal. The conformation of the central core of catalase A, about 300 residues, remains similar to the structure of catalases from distantly related organisms. In contrast, catalase A lacks a carboxy-terminal domain equivalent to that found in catalase from Penicillium vitalae, the only other fungal catalase structure available. Structural peculiarities related with the heme and NADP(H) binding pockets can be correlated with biochemical characteristics of the catalase A enzyme. The network of molecular cavities and channels, filled with solvent molecules, supports the existence of one major substrate entry and at least two possible alternative pathways to the heme active site. The structure of the variant protein Val111Ala, also determined by X-ray crystallography at 2.8 A resolution, shows a few, well-localized, differences with respect to the wild-type enzyme. These differences, that include the widening of the entry channel in its narrowest point, provide an explanation for both the increased peroxidatic activity and the reduced catalatic activity of this mutant.

Alanine↗

PubMed1999

Structure of catalase HPII from Escherichia coli at 1.9 A resolution.

Catalase HPII from Escherichia coli, a homotetramer of subunits with 753 residues, is the largest known catalase. The structure of native HPII has been refined at 1.9 A resolution using X-ray synchrotron data collected from crystals flash-cooled with liquid nitrogen. The crystallographic agreement factors R and R(free) are respectively 16.6% and 21.0%. The asymmetric unit of the crystal contains a whole molecule that shows accurate 222-point group symmetry. The structure of the central part of the HPII subunit gives a root mean square deviation of 1.5 A for 477 equivalencies with beef liver catalase. Most of the additional 276 residues of HPII are located in either an extended N-terminal arm or in a C-terminal domain organized with a flavodoxin-like topology. A small number of mostly hydrophilic interactions stabilize the relative orientation between the C-terminal domain and the core of the enzyme. The heme component of HPII is a cis-hydroxychlorin gamma-spirolactone in an orientation that is flipped 180 degrees with respect to the orientation of the heme found in beef liver catalase. The proximal ligand of the heme is Tyr415 which is joined by a covalent bond between its Cbeta atom and the Ndelta atom of His392. Over 2,700 well-defined solvent molecules have been identified filling a complex network of cavities and channels formed inside the molecule. Two channels lead close to the distal side heme pocket of each subunit suggesting separate inlet and exhaust functions. The longest channel, that begins in an adjacent subunit, is over 50 A in length, and the second channel is about 30 A in length. A third channel reaching the heme proximal side may provide access for the substrate needed to catalyze the heme modification and His-Tyr bond formation. HPII does not bind NADPH and the equivalent region to the NADPH binding pocket of bovine catalase, partially occluded in HPII by residues 585-590, corresponds to the entrance to the second channel. The heme distal pocket contains two solvent molecules, and the one closer to the iron atom appears to exhibit high mobility or low occupancy compatible with weak coordination.

Amino Acid Sequence↗

PubMed1999

Organising multi-dimensional biological image information: the BioImage Database.

Nowadays it is possible to unravel complex information at all levels of cellular organization by obtaining multi-dimensional image information. At the macromolecular level, three-dimensional (3D) electron microscopy, together with other techniques, is able to reach resolutions at the nanometer or subnanometer level. The information is delivered in the form of 3D volumes containing samples of a given function, for example, the electron density distribution within a given macromolecule. The same situation happens at the cellular level with the new forms of light microscopy, particularly confocal microscopy, all of which produce biological 3D volume information. Furthermore, it is possible to record sequences of images over time (videos), as well as sequences of volumes, bringing key information on the dynamics of living biological systems. It is in this context that work on BioImage started two years ago, and that its first version is now presented here. In essence, BioImage is a database specifically designed to contain multi-dimensional images, perform queries and interactively work with the resulting multi-dimensional information on the World Wide Web, as well as accomplish the required cross-database links. Two sister home pages of BioImage can be accessed at http://www. bioimage.org and http://www-embl.bioimage.org

Bacterial Proteins↗

PubMed1999

Biochemical and structural studies with neutralizing antibodies raised against foot-and-mouth disease virus.

The function of a loop exposed on the aphthovirus capsid (the G-H loop of protein VP1) has been explored by combining genetic and structural studies with viral mutants. The loop displays a dual function of receptor recognition and interaction with neutralizing antibodies. Remarkably, some amino acid residues play a critical role in both such disparate functions. Therefore residues subjected to antibody pressure for variation may nevertheless maintain a role in receptor recognition for which invariance is a requirement. Evolution of FMDV in cell culture may relax the requirements at this site and allow further increase of antigenic diversification. Essential residues at one stage of virus evolution may become dispensable at another not very distant point in the evolutionary landscape. Implications for FMDV evolution and vaccine design are discussed.

Animals↗