Oral ganciclovir versus valganciclovir for cytomegalovirus prophylaxis in high-risk liver transplant recipients.

This retrospective review compared oral valganciclovir (VGCV) 450 mg daily for 6 months versus oral ganciclovir (GCV) 1000 mg 3 times daily for 3 months in preventing cytomegalovirus (CMV) disease in high-risk liver transplant recipients.We evaluated all CMV donor positive-recipient negative liver transplant recipients managed at University Health System in San Antonio,Texas from August 1996 to September 2006. CMV disease was confirmed by polymerase chain-reaction or antigenemia assay, and CMV invasive disease by tissue biopsy. Patient demographics, laboratory results, complications, and therapies were collected via retrospective chart review. Patients < 18 years of age or those who died during transplant admission were excluded. Primary endpoints included incidence, onset, and severity of CMV disease up to 1 year post transplant. Data collection also included patient demographics, immunosuppression, CMV treatment regimens, and relevant lab results. A total of 64 patients (43 VGCV and 21 GCV) were identified. Four patients developed CMV disease:VGCV (3/43,7%) versus GCV (1/21, 5%) (P=1.0), with 1 VGCV patient experiencing tissue-invasive CMV. In all cases, onset of CMV disease occurred after prophylaxis was discontinued. Onset occurred at 24, 27, and 45 weeks post transplant in the VGCVgroup, and at 26 weeks in the 1 patient on GCV. Four patients received rabbit antithymocyte globulin (rATG) induction; 1 patient received rATG and developed CMV disease, with no statistical difference compared with the 3 remaining patients who received rATG but did not develop CMV disease (P=0.09). No difference was found in incidence of CMV disease between patients who received GCV and those who received VGCV at our institution.

A preliminary account of the properties of recombinant human Glyoxylate reductase (GRHPR), LDHA and LDHB with glyoxylate, and their potential roles in its metabolism.

Human lactate dehydrogenase (LDH) is thought to contribute to the oxidation of glyoxylate to oxalate and thus to the pathogenesis of disorders of endogenous oxalate overproduction. Glyoxylate reductase (GRHPR) has a potentially protective role metabolising glyoxylate to the less reactive glycolate. In this paper, the kinetic parameters of recombinant human LDHA, LDHB and GR have been compared with respect to their affinity for glyoxylate and related substrates. The Km values and specificity constants (Kcat/K(M)) of purified recombinant human LDHA, LDHB and GRHPR were determined for the reduction of glyoxylate and hydroxypyruvate. K(M) values with glyoxylate were 29.3 mM for LDHA, 9.9 mM for LDHB and 1.0 mM for GRHPR. For the oxidation of glyoxylate, K(M) values were 0.18 mM and 0.26 mM for LDHA and LDHB respectively with NAD+ as cofactor. Overall, under the same reaction conditions, the specificity constants suggest there is a fine balance between the reduction and oxidation reactions of these substrates, suggesting that control is most likely dictated by the ambient concentrations of the respective intracellular cofactors. Neither LDHA nor LDHB utilised glycolate as substrate and NADPH was a poor cofactor with a relative activity less than 3% that of NADH. GRHPR had a higher affinity for NADPH than NADH (K(M) 0.011 mM vs. 2.42 mM). The potential roles of LDH isoforms and GRHPR in oxalate synthesis are discussed.

Overcoming cloning problems by staining agarose gels with crystal violet instead of ethidium bromide in lactate dehydrogenase gene from Plasmodium vivax and Plasmodium falciparum.

In this study, lactate dehydrogenase gene from Plasmodium vivax has been tried to subclone into an expression vector. Some of the Plasmodium falciparum lactate dehydrogenase mutant genes have also been tried to clone and subclone into a vector, but we failed to clone or subclone either of the genes. DNA visualisation in electrophoretic gels typically requires UV radiation and the fluorecent dye ethidium bromide. A crystal violet-stained gel was run instead of an ethidium bromide gel and so avoided the use of UV radiation. This enabled us to clone or subclone both Plasmodium vivax lactate dehydrogenase gene and Plasmodium falciparum lactate dehydrogenase mutant genes into any desired vector.

Crystal structure of Plasmodium berghei lactate dehydrogenase indicates the unique structural differences of these enzymes are shared across the Plasmodium genus.

As Plasmodium rely extensively on homolactic fermentation for energy production, Plasmodium falciparum lactate dehydrogenase (PfLDH)--the key enzyme in this process--has previously been suggested as a novel target for antimalarials. This enzyme has distinctive kinetic and structural properties that distinguish it from its human homologues. In this study, we now describe the expression, kinetic characterisation and crystal structure determination of the LDH from Plasmodium berghei. This enzyme is seen to have a similar kinetic profile to its P. falciparum counterpart, exhibiting the characteristic lack of substrate inhibition that distinguishes plasmodial from human LDHs. The crystal structure of P. berghei lactate dehydrogenase (PbLDH) shows a very similar active site arrangement to the P. falciparum enzyme. In particular, an insertion of five amino acid residues in the active site loop creates an enlarged volume in the substrate binding site, and characteristic changes in the residues lining the NADH cofactor binding pocket result in displacement of the cofactor relative to its observed position in mammalian and all other LDH structures. These results imply the special features previously described for PfLDH may be shared across the Plasmodium genus, supporting the universal application of therapeutics targeting this enzyme.

The crystal structure of PEBP-2, a homologue of the PEBP/RKIP family.

Proteins from the PEBP (phosphatidylethanolamine-binding protein) family have been identified in a wide variety of species and are thought to regulate a range of intracellular signalling cascades. The rat homologue (known as RKIP; Raf-1 kinase inhibitor protein) has been shown to negatively regulate the MAP kinase pathway through formation of inhibitory complexes with Raf-1 and MEK. The crystal structure of a new, murine member of the PEBP family, termed mPEBP-2, has been determined. On the basis of amino-acid homology, mPEBP-2 belongs to a distinct subset of the mammalian PEBP proteins. Nonetheless, mPEBP-2 is seen to be very similar in structure to other PEBP proteins from human, bovine and plant sources. Regions of distinctive sequence associated with the PEBP-2 subset are discussed with reference to this structure.

A reassessment of the MAdCAM-1 structure and its role in integrin recognition.

Mucosal addressin cell-adhesion molecule (MAdCAM-1) is a membrane-bound leukocyte receptor regulating both the passage and retention of leukocytes in mucosal tissues. A crystal structure for the two extracellular amino-terminal domains of human MAdCAM-1 has previously been reported, confirming their expected immunoglobulin superfamily topology. In this study, a second crystal structure of this fragment is described. Although the overall structure is similar to that previously reported, one edge strand in the amino-terminal domain is instead located on the opposite sheet. This alters the arrangement and conformation of amino acids in this region that have previously been shown to be crucial for ligand binding. MAdCAM-1 is also seen to form dimers within the crystal lattice, raising the possibility that oligomerization may influence the biological role of this adhesion molecule.

Specificity in Trk receptor:neurotrophin interactions: the crystal structure of TrkB-d5 in complex with neurotrophin-4/5.

BACKGROUND: The binding of neurotrophin ligands to their respective Trk cellular receptors initiates intracellular signals essential for the growth and survival of neurons. The site of neurotrophin binding has been located to the fifth extracellular domain of the Trk receptor, with this region regulating both the affinity and specificity of Trk receptor:neurotrophin interaction. Neurotrophin function has been implicated in a number of neurological disorders, including Alzheimer's disease and Parkinson's disease. RESULTS: We have determined the 2.7 A crystal structure of neurotrophin-4/5 bound to the neurotrophin binding domain of its high-affinity receptor TrkB (TrkB-d5). As previously seen in the interaction of nerve growth factor with TrkA, neurotrophin-4/5 forms a crosslink between two spatially distant receptor molecules. The contacts formed in the TrkB-d5:neurotrophin-4/5 complex can be divided into a conserved area similar to a region observed in the TrkA-d5:NGF complex and a second site-unique in each ligand-receptor pair-formed primarily by the ordering of the neurotrophin N terminus. CONCLUSIONS: Together, the structures of the TrkB-d5:NT-4/5 and TrkA-d5:NGF complexes confirm a consistent pattern of recognition in Trk receptor:neurotrophin complex formation. In both cases, the N terminus of the neurotrophin becomes ordered only on complex formation. This ordering appears to be directed largely by the receptor surface, with the resulting complementary surfaces providing the main determinant of receptor specificity. These features provide an explanation both for the limited crossreactivity observed between the range of neurotrophins and Trk receptors and for the high-affinity binding associated with respective ligand-receptor pairs.

Stability and folding rates of domains spanning the large A-band super-repeat of titin.

Titin is a very large (>3 MDa) protein found in striated muscle where it is believed to participate in myogenesis and passive tension. A prominent feature in the A-band portion of titin is the presence of an 11-domain super-repeat of immunoglobulin superfamily and fibronectin-type-III-like domains. Seven overlapping constructs from human cardiac titin, each consisting of two or three domains and together spanning the entire 11-domain super-repeat, have been expressed in Escherichia coli. Fluorescence unfolding experiments and circular dichroism spectroscopy have been used to measure folding stabilities for each of the constructs and to assign unfolding rates for each super-repeat domain. Immunoglobulin superfamily domains were found to fold correctly only in the presence of their C-terminal fibronectin type II domain, suggesting close and possibly rigid association between these units. The domain stabilities, which range from 8.6 to 42 kJ mol(-1) under physiological conditions, correlate with previously reported mechanical forces required to unfold titin domains. Individual domains vary greatly in their rates of unfolding, with a range of unfolding rate constants between 2.6 x 10(-6) and 1.2 s(-1). This variation in folding behavior is likely to be an important determinant in ensuring independent folding of domains in multi-domain proteins such as titin.

Intercellular adhesion molecule-4 binds alpha(4)beta(1) and alpha(V)-family integrins through novel integrin-binding mechanisms.

The LW blood group glycoprotein, ICAM-4, is a member of the intercellular adhesion molecule (ICAM) family expressed in erythroid cells. To begin to address the function of this molecule, ligands for ICAM-4 on hemopoietic and nonhemopoietic cell lines were identified. Peptide inhibition studies suggest that adhesion of cell lines to an ICAM-4-Fc construct is mediated by an LDV-inhibitable integrin on hemopoietic cells and an RGD-inhibitable integrin on nonhemopoietic cells. Antibody inhibition studies identified the hemopoietic integrin as alpha(4)beta(1.) Antibody inhibition studies on alpha(4)beta(1)-negative, nonhemopoietic cell lines suggested that adhesion of these cells is mediated by alpha(V) integrins (notably alpha(V)beta(1) and alpha(V)beta(5)). The structure of ICAM-4 modeled on the crystal structure of ICAM-2 was used to identify surface-exposed amino acid residues for site-directed mutagenesis. Neither an unusual LETS nor an LDV motif in the first domain of ICAM-4 was critical for integrin binding. ICAM-4 is the first ICAM family member shown to be a ligand for integrins other than those of the beta(2) family, and the data suggest that ICAM-4 has a novel integrin-binding site(s). These findings suggest a role for ICAM-4 in normal erythropoiesis and may also be relevant to the adhesive interactions of sickle cells.

Identification and structure of the nerve growth factor binding site on TrkA.

Nerve growth factor (NGF) is involved in the development and maintenance of the nervous system and has been implicated as a possible therapeutic target molecule in a number of neurodegenerative diseases, especially Alzheimer's disease. NGF binds with high affinity to the extracellular region of a tyrosine kinase receptor, TrkA, which comprises three leucine-rich motifs (LRMs), flanked by two cysteine-rich clusters, followed by two immunoglobulin-like (Ig-like) domains. We have expressed the second Ig-like domain as a recombinant protein in E. coli and demonstrate that NGF binds to this domain with similar affinity to the native receptor. This domain (TrkAIg(2)) has the ability to sequester NGF in vitro, preventing NGF-induced neurite outgrowth, and in vivo, inhibiting NGF-induced plasma extravasation. We also present the three-dimensional structure of the TrkAIg(2) domain in a new crystal form, refined to 2.0 A resolution.

Structural basis for altered activity of M- and H-isozyme forms of human lactate dehydrogenase.

Lactate dehydrogenase (LDH) interconverts pyruvate and lactate with concomitant interconversion of NADH and NAD(+). Although crystal structures of a variety of LDH have previously been described, a notable absence has been any of the three known human forms of this glycolytic enzyme. We have now determined the crystal structures of two isoforms of human LDH-the M form, predominantly found in muscle; and the H form, found mainly in cardiac muscle. Both structures have been crystallized as ternary complexes in the presence of the NADH cofactor and oxamate, a substrate-like inhibitor. Although each of these isoforms has different kinetic properties, the domain structure, subunit association, and active-site regions are indistinguishable between the two structures. The pK(a) that governs the K(M) for pyruvate for the two isozymes is found to differ by about 0.94 pH units, consistent with variation in pK(a) of the active-site histidine. The close similarity of these crystal structures suggests the distinctive activity of these enzyme isoforms is likely to result directly from variation of charged surface residues peripheral to the active site, a hypothesis supported by electrostatic calculations based on each structure. Proteins 2001;43:175-185.

Effector sites in the three-dimensional structure of mammalian sperm beta-acrosin.

BACKGROUND: Proacrosin is a serine protease found specifically within the acrosomal vesicle of all mammalian spermatozoa. During fertilization proacrosin autoactivates to form beta-acrosin, in which there is a "light" chain cross-linked to a "heavy" chain by two disulphide bonds. beta-acrosin is thought to be multifunctional with roles in acrosomal exocytosis, as a receptor for zona pellucida proteins, and as a protease to facilitate penetration of spermatozoa into the egg. RESULTS: The crystal structures of both ram and boar beta-acrosins have been solved in complex with p-aminobenzamidine to 2.1 A and 2.9 A resolution, respectively. Both enzymes comprise a heavy chain with structural homology to trypsin, and a light chain covalently associated in a similar manner to blood coagulation enzymes. In crystals of boar beta-acrosin, the carboxyl terminus of the heavy chain is inserted into the active site of the neighboring molecule. In both enzyme structures, there are distinctive positively charged surface "patches" close to the active site, which associate with carbohydrate from adjacent molecules and also bind sulfate ions. CONCLUSIONS: From the three-dimensional structure of beta-acrosin, two separate effector sites are evident. First, proteolytic activity, believed to be important at various stages during fertilization, arises from the trypsin-like active site. Activity of this site may be autoregulated through intermolecular associations. Second, positively charged regions on the surface adjacent to the active site may act as receptors for binding zona pellucida glycoproteins. The spatial proximity of these two effector sites suggests there may be synergy between them.

Recognition of structurally diverse substrates by type II 3-hydroxyacyl-CoA dehydrogenase (HADH II)/amyloid-beta binding alcohol dehydrogenase (ABAD).

Human type II hydroxyacyl-CoA dehydrogenase/amyloid-beta binding alcohol dehydrogenase (HADH II/ABAD) is an oxidoreductase whose salient features include broad substrate specificity, encompassing 3-hydroxyacyl-CoA derivatives, hydroxysteroids, alcohols and beta-hydroxybutyrate, and the capacity to bind amyloid-beta peptide, leading to propagation of amyloid-induced cell stress. In this study, we examine the structure and enzymatic activity of the homologous rat HADH II/ABAD enzyme. We report the crystal structure of rat HADH II/ABAD as a binary complex with its NADH cofactor to 2.0 A resolution, as a ternary complex with NAD(+) and 3-ketobutyrate (acetoacetate) to 1.4 A resolution, and as a ternary complex with NADH and 17 beta-estradiol to 1.7 A resolution. This first crystal structure of an HADH II confirms these enzymes are closely related to the short-chain hydroxysteroid dehydrogenases and differ substantially from the classic, type I 3-hydroxyacyl-CoA dehydrogenases. Binding of the ketobutyrate substrate is accompanied by closure of the active site specificity loop, whereas the steroid substrate does not appear to require closure for binding. Despite the different chemical nature of the two bound substrates, the presentation of chemical groups within the active site of each complex is remarkably similar, allowing a general mechanism for catalytic activity to be proposed. There is a characteristic extension to the active site that is likely to accommodate the CoA moiety of 3-hydroxyacyl-CoA substrates. Rat HADH II/ABAD also binds amyloid-beta (1-40) peptide with a K(D) of 21 nM, which is similar to the interaction exhibited between this peptide and human HADH II/ABAD. These studies provide the first structural insights into HADH II/ABAD interaction with its substrates, and indicate the relevance of the rodent enzyme and associated rodent models for analysis of HADH II/ABAD's physiologic and pathophysiologic properties.

The structure of Antirrhinum centroradialis protein (CEN) suggests a role as a kinase regulator.

Expression of the plant protein centroradialis (CEN) leads to a morphological switch between shoot growth and the development of flower structures (inflorescence). We have determined the crystal structure of Antirrhinum CEN to 1.9 A resolution. This structure confirms the CEN proteins as a subset of the family of phosphatidylethanolamine-binding proteins (PEBP), as predicted from sequence homology. Mammalian forms of PEBP have been found to act as inhibitors of MAP kinase signalling, a central signalling cascade regulating cell differentiation. CEN and PEBP proteins share a similar topology dominated by a large central beta-sheet. The strong conservation of a binding pocket at one end of this sheet which is capable of binding phosphoryl ligands, suggests the biological effects of CEN, like PEBP, arise from the ability of this region to form complexes with phosphorylated ligands, hence interfering with kinases and their effectors.

Structural analysis of a mutational hot-spot in the EcoRV restriction endonuclease: a catalytic role for a main chain carbonyl group.

Following random mutagenesis of the Eco RV endonuclease, a high proportion of the null mutants carry substitutions at Gln69. Such mutants display reduced rates for the DNA cleavage step in the reaction pathway, yet the crystal structures of wild-type Eco RV fail to explain why Gln69 is crucial for activity. In this study, crystal structures were determined for two mutants of Eco RV, with Leu or Glu at residue 69, bound to specific DNA. The structures of the mutants are similar to the native protein and no function can be ascribed to the side chain of the amino acid at this locus. Instead, the structures of the mutant proteins suggest that the catalytic defect is due to the positioning of the main chain carbonyl group. In the enzyme-substrate complex for Eco RV, the main chain carbonyl of Gln69 makes no interactions with catalytic functions but, in the enzyme-product complex, it coordinates a metal ion bound to the newly liberated 5'-phosphate. This re-positioning may be hindered in the mutant proteins. Molecular dynamics calculations indicate that the metal on the phosphoryl oxygen interacts with the carbonyl group upon forming the pentavalent intermediate during phosphodiester hydrolysis. A main chain carbonyl may thus play a role in catalysis by Eco RV.

High-resolution structure of a potent, cyclic proteinase inhibitor from sunflower seeds.

Proteinaceous serine proteinase inhibitors are widespread throughout the plant kingdom where they play an important role in protection against pests and pathogens. Here, we describe the isolation and characterisation of a novel 14 amino acid residue cyclic peptide from sunflower seeds, which is a potent inhibitor of trypsin (Ki=100 pM). The crystal structure of this peptide in complex with bovine beta-trypsin shows both sequence and conformational similarity with the trypsin-reactive loop of the Bowman-Birk family of serine proteinase inhibitors. This inhibitor, however, is unique in being monofunctional, cyclic and far shorter (14 amino acid residues) than inhibitors belonging to this family (typically 60-70 amino acid residues). The high potency of this peptide is likely to arise from the considerable structural rigidity achieved through its cyclic nature which is further stabilised by a single internal disulphide bond. This study helps delineate the minimal unit required for effective peptide inhibitors of serine proteinases, and will assist in the further design of inhibitors to this widespread class of enzymes.

One sequence, four folds: transitions between an ensemble of metastable folds for the N-terminal domain of CD2.

Recombinant forms of the N-terminal domain of the cell adhesion receptor CD2 adopt a variety of olds by exchange of beta-sheets between adjacent polypeptide chains. Although these interdigitated forms are normally metastable, we have used site-directed mutagenesis to alter the kinetics of formation and relative stabilities of these states, leading to spontaneous formation of monomeric, dimeric, trimeric and tetrameric intertwined folded states. A characteristic feature of these fold-disorder-alternative fold transitions is the independence of each domain folding event, as deduced from kinetic analysis of folding data. Structures for fully interdigitated trimeric and tetrameric forms have been modelled, consistent with both the crystallographic and kinetic data. Although the biological role of these alternative folded states remains unclear, these structures form a remarkable demonstration of the fluidity of structure generated from a single polypeptide chain.

Chloroquine binds in the cofactor binding site of Plasmodium falciparum lactate dehydrogenase.

Although the molecular mechanism by which chloroquine exerts its effects on the malarial parasite Plasmodium falciparum remains unclear, the drug has previously been found to interact specifically with the glycolytic enzyme lactate dehydrogenase from the parasite. In this study we have determined the crystal structure of the complex between chloroquine and P. falciparum lactate dehydrogenase. The bound chloroquine is clearly seen within the NADH binding pocket of the enzyme, occupying a position similar to that of the adenyl ring of the cofactor. Chloroquine hence competes with NADH for binding to the enzyme, acting as a competitive inhibitor for this critical glycolytic enzyme. Specific interactions between the drug and amino acids unique to the malarial form of the enzyme suggest this binding is selective. Inhibition studies confirm that chloroquine acts as a weak inhibitor of lactate dehydrogenase, with mild selectivity for the parasite enzyme. As chloroquine has been shown to accumulate to millimolar concentrations within the food vacuole in the gut of the parasite, even low levels of inhibition may contribute to the biological efficacy of the drug. The structure of this enzyme-inhibitor complex provides a template from which the quinoline moiety might be modified to develop more efficient inhibitors of the enzyme.

Structural basis of substrate specificity in malate dehydrogenases: crystal structure of a ternary complex of porcine cytoplasmic malate dehydrogenase, alpha-ketomalonate and tetrahydoNAD.

The structural basis for the extreme discrimination achieved by malate dehydrogenases between a variety of closely related substrates encountered within the cell has been difficult to assess because of the lack of an appropriate catalytically competent structure of the enzyme. Here, we have determined the crystal structure of a ternary complex of porcine cytoplasmic malate dehydrogenase with the alternative substrate alpha-ketomalonate and the coenzyme analogue 1,4,5,6-tetrahydronicotinamide. Both subunits of the dimeric porcine heart, and from the prokaryotes Escherichia coli and Thermus flavus. However, large changes are noted around the active site, where a mobile loop now closes to bring key residues into contact with the substrate. This observation substantiates a postulated mechanism in which the enzyme achieves high levels of substrate discrimination through charge balancing in the active site. As the activated cofactor/substrate complex has a net negative charge, a positive counter-charge is provided by a conserved arginine in the active site loop. The enzyme must, however, also discriminate against smaller substrates, such as pyruvate. The structure shows in the closed (loop down) catalytically competent complex two arginine residues (91 and 97) are driven into close proximity. Without the complimentary, negative charge of the substrate side-chain of oxaloacetate or alpha-ketomalonate, charge repulsion would resist formation production of this catalytically productive conformation, hence minimising the effectiveness of pyruvate as a substrate. By this mechanism, malate dehydrogenase uses charge balancing to achieve fivefold orders of magnitude in discrimination between potential substrates.

Engineered assembly of intertwined oligomers of an immunoglobulin chain.

Domain 1 of CD2 (CD2.D1) forms a conventional Ig fold stabilised by non-covalent antiparallel contacts between beta-strands. Removing two residues from the middle of the protein sequence, where the polypeptide chain normally folds back upon itself, stabilises an open conformation. In this modified molecule, the optimum evolved contacts between side-chains can only be satisfied through the antiparallel association of two chains to create a symmetrical pair of pseudo-domains. Here, we describe the dynamics of the switch between monomeric and dimeric states and demonstrate the extension of this novel underlying principle to trimer and tetramer formation. The ability of a protein molecule to form higher-order antiparallel structures is reminiscent of the behaviour of hairpins, duplexes, three-way and Holliday junctions in DNA.