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PubMed1995

[Conformational differences in the sorption of choline ligands at the active site of acetylcholinesterase].

All the relatively stable conformers of acetylcholine, acetylthiocholine, and Rp- and Sp-enantiomers of the thiocholine-containing inhibitor of acetylcholine esterase, (CH3)2CHO(CH3)P(O)SCH2CH2N+(CH3)3, were calculated by the method of molecular mechanics. The population and the distances between functional atoms were determined for the relatively stable conformers. For the inhibitors, the accessibility of the phosphorus atom for interaction with the hydroxyl group of the Ser200 residue was determined. A computer model is proposed for the productive sorption of acetylcholine. The model assumes the contact of acetylcholine in the active center the hydroxyl group of the Ser200 residue, with the group behaving as a donor of H-bond, and also with the trimethylammonium sorptive segment. Among the organophosphorus inhibitors studied there are no relatively stable conformers that would be complementary to the sorptive site of the substrate, the choline head of the inhibitor cannot be sorbed at the trimethylammonium segment of the active center. An explanation was given for the stereospecificity of the studied enantiomers of an organophosphorus inhibitor within the limits of the proposed model.

Acetylcholinesterase↗
PubMed1997

The crystal structure of a triacylglycerol lipase from Pseudomonas cepacia reveals a highly open conformation in the absence of a bound inhibitor.

BACKGROUND: . Lipases, a family of enzymes which catalyze the hydrolysis of triglycerides, are widely distributed in many organisms. True lipases are distinguished from esterases by the characteristic interfacial activation they exhibit at an oil-water interface. Lipases are one of the most frequently used biocatalysts for organic reactions performed under mild conditions. Their biotechnological applications include food and oil processing and the preparation of chiral intermediates for the synthesis of enantiomerically pure pharmaceuticals. Recent structural studies on several lipases have provided some clues towards understanding the mechanisms of hydrolytic activity, interfacial activation, and stereoselectivity. This study was undertaken in order to provide structural information on bacterial lipases, which is relatively limited in comparison to that on the enzymes from other sources. RESULTS: . We have determined the crystal structure of a triacylglycerol lipase from Pseudomonas cepacia (PcL) in the absence of a bound inhibitor using X-ray crystallography. The structure shows the lipase to contain an alpha/beta-hydrolase fold and a catalytic triad comprising of residues Ser87, His286 and Asp264. The enzyme shares several structural features with homologous lipases from Pseudomonas glumae (PgL) and Chromobacterium viscosum (CvL), including a calcium-binding site. The present structure of PcL reveals a highly open conformation with a solvent-accessible active site. This is in contrast to the structures of PgL and PcL in which the active site is buried under a closed or partially opened 'lid', respectively. CONCLUSIONS: . PcL exhibits some structural features found in other lipases. The presence of the Ser-His-Asp catalytic triad, an oxyanion hole, and the opening of a helical lid suggest that this enzyme shares the same mechanisms of catalysis and interfacial activation as other lipases. The highly open conformation observed in this study is likely to reflect the activated form of the lipase at an oil-water interface. The structure suggests that the interfacial activation of bacterial lipases involves the reorganization of secondary structures and a large movement of the lid to expose the active site. This is similar to the mechanism described for other well characterized fungal and mammalian lipases.

Amino Acid Sequence↗
PubMed2007

Trafficking of phosphatidylinositol by phosphatidylinositol transfer proteins.

PtdIns is synthesized at the endoplasmic reticulum and its intracellular distribution to other organelles can be facilitated by lipid transfer proteins [PITPs (phosphatidylinositol transfer proteins)]. In this review, I summarize the current understanding of how PITPs are regulated by phosphorylation, how can they dock to membranes to exchange their lipid cargo and how cells use PITPs in signal transduction and membrane delivery. Mammalian PITPs, PITPalpha and PITPbeta, are paralogous genes that are 94% similar in sequence. Their structural design demonstrates that they can sequester PtdIns or PtdCho (phosphatidylcholine) in their hydrophobic cavity. To deliver the lipid cargo to a membrane, PITP has to undergo a conformational change at the membrane interface. PITPs have a higher affinity for PtdIns than PtdCho, which is explained by hydrogen-bond contacts between the inositol ring of PtdIns and the side-chains of four amino acid residues, Thr59, Lys61, Glu86 and Asn90, in PITPs. Regardless of species, these residues are conserved in all known PITPs. PITP transfer activity is regulated by a conserved serine residue (Ser166) that is phosphorylated by protein kinase C. Ser166 is only accessible for phosphorylation when a conformational change occurs in PITPs while docking at the membrane interface during lipid transfer, thereby coupling regulation of activity with lipid transfer function. Biological roles of PITPs include their ability to couple phospholipase C signalling to neurite outgrowth, cell division and stem cell growth.

Animals↗
PubMed1991

Beta-breakers: an aperiodic secondary structure.

We have studied the architecture of parallel beta-sheets in proteins and focused on the residues that initiate and terminate the beta-strands. These beta-breaker residues are at the origin of the kink between the beta-strand and the turn that precedes or follows it. beta-Breakers can be located automatically using a consensus approach based on algorithmic secondary structure assignment, solvent accessibility and backbone dihedral angles. These beta-breakers are conformationally homogeneous with respect to side-chain solvent accessibility and backbone dihedral angle profile. A sequence-structure correlation is noted: a restricted subset of amino acids is observed at these positions. Analysis of homologous protein sequences shows that these residues are more highly conserved than other residues in the loop. We conclude that beta-breakers are the structural analogs of the N and C-terminal caps of alpha-helices. The identification of this aperiodic substructure suggests a strategy for improving secondary structure prediction and may guide site-directed mutagenesis experiments.

Amino Acid Sequence↗
PubMed2006

Access of extracellular cations to their binding sites in Na,K-ATPase: role of the second extracellular loop of the alpha subunit.

Na,K-ATPase, the main active transport system for monovalent cations in animal cells, is responsible for maintaining Na(+) and K(+) gradients across the plasma membrane. During its transport cycle it binds three cytoplasmic Na(+) ions and releases them on the extracellular side of the membrane, and then binds two extracellular K(+) ions and releases them into the cytoplasm. The fourth, fifth, and sixth transmembrane helices of the alpha subunit of Na,K-ATPase are known to be involved in Na(+) and K(+) binding sites, but the gating mechanisms that control the access of these ions to their binding sites are not yet fully understood. We have focused on the second extracellular loop linking transmembrane segments 3 and 4 and attempted to determine its role in gating. We replaced 13 residues of this loop in the rat alpha1 subunit, from E314 to G326, by cysteine, and then studied the function of these mutants using electrophysiological techniques. We analyzed the results using a structural model obtained by homology with SERCA, and ab initio calculations for the second extracellular loop. Four mutants were markedly modified by the sulfhydryl reagent MTSET, and we investigated them in detail. The substituted cysteines were more readily accessible to MTSET in the E1 conformation for the Y315C, W317C, and I322C mutants. Mutations or derivatization of the substituted cysteines in the second extracellular loop resulted in major increases in the apparent affinity for extracellular K(+), and this was associated with a reduction in the maximum activity. The changes produced by the E314C mutation were reversed by MTSET treatment. In the W317C and I322C mutants, MTSET also induced a moderate shift of the E1/E2 equilibrium towards the E1(Na) conformation under Na/Na exchange conditions. These findings indicate that the second extracellular loop must be functionally linked to the gating mechanism that controls the access of K(+) to its binding site.

Amino Acid Substitution↗
PubMed1998

Comparison of oriented and random antibody immobilization in immunoaffinity chromatography of cytokinins.

Immunosorbents for the plant hormones cytokinins prepared by random antibody immobilization (to Affi-Gel 10) and by oriented approach via oxidized carbohydrate moieties on the Fc region (to Affi-Gel Hz or hydrazide derivative of Perloza MT 200) have been compared. Both approaches yielded immunosorbents with high dynamic capacity (ca. 5-10 nmol ml gel-1). Oriented antibody immobilization did not exhibit crucial effects in the case of low-molecular-mass cytokinins. Antibodies immobilized via a spacer to Affi-Gel 10 have probably enough conformational freedom to enable good accessibility to cytokinins. The sorbents were used in analysis of endogenous cytokinins in maize seeds. In phosphatase treated samples trans-zeatin and its riboside were predominant.

Antibodies↗
PubMed2004

DNA charge transport: conformationally gated hopping through stacked domains.

The role of base motions in delocalization and propagation of charge through double helical DNA must be established experimentally and incorporated into mechanistic descriptions of DNA-mediated charge transport (CT). Here, we address these fundamental issues by examining the temperature dependence of the yield of CT between photoexcited 2-aminopurine (Ap) and G through DNA bridges of varied length and sequence. DNA assemblies (35-mers) were constructed containing adenine bridges Ap(A)(n)()G (n = 0-9, 3.4-34 A) and mixed bridges, ApAAIAG and ApATATG. CT was monitored through fluorescence quenching of Ap by G and through HPLC analysis of photolyzed DNA assemblies containing Ap and the modified guanine, N(2)-cyclopropylguanosine ((CP)G); upon oxidation, the (CP)G radical cation undergoes rapid ring opening. First, we find that below the duplex melting temperature ( approximately 60 degrees C), the yield of CT through duplex DNA increases with increasing temperature governed by the length and sequence of the DNA bridge. Second, the distance dependence of CT is regulated by temperature; enhanced DNA base fluctuations within duplex DNA extend CT to significantly longer distances, here up to 34 A in <10 ns. Third, at all temperatures the yield of CT does not exhibit a simple distance dependence; an oscillatory component, with a period of approximately 4-5 base pairs, is evident. These data cannot be rationalized by superexchange, hopping of a localized charge injected into the DNA bridge, a temperature-induced transition from superexchange to thermally induced hopping, or by phonon-assisted polaron hopping. Instead, we propose that CT occurs within DNA assemblies possessing specific, well-coupled conformations of the DNA bases, CT-active domains, accessed through base motion. CT through DNA is described as conformationally gated hopping among stacked domains. Enhanced DNA base motions lead to longer range CT with a complex distance dependence that reflects the roles of coherent dynamics and charge delocalization through transient domains. Consequently, DNA CT is not a simple function of distance but is intimately related to the dynamical structure of the DNA bridge.

2-Aminopurine↗
PubMed2000

Probing ligand-induced conformational changes of human CD38.

The lymphoid surface antigen CD38 is basically a NAD+glycohydrolase, which is also involved in the metabolism of cyclic ADP-ribose. Besides, this ecto-enzyme has potential signalling roles in T- and B-cells. Such multiple functions prompted us to study the molecular dynamics of the CD38 protein and especially the relationship between its ecto-enzymatic active site and its epitope, i.e. the binding site of most known anti-CD38 monoclonal antibodies. Both epitopic and enzymatic sites were shown to be degraded by proteases, such as trypsin or chymotrypsin. This sensitivity was almost entirely suppressed in the presence of substrates or inhibitors. Both sites were also degraded in the presence of reducing agents, as dithiothreitol. Inhibitory ligands induced the same resistance of both sites against reducing attack. The binding of CD38 ligands to the active site triggers therefore conformational changes that shield some backbone bonds and disulfide bridges against, respectively, proteolytic cleavage or reduction. This transconformation was found moreover to irreversibly take place after incubation with substrates such as NAD+ in the presence of dithiothreitol. The epitope remained preserved, while the enzymatic activity was lost. This inactivation probably resulted from the covalent trapping of the catalytically reactive intermediate in the active site (i.e. paracatalytic inactivation). These data have major implications in the knowledge of the CD38 structure, especially with regard to the location of disulfide bridges and their accessibility. Potential consequences of the conformational plasticity of CD38 should also be considered in its physiological functions such as signalling.

ADP-ribosyl Cyclase↗
PubMed2005

Loop conformation and dynamics of the Escherichia coli HPPK apo-enzyme and its binary complex with MgATP.

Comparison of the crystallographic and NMR structures of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) suggests that the enzyme may undergo significant conformational change upon binding to its first substrate, ATP. Two of the three surface loops (loop 2 and loop 3) accounting for most of the conformational differences appear to be confined by crystal contacts, raising questions about the putative large-scale induced-fit conformational change of HPPK and the functional roles of the conserved side-chain residues on the loops. To investigate the loop dynamics in crystal-free environment, we carried out molecular dynamics and locally enhanced sampling simulations of the apo-enzyme and the HPPK.MgATP complex. Our simulations showed that the crystallographic B-factors underestimated the loop dynamics considerably. We found that the open-conformation of loop 3 in the binary complex is accessible to the apo-enzyme and is the favored conformation in solution phase. These results revise our previous view of HPPK-substrate interactions and the associated functional mechanism of conformational change. The lessons learned here offer valuable structural insights into the workings of HPPK and should be useful for structure-based drug design.

Adenosine Triphosphate↗
PubMed1986

Spectroscopic study of environment-dependent changes in the conformation of the isolated carboxy-terminal telopeptide of type I collagen.

The C-terminal telopeptide of the alpha 1 chain of type I collagen from bovine skin was isolated from a bacterial collagenase digest. Two forms of the telopeptide were obtained, one with two and the other with three residues of tyrosine. In both of these, the single lysyl residue had been oxidized to alpha-aminoadipic delta-semialdehyde. Circular dichroism spectra of the telopeptide in aqueous solution at neutral pH were interpreted as indicating the presence of little regular secondary structure. However, sodium dodecyl sulfate at a concentration of 40 mM induced some alpha helix, as predicted from the sequence, and trifluoroethanol also induced secondary structure, probably a mixture of alpha helix and beta sheet. A major feature of the circular dichroism spectra of the telopeptide in sodium dodecyl sulfate, in denaturing agents, and in sodium phosphate buffer at low temperature was a positive band at 227 nm due to tyrosine side-chain chromophores. The disappearance of this band on heating and at high pH was ascribed to the adoption by the telopeptide of a specific tertiary structure. Poly(ethylene glycol) 1000 used as a perturbant in UV difference spectroscopy caused conformational changes resulting in decreased accessibility of tyrosine side chains and transfer of these to a less polar environment. A structural model in which the four aromatic side chains of the telopeptide are arranged in two pairs with the rings antiparallel is proposed to account for these results.

Amino Acid Sequence↗
PubMed1996

Conformational dynamics in cytochrome P450-substrate interactions.

There now are four known cytochrome P450 crystal structures. Two of these, P450cam and P450eryF, are substrate-bound while P450terp and the heme domain of P450BM-3 are substrate-free. Here we describe a preliminary analysis of the P450BM-3 heme domain complexed with the 16-carbon fatty acid substrate, palmitoleic acid. A comparison of the substrate-free and -bound structures shows that a large conformational change in the substrate access channel accompanies substrate binding. This new information, together with the substrate-bound structures of P450cam and P450eryF, reveals which regions of P450 are the most important in controlling the dynamics of substrate binding and recognition.

Bacterial Proteins↗
PubMed2004

Crystallization of cytochromes P450 and substrate-enzyme interactions.

Our understanding of structure-function relationships have made considerable advances owing to the increasing number of new P450 crystal structures. This is especially true with mammalian P450s. As always, the main bottleneck in a structure determination project is crystallization. While the crystallization techniques used for P450 crystal growth are not much different from that utilized for other proteins, special protein engineering strategies have been developed in order to generate soluble, homogeneous membrane-bound P450 samples amendable for crystallization. Newly determined P450 structures also provide convincing evidence that P450 enzymes are highly dynamic and flexible. Common structural elements found in all P450s have been identified that undergo large conformational changes to allow substrate access and product release. In addition, flexible regions may enable the active site to adapt to the binding of substrates of different size, shape, and polarity. This review will focus on the successful membrane P450 crystallization techniques and the new structural insights based on the growing P450 structure database.

Binding Sites↗
PubMed1998

A monoclonal antibody recognizing the activation domain of protein C in its calcium-free conformation.

A monoclonal antibody (mAb) binding to protein C (PC) heavy chain but not to activated PC was found to inhibit PC activation by free thrombin, suggesting that epitope involved the activation site. Using a set of overlapping synthetic peptides, we confirmed that this mAb recognizes the sequence encompassing the thrombin cleavage site (165QVDPRLI(171)). Surprisingly, epitope was only accessible in the absence of calcium, half-maximal inhibition of mAb binding occurring at 100 microM Ca2+. Thus, our antibody provides direct evidence that conformation and/or accessibility of the activation site differ between the apo and Ca2+-stabilized conformers of PC.

Animals↗
PubMed1993

Investigations of the thermostability of rubredoxin models using molecular dynamics simulations.

The affects of differences in amino acid sequence on the temperature stability of the three-dimensional structure of the small beta-sheet protein, rubredoxin (Rd), was revealed when a set of homology models was subjected to molecular dynamics simulations at relatively high temperatures. Models of Rd from the hyperthermophile, Pyrococcus furiosus (Pf), an organism that grows optimally at 100 degrees C, were compared to three mesophilic Rds of known X-ray crystal structure. Simulations covering the limits of known Rd thermostabilities were carried out at temperatures of 300 K, 343 K, 373 K, and 413 K. They suggest that Rd stability is correlated with structural dynamics. Because the dynamic behavior of three Pf Rd models was consistently different from the dynamic behavior of the three mesophilic Rd structures, detailed analysis of the temperature-dependent dynamic behavior was carried out. The major differences between the models of the protein from the hyperthermophile and the others were: (1) an obvious temperature-dependent transition in the mesophilic structures not seen with the Pf Rd models, (2) consistent AMBER energy for the Pf Rd due to differences in nonbonded interaction terms, (3) less variation in the average conformations for the Pf Rd models with temperature, and (4) the presence of more extensive secondary structure for the Pf Rd models. These unsolvated dynamics simulations support a simple, general hypothesis to explain the hyperthermostability of Pf Rd. Its structure simplifies the conformational space to give a single minimum accessible over an extreme range of temperatures, whereas the mesophilic proteins sample a more complex conformational space with two or more minima over the same temperature range.

Computer Simulation↗
PubMed1992

Disulfide bonds are essential for the stability of the Sindbis virus envelope.

Sindbis virus is a membrane-containing virus which has two glycoproteins organized in an icosahedral lattice. Protein-protein associations have been identified which participate in the formation of the icosahedron and these associations are stabilized by intramolecular disulfide bridges (Anthony, R. P., and Brown, D. T., 1990, J. Virol. 65, 1187-1194). The present study further examines the role of disulfides in the structure and function of Sindbis virus by following the effect of dithiothreitol on the protease sensitivity of envelope proteins as well as the electron microscopic appearance and infectivity of Sindbis virus. Treatment of isolated virus with 5 mM dithiothreitol for 6 hr causes a marked increase in trypsin sensitivity of both E1 and E2, profound morphological alterations in the viral envelope, increased susceptibility of the nucleocapsid to RNase, and 95% loss of infectivity. These effects are greatly enhanced and accelerated when treatment with DTT is preceded by a brief exposure of the virus to pH 5.3, suggesting that acid-induced conformational changes render structurally critical disulfides more accessible to reductive cleavage by DTT. When compared to other manipulations known to change the conformation of the viral envelope, such as heating to 51 or 60 degrees or exposure to acid pH, only the exposure to DTT with or without prior acid treatment caused marked structural changes correlated with a loss of infectivity. These data provide electron microscopic and functional evidence that intact disulfide bonds are critical for the stability of the virus envelope and suggest that the cleavage of critical disulfide(s) may play a role in the process of virus infection.

Animals↗
PubMed2000

[CT- and MRI-guided conformal interstitial and external beam radiotherapy of primary brain tumors: prospects in Hungary].

The technical improvements gained over the last decade in neuroradiology and radiotherapy have brought significant theoretical and experimental changes in the treatment of primary glial brain tumours. The follow-up of patients with CT, MRI and isotope diagnostic examinations proves that the relapses take place in the vicinity of the primary site in most cases. Consequently, the conventional large fields or whole brain irradiation techniques have been phased out and the conformal irradiation methods focused on the tumorous targets have gained ground. The exact neuroanatomic projection, the image based 3-dimensional treatment planning methods and the conformal irradiation techniques ensure that only the tumorous or the potentially infiltrated regions become irradiated with maximal protection of the normal brain tissues. The increased protection of the normal tissues makes a dose escalation possible, which may result in the augmentation of the therapeutic benefits. In Hungary both the interstitial and the external beam conformal radiotherapies of the CNS have become accessible in practice over the recent years. In possession of manifold treatment modalities (percutaneous fractionated conformal radiotherapy, stereotactic radiosurgery, high dose rate after-loading and low dose rate interstitial irradiation), it is found necessary to overview the indicative territories, advantages, limitations and possible complications of different interventions. The authors describe the possible routes of further improvements and ways of dose escalation. Nevertheless, it is emphasised that gliomas--with reduced radiosensitivity and high potential to infiltrate the adjacent brain tissues-represent the illness in the whole CNS. It implies that in the future it will probably be necessary to initiate systemic therapeutic modalities in the course of routine treatment strategies in addition to the focused and more effective radiotherapy regimens.

Brain Neoplasms↗
PubMedDate not supplied

Cell-binding domain context affects cell behavior on engineered proteins.

A family of artificial extracellular matrix proteins developed for application in small-diameter vascular grafts is used to examine the importance of cell-binding domain context on cell adhesion and spreading. The engineered protein sequences are derived from the naturally occurring extracellular matrix proteins elastin and fibronectin. While each engineered protein contains identical CS5 cell-binding domain sequences, the lysine residues that serve as cross-linking sites are either (i) within the elastin cassettes or (ii) confined to the ends of the protein. Endothelial cells adhere specifically to the CS5 sequence in both of these proteins, but cell adhesion and spreading are more robust on proteins in which the lysine residues are confined to the terminal regions of the chain. These results may be due to altered protein conformations that affect either the accessibility of the CS5 sequence or its affinity for the alpha(4)beta(1) integrin receptor on the endothelial cell surface. Amino acid choice outside the cell-binding domain can thus have a significant impact on the behavior of cells cultured on artificial extracellular matrix proteins.

Amino Acid Sequence↗
PubMed1997

Free energies of amino acid side-chain rotamers in alpha-helices, beta-sheets and alpha-helix N-caps.

Scales have previously been determined for the entropic cost of restricting amino acid side-chain rotations upon protein folding, giving the rule of thumb that the entropic cost of restricting a single side-chain bond is approximately 0.5 kcal mol-1. However, this result does not consider the distinct preferences shown by amino acid side-chains for particular side-chain chi1 angles in the folded protein. For example, Glu in an alpha-helix has chi1 4% gauche- (g-), 39% trans (t) and 58% gauche+ (g+) showing that it is most favourable to restrict Glu chi1 as g+ in a helix while g- is least favoured. The change in side-chain conformational entropy is the same in both cases, but the free energy of each rotamer is different. Here, we determine the energies of every amino acid chi1 rotamer in alpha-helices, beta-sheets and alpha-helix N-caps and each chi1chi2 rotamer pair in helices and sheets. The calculation uses observed rotamer distributions in secondary structure and the coil state, together with experimentally determined free energy changes for secondary structure formation. The results are sets of rotamer energies within a secondary structure that can be directly compared to each other. For example, we conclude that Tyr is the most stable residue in a beta-sheet if only the trans rotamer is accessible; if only the gauche- conformation is available, Thr would be the most stabilising. Previously published scales of amino acid preferences for secondary structure are weighted averages of rotamer energies and therefore imply that Thr is the most stabilising substitution in a beta-sheet in any side-chain conformation. Both side-chain conformational entropies and intrinsic secondary structure preferences are subsumed within our data; the results presented here should therefore be used in preference to both side-chain conformational entropies and intrinsic secondary structure preferences when the rotamer occupied in the folded state is known. The results may be useful in protein engineering, simulations of binding and folding, prediction of protein stability and peptide binding energies, and identification of incorrectly folded structures.

Algorithms↗