Biomedical subjects

Oded Livnah

Publications and source records attributed to Oded Livnah.

At least 19 recordsLinked to original sources

PubMed2006

Hyperactive variants of p38alpha induce, whereas hyperactive variants of p38gamma suppress, activating protein 1-mediated transcription.

The p38 family of kinases is a subgroup of the mitogen-activated protein kinase family. It is composed of four isoforms and is involved in critical biological processes as well as in inflammatory diseases. The exact unique role of each p38 isoform in these processes is not understood well. To approach this question we have been developing intrinsically active variants of p38s. Recently we described a series of mutants of the human p38alpha, which were spontaneously active as recombinant proteins purified from Escherichia coli cells. We show here that some of these mutants are spontaneously active in several mammalian cells in culture. The spontaneous activity of some mutants is higher than the activity of the fully activated wild type counterpart. We further produced mutants of the other p38 isoforms and found that p38beta(D176A), p38gamma(D179A), p38delta(D176A), and p38delta(F324S) are spontaneously active in vivo. The active mutants are also spontaneously phosphorylated. To test whether the mutants actually fulfill downstream duties of p38 proteins, we tested their effect on activating protein 1(AP-1)-mediated transcription. Active mutants of p38alpha induced AP-1-driven reporter genes, as well as the c-jun and c-fos promoters. An active variant of p38gamma suppressed AP-1-mediated transcription. When active variants of p38alpha and p38gamma were co-expressed, AP-1 activity was not induced, showing that p38gamma is dominant over p38alpha with respect to AP-1 activation. Thus, intrinsically active variants that are spontaneously active in vivo have been obtained for all p38 isoforms. These variants have disclosed different effects of each isoform on AP-1 activity.

Amino Acid Sequence↗

PubMed2006

Structures of p38alpha active mutants reveal conformational changes in L16 loop that induce autophosphorylation and activation.

p38 mitogen-activated protein (MAP) kinases function in numerous signaling processes and are crucial for normal functions of cells and organisms. Abnormal p38 activity is associated with inflammatory diseases and cancers making the understanding of its activation mechanisms highly important. p38s are commonly activated by phosphorylation, catalyzed by MAP kinase kinases (MKKs). Moreover, it was recently revealed that the p38alpha is also activated via alternative pathways, which are MKK independent. The structural basis of p38 activation, especially in the alternative pathways, is mostly unknown. This lack of structural data hinders the study of p38's biology as well as the development of novel strategies for p38 inhibition. We have recently discovered and optimized a novel set of intrinsically active p38 mutants whose activities are independent of any upstream activation. The high-resolution crystal structures of the intrinsically active p38alpha mutants reveal that local alterations in the L16 loop region promote kinase activation. The L16 loop can be thus regarded as a molecular switch that upon conformational changes promotes activation. We suggest that similar conformational changes in L16 loop also occur in natural activation mechanisms of p38alpha in T-cells. Our biochemical studies reveal novel mechanistic insights into the activation process of p38. In this regard, the results indicate that the activation mechanism of the mutants involves dimerization and subsequent trans autophosphorylation on Thr180 (on the phosphorylation lip). Finally, we suggest a model of in vivo p38alpha activation induced by the L16 switch with auto regulatory characteristics.

Amino Acid Sequence↗

PubMed2006

Butyrylcholinesterase attenuates amyloid fibril formation in vitro.

In Alzheimer's disease, both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) colocalize with brain fibrils of amyloid-beta (Abeta) peptides, and synaptic AChE-S facilitates fibril formation by association with insoluble Abeta fibrils. Here, we report that human BChE and BSP41, a synthetic peptide derived from the BChE C terminus, inversely associate with the soluble Abeta conformers and delay the onset and decrease the rate of Abeta fibril formation in vitro, at a 1:100 BChE/Abeta molar ratio and in a dose-dependent manner. The corresponding AChE synthetic peptide (ASP)40 peptide, derived from the homologous C terminus of synaptic human (h)AChE-S, failed to significantly affect Abeta fibril formation, attributing the role of enhancing this process to an AChE domain other than the C terminus. Circular dichroism and molecular modeling confirmed that both ASP40 and BChE synthetic peptide (BSP)41 are amphipathic alpha-helices. However, ASP40 shows symmetric amphipathicity, whereas BSP41 presented an aromatic tryptophan residue in the polar side of the C terminus. That this aromatic residue is causally involved in the attenuating effect of BChE was further supported by mutagenesis experiments in which (W8R) BSP41 showed suppressed capacity to attenuate fibril formation. In Alzheimer's disease, BChE may have thus acquired an inverse role to that of AChE by adopting imperfect amphipathic characteristics of its C terminus.

Acetylcholinesterase↗

PubMed2006

MAP-quest: could we produce constitutively active variants of MAP kinases?

Constitutively active mutants that acquired intrinsic activity and escaped regulation, serve as powerful tools for revealing the biochemical, biological and pathological functions of proteins. Such mutants are not available for mitogen-activated protein kinases (MAPKs). It is not known how to mimic the unusual mode of MAPK activation and to enforce, by mutations, their active conformation. In this review we describe the strategies employed in attempts to overcome this obstacle. We focus on a recent breakthrough with the p38 family that suggests that active variants of all MAPKs will soon be available.

Amino Acid Sequence↗

PubMed2006

Factors dictating the pseudocatalytic efficiency of avidins.

The hydrolysis of biotinyl p-nitrophenyl ester (BNP) by a series of avidin derivatives was examined. Surprisingly, a hyperthermostable avidin-related protein (AVR4) was shown to display extraordinary yet puzzling hydrolytic activity. In order to evaluate the molecular determinants that contribute to the reaction, the crystal structure of AVR4 was compared with those of avidin, streptavidin and key mutants of the two proteins in complex with biotinyl p-nitroanilide (BNA), the inert amide analogue of BNP. The structures revealed that a critical lysine residue contributes to the hydrolysis of BNP by avidin but has only a minor contribution to the AVR4-mediated reaction. Indeed, the respective rates of hydrolysis among the different avidins reflect several molecular parameters, including binding-site architecture, the availability of the ligand to solvent and the conformation of the ligand and consequent susceptibility to efficient nucleophilic attack. In avidin, the interaction of BNP with Lys111 and disorder of the L3,4 loop (and consequent solvent availability) together comprise the major driving force behind the hydrolysis, whereas in AVR4 the status of the ligand (the pseudo-substrate) is a major distinguishing feature. In the latter protein, a unique conformation of the L3,4 loop restrains the pseudo-substrate, thereby exposing the carbonyl carbon atom to nucleophilic attack. In addition, due to its conformation, the pseudo-substrate in the AVR4 complex cannot interact with the conserved lysine analogue (Lys109); instead, this function is superseded by polar interactions with Arg112. The results demonstrate that, in highly similar proteins, different residues can perform the same function and that subtle differences in the active-site architecture of such proteins can result in alternative modes of reaction.

Avidin↗

PubMed2006

Isolation of intrinsically active mutants of MAP kinases via genetic screens in yeast.

Intrinsically active variants of a protein are powerful tools for deciphering the specific functions of that protein. Since the catalytic activity of such variants is spontaneously active in vivo, they can disclose very accurately biochemical and biological functions of the parental protein. It is particularly important to obtain intrinsically active variants of individual MAP kinases. This is because in response to extracellular signals, more than one MAPK is typically concomitantly activated making it difficult to reveal their individual functions and downstream targets. Until very recently intrinsically active variants were not available for MAP kinases because of their unusual mechanism of activation that requires dual phosphorylation on neighboring Thr and Tyr residues. It is not known how to mimic the phospho-Thr-Xaa-phospho-Tyr motif by mutagenesis. We describe here a genetic screen in yeast that we successfully used to isolate bona fide intrinsically active variants of the yeast MAP kinase Hog1 and all isoforms of the human p38 family. We further established a screen for isolation of intrinsically active ERKs. The rationale of our screening approach is to search for MAPK molecules that are active in the absence of their activators. The method could be applied to the discovery of intrinsically active variants of any MAP kinase of any organism. We describe in detail the rationale, the steps that should be taken for establishment of such a screen and a step-by-step protocol for carrying out the screen.

Fungal Proteins↗

PubMed2005

Essentials of biorecognition: the (strept)avidin-biotin system as a model for protein-protein and protein-ligand interaction.

Molecular recognition or biorecognition is as the heart of all biological interactions. These interactions are characterized by a collection of noncovalent bonds, namely ionic, hydrogen-bonding and hydrophobic interactions. In addition, shape complementarity appears to play a pivotal role in the process of biorecognition. In this review, we examine the versatile avidin-biotin complex as a model system for study of the biorecognition phenomenon with respect to protein-protein, protein-peptide, protein-ligand and protein-DNA interactions.

Bacterial Proteins↗

PubMed2005

Structural basis for the thermostability of ferredoxin from the cyanobacterium Mastigocladus laminosus.

Plant-type ferredoxins (Fds) carry a single [2Fe-2S] cluster and serve as electron acceptors of photosystem I (PSI). The ferredoxin from the thermophilic cyanobacterium Mastigocladus laminosus displays optimal activity at 65 degrees C. In order to reveal the molecular factors that confer thermostability, the crystal structure of M.laminosus Fd (mFd) was determined to 1.25 A resolution and subsequently analyzed in comparison with four similar plant-type mesophilic ferredoxins. The topologies of the plant-type ferredoxins are similar, yet two structural determinants were identified that may account for differences in thermostability, a salt bridge network in the C-terminal region, and the flexible L1,2 loop that increases hydrophobic accessible surface area. These conclusions were verified by three mutations, i.e. substitution of L1,2 into a rigid beta-turn ((Delta)L1,2) and two point mutations (E90S and E96S) that disrupt the salt bridge network at the C-terminal region. All three mutants have shown reduced electron transfer (ET) capabilities and [2Fe-2S] stability at high temperatures in comparison to the wild-type mFd. The results have also provided new insights into the involvement of the L1,2 loop in the Fd interactions with its electron donor, the PSI complex.

Amino Acid Sequence↗

PubMed2005

High-resolution crystal structure of an avidin-related protein: insight into high-affinity biotin binding and protein stability.

The chicken avidin gene belongs to an extended gene family encoding seven avidin-related genes (AVRs), of which only avidin is expressed in the chicken. The sequences of AVR4 and AVR5 are identical and the common protein (AVR4) has been expressed both in insect and bacterial systems. The recombinant proteins are similarly hyperthermostable and bind biotin with similarly high affinities. AVR4 was crystallized in the apo and biotin-complexed forms and their structures were determined at high resolution. Its tertiary and quaternary structures are very similar to those of avidin and streptavidin. Its biotin-binding site shows only a few alterations compared with those of avidin and streptavidin, which account for the observed differences in binding affinities. The increased hyperthermostability can be attributed to the conformation of the critical L3,4 loop and the extensive network of 1-3 inter-monomeric interactions. The loop contains a tandem Pro-Gly sequence and an Asp-Arg ion pair that collectively induce rigidity, thus maintaining its closed and ordered conformation in both the apo and biotin-complexed forms. In addition, Tyr115 is present on the AVR4 1-3 monomer-monomer interface, which is absent in avidin and streptavidin. The interface tyrosine generates inter-monomeric interactions, i.e. a tyrosine-tyrosine pi-pi interaction and a hydrogen bond with Lys92. The resultant network of interactions confers a larger 1-3 dimer-dimer contact surface on AVR4, which correlates nicely with its higher thermostability compared with avidin and streptavidin. Several of the proposed thermostability-determining factors were found to play a role in strengthening the tertiary and quaternary integrity of AVR4.

Animals↗

PubMed2005

Design and construction of highly stable, protease-resistant chimeric avidins.

The chicken avidin gene family consists of avidin and seven separate avidin-related genes (AVRs) 1-7. Avidin protein is a widely used biochemical tool, whereas the other family members have only recently been produced as recombinant proteins and characterized. In our previous study, AVR4 was found to be the most stable biotin binding protein thus far characterized (T(m) = 106.4 degrees C). In this study, we studied further the biotin-binding properties of AVR4. A decrease in the energy barrier between the biotin-bound and unbound state of AVR4 was observed when compared with that of avidin. The high resolution structure of AVR4 facilitated comparison of the structural details of avidin and AVR4. In the present study, we used the information obtained from these comparative studies to transfer the stability and functional properties of AVR4 to avidin. A chimeric avidin protein, ChiAVD, containing a 21-amino acid segment of AVR4 was found to be significantly more stable (T(m) = 96.5 degrees C) than native avidin (T(m) = 83.5 degrees C), and its biotin-binding properties resembled those of AVR4. Optimization of a crucial subunit interface of avidin by an AVR4-inspired point mutation, I117Y, significantly increased the thermostability of the avidin mutant (T(m) = 97.5 degrees C) without compromising its high biotin-binding properties. By combining these two modifications, a hyperthermostable ChiAVD(I117Y) was constructed (T(m) = 111.1 degrees C). This study provides an example of rational protein engineering in which another member of the protein family has been utilized as a source in the optimization of selected properties.

Amino Acid Sequence↗

PubMed2005

Inherited and acquired interactions between ACHE and PON1 polymorphisms modulate plasma acetylcholinesterase and paraoxonase activities.

The 5.5 Mb chromosome 7q21-22 ACHE/PON1 locus harbours the ACHE gene encoding the acetylcholine hydrolyzing, organophosphate (OP)-inhibitable acetylcholinesterase protein and the paraoxonase gene PON1, yielding the OP-hydrolyzing PON1 enzyme which also displays arylesterase activity. In search of inherited and acquired ACHE-PON1 interactions we genotyped seven polymorphic sites and determined the hydrolytic activities of the corresponding plasma enzymes and of the AChE-homologous butyrylcholinesetrase (BChE) in 157 healthy Israelis. AChE, arylesterase, BChE and paraoxonase activities in plasma displayed 5.4-, 6.5-, 7.2- and 15.5-fold variability, respectively, with genotype-specific differences between carriers of distinct compound polymorphisms. AChE, BChE and arylesterase but not paraoxonase activity increased with age, depending on leucine at PON1 position 55. In contrast, carriers of PON1 M55 displayed decreased arylesterase activity independent of the - 108 promoter polymorphism. Predicted structural consequences of the PON1 L55M substitution demonstrated spatial shifts in adjacent residues. Molecular modelling showed substrate interactions with the enzyme variants, explaining the changes in substrate specificity induced by the Q192R substitution. Intriguingly, PON1, but not BChE or arylesterase, activities displayed inverse association with AChE activity. Our findings demonstrate that polymorphism(s) in the adjacent PON1 and ACHE genes affect each other's expression, predicting for carriers of biochemically debilitating ACHE/PON1 polymorphisms adverse genome-environment interactions.

Acetylcholinesterase↗

PubMed2004

Active mutants of the human p38alpha mitogen-activated protein kinase.

Mitogen-activated protein (MAP) kinases compose a family of serine/threonine kinases that function in many signal transduction pathways and affect various cellular phenotypes. Despite the abundance of available data, the exact role of each MAP kinase is not completely defined, in part because of the inability to activate MAP kinase molecules individually and specifically. Based on activating mutations found in the yeast MAP kinase p38/Hog1 (Bell, M., Capone, R., Pashtan, I., Levitzki, A., and Engelberg, D. (2001) J. Biol. Chem. 276, 25351-25358), we designed and constructed single and multiple mutants of human MAP kinase p38alpha. Single (p38D176A, p38F327L, and p38F327S) and subsequent double (p38D176A/F327L and p38D176A/F327S) mutants acquired high intrinsic activity independent of any upstream regulation and reached levels of 10 and 25%, respectively, in reference to the dually phosphorylated wild type p38alpha. The active p38 mutants have retained high specificity toward p38 substrates and were inhibited by the specific p38 inhibitors SB-203580 and PD-169316. We also show that similar mutations can render p38gamma active as well. Based on the available structures of p38 and ERK2, we have analyzed the p38 mutants and identified a hydrophobic core stabilized by three aromatic residues, Tyr-69, Phe-327, and Trp-337, in the vicinity of the L16 loop region. Upon activation, a segment of the L16 loop, including Phe-327, becomes disordered. Structural analysis suggests that the active p38 mutants emulate the conformational changes imposed naturally by dual phosphorylation, namely, destabilization of the hydrophobic core. Essentially, the hydrophobic core is an inherent stabilizer that maintains low basal activity level in unphosphorylated p38.

Amino Acid Sequence↗

PubMed2004

Structural elements responsible for conversion of streptavidin to a pseudoenzyme.

Avidin enhances the hydrolysis of biotinyl p-nitrophenyl ester (BNP) under mild alkaline conditions, whereas streptavidin prevents hydrolysis of BNP up to pH 12. Recently, we imposed hydrolytic activity on streptavidin by rational mutagenesis, based on the molecular elements responsible for the hydrolysis by avidin. Three mutants were designed, whereby the desired features, the distinctive L124R point mutation (M1), the L3,4 loop replacement (M2), and the combined mutation (M3), were transferred from avidin to streptavidin. The crystal structures of the mutants, in complex with biotinyl p-nitroanilide (BNA), the stable amide analogue of BNP, were determined. The results demonstrate that the point mutation alone has little effect on hydrolysis, and BNA exhibits a conformation similar to that of streptavidin. Substitution of a lengthier L3,4 loop (from avidin to streptavidin), resulted in an open conformation, thus exposing the ligand to solvent. Moreover, the amide bond of BNA was flipped relative to that of the streptavidin and M1 complexes, thus deflecting the nitro group toward Lys-121. Consequently, the leaving group potential of the nitrophenyl group of BNP is increased, and M2 hydrolyzes BNP at pH values >8.5. To better emulate the hydrolytic potential of avidin, M3 was required. The combination of loop replacement and point mutation served to further increase the leaving group potential by interaction of the nitro group with Arg-124 and Lys-121. The information derived from this study may provide insight into the design of enzymes and transfer of desired properties among homologous proteins.

Hydrogen-Ion Concentration↗

PubMed2003

Purification, crystallization and preliminary X-ray analysis of ferredoxin isolated from thermophilic cyanobacterium Mastigocladus laminosus.

Ferredoxins are soluble iron-sulfur proteins that are involved in numerous electron-transfer reactions. Plant-type ferredoxins, which carry a single [2Fe-2S] cluster, serve as the electron acceptors of Photosystem I. The ferredoxin from the thermophilic cyanobacterium Mastigocladus laminosus has unique thermostable properties. The isolated protein is active at temperatures of higher than 338 K. The gene encoding the ferredoxin from M. laminosus was subcloned into an expression vector and overproduced in Escherichia coli. The recombinant protein was purified to near-homogeneity and crystallized using the hanging-drop method. Thin needle-like crystals were grown in several crystallization conditions but were unsuitable for X-ray analysis owing to poor scattering. In order to obtain better diffracting crystals, ferredoxin was purified directly from M. laminosus cells. Single crystals were obtained using 30% PEG 4000, 0.32 M Mg(NO(3))(2), 20 mM Tris-HCl pH 8.2. These crystals diffracted to 1.25 A resolution using synchrotron radiation and were found to belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 28.45, b = 50.93, c = 110.91 A. The asymmetric unit was found to contain two ferredoxin molecules, with a corresponding V(M) of 1.9 A(3) Da(-1) and a solvent content of 34%. The present study indicates that overcoming the poor diffraction of crystals obtained from recombinant protein can be achieved by producing crystals from protein purified from the native host.

Cloning, Molecular↗

PubMed2003

The pp60c-Src inhibitor PP1 is non-competitive against ATP.

Glutathione-S-transferase (GST)-pp60(c-Src) (GST-Src) expressed in Escherichia coli is as catalytically active as purified, activated pp60(c-Src) protein derived from human platelets. We utilized the bacterially expressed enzyme, together with information about the structures of Src family kinases in complex with their inhibitors PP1 and PP2, to modify PP1 in a quest for improved inhibitors. Despite the detailed structural information on Hck-PP1 and Lck-PP2 complexes, which shows that PP1 and PP2 bind to the adenosine triphosphate (ATP) pocket, we were unable to improve the affinity between modified PP1 and Src. Puzzled, we examined in detail the mechanism by which PP1 inhibits the kinase activity of Src. Here we report that PP1 is non-competitive with ATP for the inhibition of Src, at variance with what is currently accepted, and is a 'mixed competitive inhibitor' vis-à-vis the substrate. These findings shed new light on the mechanism whereby PP1-like molecules inhibit Src. Examination of the homology between the kinase domain of Src and those of Hck and Lck reveals significant differences outside the ATP binding pocket, whereas they are identical within the ATP binding domain. These results suggest that PP1 may be a leading compound for ATP non-competitive inhibitors of Src family kinases. Since Src in its active form is the hallmark of numerous cancers, understanding how PP1 inhibits activated Src will aid in the discovery of potent and selective Src kinase inhibitors.

Adenosine Triphosphate↗

PubMed2003

Dimer-tetramer transition between solution and crystalline states of streptavidin and avidin mutants.

The biotin-binding tetrameric proteins, streptavidin from Streptomyces avidinii and chicken egg white avidin, are excellent models for the study of subunit-subunit interactions of a multimeric protein. Efforts are thus being made to prepare mutated forms of streptavidin and avidin, which would form monomers or dimers, in order to examine their effect on quaternary structure and assembly. In the present communication, we compared the crystal structures of binding site W-->K mutations in streptavidin and avidin. In solution, both mutant proteins are known to form dimers, but upon crystallization, both formed tetramers with the same parameters as the native proteins. All of the intersubunit bonds were conserved, except for the hydrophobic interaction between biotin and the tryptophan that was replaced by lysine. In the crystal structure, the binding site of the mutated apo-avidin contains 3 molecules of structured water instead of the 5 contained in the native protein. The lysine side chain extends in a direction opposite that of the native tryptophan, the void being partially filled by an adjacent lysine residue. Nevertheless, the binding-site conformation observed for the mutant tetramer is an artificial consequence of crystal packing that would not be maintained in the solution-phase dimer. It appears that the dimer-tetramer transition may be concentration dependent, and the interaction among subunits obeys the law of mass action.

Animals↗

PubMed2002

Structure-based rational design of streptavidin mutants with pseudo-catalytic activity.

Introduction of enzymatic activity into proteins or other types of polymers by rational design is a major objective in the life sciences. To date, relatively low levels of enzymatic activity could be introduced into antibodies by using transition-state analogues of haptens. In the present study, we identify the structural elements that contribute to the observed hydrolytic activity in egg white avidin, which promote the cleavage of active biotin esters (notably biotinyl p-nitrophenyl ester). The latter elements were then incorporated into bacterial streptavidin via genetic engineering. The streptavidin molecule was thus converted from a protector to an enhancer of hydrolysis of biotin esters. The conversion was accomplished by the combined replacement of a "lid-like loop" (L3,4) and a leucine-to-arginine point mutation in streptavidin. Interestingly, neither of these elements play a direct role in the hydrolytic reaction. The latter features were thus shown to be responsible for enhanced substrate hydrolysis. This work indicates that structural and non-catalytic elements of a protein can be modified to promote the induced fit of a substrate for subsequent interaction with either a catalytic residue or water molecules. This approach complements the conventional design of active sites that involves direct modifications of catalytic residues.

Arginine↗

PubMed2002

Rational design of an active avidin monomer.

Homotetrameric chicken avidin that binds four molecules of biotin was converted to a monomeric form (monoavidin) by mutations of two interface residues: tryptophan 110 in the 1 --> 2 interface was mutated to lysine and asparagine 54 in the 1 --> 4 interface was converted to alanine. The affinity for biotin binding of the mutant decreased from K(d) approximately 10(-15) m of the wild-type tetramer to K(d) approximately 10(-7) m, which was studied by an optical biosensor IAsys and by a fluorescence spectroscopical method in solution. The binding was completely reversible. Conversion of the tetramer to a monomer results in increased sensitivity to proteinase K digestion. The antigenic properties of the mutated protein were changed, such that monoavidin was only partially recognized by a polyclonal antibody whereas two different monoclonal antibodies entirely failed to recognize the avidin monomer. This new monomeric avidin, which binds biotin reversibly, may be useful for applications both in vitro and in vivo. It may also shed light on the effect of intersubunit interactions on the binding of ligands.

Animals↗