Human recombinant butyrylcholinesterase purified from the milk of transgenic goats interacts with beta-amyloid fibrils and suppresses their formation in vitro.

BACKGROUND: In Alzheimer's disease (AD), brain butyrylcholinesterase (BChE) co-localizes with beta-amyloid (Abeta) fibrils. AIMS: In vitro testing of the significance of this phenomenon to AD progress. METHODS: A thioflavine T (ThT) fluorogenic assay, photo-induced cross-linking and quantifiable electron microscopy served to compare the effect on Abeta fibril formation induced by highly purified recombinant human BChE (rBChE) produced in the milk of transgenic goats with that of serum-derived human BChE. RESULTS: Both proteins at 1:50 and 1:25 ratios to Abeta dose-dependently prolonged the ThT lag time and reduced the apparent rate of Abeta fibril formation compared to Abeta alone. Photo-induced cross-linking tests showed that rBChE prolonged the persistence of amyloid dimers, trimers and tetramers in solution, whereas Abeta alone facilitated precipitation of such multimers from solution. Transmission electron microscopy showed that rBChE at 1:100 to Abeta prevented the formation of larger, over 150-nm-long, Abeta fibrils and reduced fibril branching compared to Abeta alone as quantified by macro programming of Image Pro Plus software. CONCLUSION: Our findings demonstrate that rBChE interacts with Abeta fibrils and can attenuate their formation, extension and branching, suggesting further tests of rBChE, with unlimited supply and no associated health risks, as a therapeutic agent for delaying the formation of amyloid toxic oligomers in AD patients.

Mapping leptin-interacting sites in recombinant leptin-binding domain (LBD) subcloned from chicken leptin receptor.

The binding domain of the chicken leptin receptor [chLBD (chicken leptin-binding domain)], subcloned from the full-size chicken leptin receptor and prepared in an Escherichia coli system, was subjected to site-directed mutagenesis to identify the amino acids involved in leptin binding. A total of 22 electrophoretically pure, >90% monomer-containing mutants were expressed, refolded and purified. The effects of the mutations were tested by the ability to form complexes with ovine leptin, and the kinetic parameters of interaction were determined by surface plasmon resonance. Six mutants were used to determine whether mutations of several amino acids that differ between chLBD and mammalian LBDs will affect affinity: none showed any such effect, except the mutant A105D (Ala(105)-->Asp), which exhibited some decrease in affinity. Surface plasmon resonance analysis identified six mutants in which binding activity was totally abolished (F73A, Y14A/F73A, V76A/F77A, L78A/L79A, V76A/F77A/L78A/L79A and A105D/D106V) and six mutants (Y14A, R41A, R41A/S42A/K43A, V103A, V135A/F136A and F136A) in which affinity for the hormone was reduced, mainly by increased dissociation rates. Gel-filtration experiments indicated the formation of a 1:1 ovine or human leptin-chLBD complex with a molecular mass of approx. 41 kDa. Gel-filtration experiments yielded 1:1 complexes with those mutants in which affinity had decreased, but not with the six mutants, which had totally lost their binding capacity. Modelling the leptin-chLBD complex indicated that the binding domain of the latter is located mainly in the L3 loop, which contributes nine amino acid residues interacting with leptin. Contact-surface analysis identified the residues having the highest contribution to the recognition site to be Phe73, Phe77 and Leu79.

Crystallization and preliminary X-ray analysis of W120K mutant of streptavidin.

Bacterial streptavidin and chicken avidin are homotetrameric proteins that share an exceptionally high affinity towards the vitamin biotin. The biotin-binding sites in both proteins contain a crucial tryptophan residue contributed from an adjacent subunit. This particular tryptophan (W110 in avidin and W120 in streptavidin) plays an important role in both biotin binding and in the quaternary stabilities of the proteins. An intriguing naturally occurring alteration of tryptophan to lysine was previously described in the C-terminal domain of sea-urchin fibropellins, which share a relatively high sequence similarity with avidin and streptavidin. Avidin (Avm-W110K) and streptavidin (Savm-W120K) mutations show substantially reduced affinities towards biotin as well as the dissociation of their tetrameric structure into stable avidin and streptavidin dimers. Savm-W120K was crystallized at 293 K using the hanging-drop vapour-diffusion method. The crystals diffract to 1.7 A resolution using synchrotron radiation and belong to the monoclinic space group P2(1), with unit-cell parameters a = 50.43, b = 100.41, c = 52.51 A, beta = 112.12 degrees. The asymmetric unit contains four molecules of Savm-W120K, with a corresponding V(M) of 2.3 A(3) Da(-1) and a solvent content of 46%.

Chicken avidin exhibits pseudo-catalytic properties. Biochemical, structural, and electrostatic consequences.

Avidin and its bacterial analogue streptavidin exhibit similarly high affinities toward the vitamin biotin. The extremely high affinity of these two proteins has been utilized as a powerful tool in many biotechnological applications. Although avidin and streptavidin have similar tertiary and quaternary structures, they differ in many of their properties. Here we show that avidin enhances the alkaline hydrolysis of biotinyl p-nitrophenyl ester, whereas streptavidin protects this reaction even under extreme alkaline conditions (pH > 12). Unlike normal enzymatic catalysis, the hydrolysis reaction proceeds as a single cycle with no turnover because of the extremely high affinity of the protein for one of the reaction products (i.e. free biotin). The three-dimensional crystal structures of avidin (2 A) and streptavidin (2.4 A) complexed with the amide analogue, biotinyl p-nitroanilide, as a model for the p-nitrophenyl ester, revealed structural insights into the factors that enhance or protect the hydrolysis reaction. The data demonstrate that several molecular features of avidin are responsible for the enhanced hydrolysis of biotinyl p-nitrophenyl ester. These include the nature of a decisive flexible loop, the presence of an obtrusive arginine 114, and a newly formed critical interaction between lysine 111 and the nitro group of the substrate. The open conformation of the loop serves to expose the substrate to the solvent, and the arginine shifts the p-nitroanilide moiety toward the interacting lysine, which increases the electron withdrawing characteristics and consequent electrophilicity of the carbonyl group of the substrate. Streptavidin lacked such molecular properties, and analogous interactions with the substrate were consequently absent. The information derived from these structures may provide insight into the action of artificial protein catalysts and the evolution of catalytic sites in general.

Mutations of ovine and bovine placental lactogens change, in different ways, the biological activity mediated through homologous and heterologous lactogenic receptors.

The biological activities of ovine (o) and bovine (b) placental lactogens (PLs) and their mutated analogues were compared using several binding and in vitro bioassays. In almost all cases, the biological activities of these analogues mediated through rat (r) prolactin receptor (PRLR) showed little or no change, despite a remarkable decrease in their capacity to bind to the extracellular domain of rPRLR and despite compromised stability of the 2:1 complexes. These results indicate that mutations impairing the ability of oPL or bPL to form stable complexes with lactogenic receptors do not necessarily lead to a decrease in the biological activity, because the transient existence of the homodimeric complex is still sufficient to initiate the signal transduction. In contrast, oPL and bPL analogues completely, or almost completely, lost their ability to activate homologous PRLRs, and some of them even acted as site-2 antagonists. To explain the difference between the activity transduced through homologous and that transduced through heterologous PRLRs, we propose the novel term 'minimal time of homodimer persistence'. This concept assumes that in order to initiate the signal transduction, the associated kinase JAK2 has to be transphosphorylated and this requires a 'minimal time' of homodimer existence. In the case of homologous interaction between ruminant PLs and homologous PRLRs, this 'minimal time' is met, though the interaction with homologous PRLRs has a shorter half-life than that with heterologous PRLRs. Therefore oPL or bPL are active in cells possessing both homologous and heterologous PRLRs. Mutations of oPL or bPL lead to reduced affinity and, consequently, the 'time of homodimer persistence' is shortened. Although in the case of heterologous interaction the 'minimal time' is still sufficient to initiate the biological activity, in homologous interactions, which are already weaker than heterologous interactions, further destabilization of the complex shortens its persistence to below the 'minimal time', leading to full or partial loss of biological activity.

Biotin induces tetramerization of a recombinant monomeric avidin. A model for protein-protein interactions.

Chicken avidin, a homotetramer that binds four molecules of biotin was converted to a monomeric form by successive mutations of interface residues to alanine. The major contribution to monomer formation was the mutation of two aspartic acid residues, which together account for ten hydrogen bonding interactions at the 1-4 interface. Mutation of these residues, together with the three hydrophobic residues at the 1-3 interface, led to stable monomer formation in the absence of biotin. Upon addition of biotin, the monomeric avidin reassociated to the tetramer, which exhibited properties similar to those of native avidin, with respect to biotin binding, thermostability, and protease resistance. To our knowledge, these unexpected results represent the first example of a small monovalent ligand that induces oligomerization of a monomeric protein. This study may suggest a biological role for low molecular weight ligands in inducing oligomerization and in maintaining the stability of multimeric protein assemblies.

Shared and unique determinants of the erythropoietin (EPO) receptor are important for binding EPO and EPO mimetic peptide.

We have shown previously that Phe93 in the extracellular domain of the erythropoietin (EPO) receptor (EPOR) is crucial for binding EPO. Substitution of Phe93 with alanine resulted in a dramatic decrease in EPO binding to the Escherichia coli-expressed extracellular domain of the EPOR (EPO-binding protein or EBP) and no detectable binding to full-length mutant receptor expressed in COS cells. Remarkably, Phe93 forms extensive contacts with a peptide ligand in the crystal structure of the EBP bound to an EPO-mimetic peptide (EMP1), suggesting that Phe93 is also important for EMP1 binding. We used alanine substitution of EBP residues that contact EMP1 in the crystal structure to investigate the function of these residues in both EMP1 and EPO binding. The three largest hydrophobic contacts at Phe93, Met150, and Phe205 and a hydrogen bonding interaction at Thr151 were examined. Our results indicate that Phe93 and Phe205 are important for both EPO and EMP1 binding, Met150 is not important for EPO binding but is critical for EMP1 binding, and Thr151 is not important for binding either ligand. Thus, Phe93 and Phe205 are important binding determinants for both EPO and EMP1, even though these ligands share no sequence or structural homology, suggesting that these residues may represent a minimum epitope on the EPOR for productive ligand binding.

Ruminant placental lactogens act as antagonists to homologous growth hormone receptors and as agonists to human or rabbit growth hormone receptors.

Growth hormone receptor (GHR)-mediated activity of ruminant placental lactogens (PLs) and ovine (o) GH was compared, using cells transfected with full size human (h), rabbit (rb), and oGHRs. All three PLs acted as agonists in heterologous bioassays, whereas in homologous bioassays in cells transfected with oGHRs they antagonized the oGH activity. Despite these differences, oGH and PLs bound with similar affinity to the oGHR extracellular domain (oGHR-ECD), indicating that the binding occurs through hormone site I. Gel filtration of complexes between oPL and oGHR-ECD showed a 1:1 stoichiometry, confirming this conclusion. The oPL T185D and bPL T188D, which exhibited weak biological activity mediated through GHRs, behaved as site I antagonists, whereas oPL G130R and bPL G133R formed a 1:1 complex with GHR-ECDs and bound to h/rb/oGHR-ECDs with affinity similar to that of wild-type oPL. They had no agonistic activity in all models transfected with h/rb and oGHRs, but were antagonistic to all of them. In conclusion, ruminant PLs antagonize the activity of oGH in homologous systems, because they cannot homodimerize oGHRs, whereas in heterologous systems they act as agonists. The structural analysis hints that minor differences in the sequence of the GHR-ECDs may account for this difference. Since the initial step in the activity transduced through cytokine/hemapoietic receptors family is receptor homodimerization or heterodimerization, we suggest that the question of homologous versus heterologous interactions should be reexamined.

Crystallographic evidence for preformed dimers of erythropoietin receptor before ligand activation.

Erythropoietin receptor (EPOR) is thought to be activated by ligand-induced homodimerization. However, structures of agonist and antagonist peptide complexes of EPOR, as well as an EPO-EPOR complex, have shown that the actual dimer configuration is critical for the biological response and signal efficiency. The crystal structure of the extracellular domain of EPOR in its unliganded form at 2.4 angstrom resolution has revealed a dimer in which the individual membrane-spanning and intracellular domains would be too far apart to permit phosphorylation by JAK2. This unliganded EPOR dimer is formed from self-association of the same key binding site residues that interact with EPO-mimetic peptide and EPO ligands. This model for a preformed dimer on the cell surface provides insights into the organization, activation, and plasticity of recognition of hematopoietic cell surface receptors.

An antagonist peptide-EPO receptor complex suggests that receptor dimerization is not sufficient for activation.

Dimerization of the erythropoietin (EPO) receptor (EPOR), in the presence of either natural (EPO) or synthetic (EPO-mimetic peptides, EMPs) ligands is the principal extracellular event that leads to receptor activation. The crystal structure of the extracellular domain of EPOR bound to an inactive (antagonist) peptide at 2.7 A resolution has unexpectedly revealed that dimerization still occurs, but the orientation between receptor molecules is altered relative to active (agonist) peptide complexes. Comparison of the biological properties of agonist and antagonist EMPs with EPO suggests that the extracellular domain orientation is tightly coupled to the cytoplasmic signaling events and, hence, provides valuable new insights into the design of synthetic ligands for EPOR and other cytokine receptors.

Identification of a 13 amino acid peptide mimetic of erythropoietin and description of amino acids critical for the mimetic activity of EMP1.

To obtain information about the functional importance of amino acids required for effective erythropoietin (EPO) mimetic action, the conserved residues of a peptide mimetic of EPO, recently discovered by phage display, were subjected to an alanine replacement strategy. Further, to identify a minimal mimetic peptide sequence, a series of truncation peptides has been generated. One EPO mimetic peptide sequence, EMP1, was targeted and more than 25 derivatives of this sequence were evaluated for their ability to compete with [125I]EPO for receptor binding and for their ability to support the proliferation of two EPO-responsive cell lines. Two hydrophobic amino acids, Tyr4 and Trp13, appear essential for mimetic action, and aromatic residues appear to be important at these sites. These findings are consistent with the previously reported X-ray crystal structure of EMP1 complexed with the extracellular domain of the EPO receptor (EPO binding protein; EBP). In our efforts to define the structural elements required for EPO mimetic action, a 13 amino acid peptide was identified which possesses mimetic properties and contains a minimal agonist epitope. The ability of this peptide to effectively serve as a mimetic capable of the induction of EPO-responsive cell proliferation appears to reside within a single residue, equivalent to position Tyr4 of EMP1, when present in a sequence that includes the cyclic core peptide structure. Although these peptides are less potent than EPO, they should serve as an excellent starting point for the design of compounds with EPO mimetic activity.

Sequence, specificity and crystallization of an oestrone-3-glucuronide antibody (3910).

We describe the specificity profile and V region sequences of a high-affinity monoclonal antibody (mAb), 3910, directed against oestrone-3-glucuronide (E3G). Inhibition studies show that the D-ring is critical for steroid specificity, while the glucuronic acid attached to the A ring is required for high binding affinity, suggesting that both 'ends' of the E3G ligand are recognized. The VH domain is encoded by a gene from the VH7183 family, while VL appears to be encoded by the Vk5.1 gene (kappa II subgroup) with a deletion of six residues from complementarity-determining region-1 (CDR1). The VH CDR3 is 10 amino acid residues in length, of which D/N contributes five residues. Comparison of VH CDR of 3910 with those of mAb against progesterone (DB3) and digoxin (26-10, 40-50), for which crystal structures have been determined, suggests that aromatic side chains are important for E3G binding and that tyrosine residues H50, H97 and H100 may interact with the ligand. The Fab fragment of 3910 has been crystallized in its native and steroid (E3G and oestriol-3-glucuronide) complexed forms. An X-ray diffraction data set to 3 A resolution has been collected for the native Fab.

Homology modeling of the structure of bacterial acetohydroxy acid synthase and examination of the active site by site-directed mutagenesis.

Acetohydroxy acid synthase (AHAS, EC 4.1.3.18) catalyzes the thiamin pyrophosphate (TPP)-dependent decarboxylation of pyruvate and condensation of the resulting two-carbon moiety with a second alpha-keto acid. It belongs to a family of homologous, TPP-dependent enzymes which catalyze different reactions which start from decarboxylation of alpha-keto acids. A model for the structure of Escherichia coli AHAS isozyme II, based on its homology with pyruvate oxidase and experimental testing of the model by site-directed mutagenesis, has been used here to study how AHAS controls the chemical fate of a decarboxylated keto acid. Because of the potential conformational freedom of the reacting substrates, residues interacting with the substrate could not be identified directly from the model of AHAS. Three residues were considered as candidates for involvement in the recognition of alpha-ketobutyrate, as the amino acids at these sites in a unique low-specificity AHAS are different from those in typical AHASs, which are highly specific for reaction with alpha-ketobutyrate as second substrate, in preference to pyruvate. These residues were altered in AHAS II by site-directed mutagenesis. Replacement of Trp464 lowers the specificity by at least 1 order of magnitude, with minor effects on the activity or stability of the enzyme, suggesting that Trp464 contributes > or = 1.3 kcal mol-1 to interaction with the "extra" methyl of alpha-ketobutyrate. Mutations of Met460 or Thr70 have small effects on specificity and do affect other properties of the protein. A model for enzyme-substrate interactions can be proposed on the basis of these results. The model of AHAS also explains previously reported spontaneous mutants of AHAS resistant to sulfonylurea herbicides, which probably bind in the narrow depression which provides access to the bound TPP. A role for the C terminus of the enzyme polypeptide in determination on the reaction pathway is also possible.

Functional mimicry of a protein hormone by a peptide agonist: the EPO receptor complex at 2.8 A.

The functional mimicry of a protein by an unrelated small molecule has been a formidable challenge. Now, however, the biological activity of a 166-residue hematopoietic growth hormone, erythropoietin (EPO), with its class 1 cytokine receptor has been mimicked by a 20-residue cyclic peptide unrelated in sequence to the natural ligand. The crystal structure at 2.8 A resolution of a complex of this agonist peptide with the extracellular domain of EPO receptor reveals that a peptide dimer induces an almost perfect twofold dimerization of the receptor. The dimer assembly differs from that of the human growth hormone (hGH) receptor complex and suggests that more than one mode of dimerization may be able to induce signal transduction and cell proliferation. The EPO receptor binding site, defined by peptide interaction, corresponds to the smaller functional epitope identified for hGH receptor. Similarly, the EPO mimetic peptide ligand can be considered as a minimal hormone, and suggests the design of nonpeptidic small molecule mimetics for EPO and other cytokines may indeed be achievable.

The structure of the complex between avidin and the dye, 2-(4'-hydroxyazobenzene) benzoic acid (HABA).

The crystal structure of the complex formed between the egg-white biotin-binding protein, avidin, and the dye, 2-(4'-hydroxyazobenzene) benzoic acid (HABA), was determined to a resolution of 2.5 A. The interaction of avidin with the benzoate ring of HABA is essentially identical to that of the complex formed between HABA and streptavidin (the bacterial analogue of the egg-white protein). This interaction emulates the definitive high-affinity interaction of both proteins with the ureido moiety of biotin. The major difference between the avidin- and streptavidin-HABA complexes lies in their interaction with the hydroxyphenyl ring of the dye molecule; in avidin, two adjacent amino acid residues (Phe72 and Ser73), which are not present in streptavidin, form additional interactions with this ring. These are suggested to account for the higher affinity of avidin for HABA. The characteristic red shift, which accompanies the interaction of both proteins with the dye, was traced to a proposed charge-transfer complex formed between the hydroxyphenyl ring of HABA and the indole ring of Trp70 in avidin (Trp79 in streptavidin). Comparison of binding site residues of two such similar proteins versus their markedly different affinities for two such different substrates should eventually contribute to a better design of biomimetic reagents and drugs.

Three-dimensional structures of avidin and the avidin-biotin complex.

The crystal structures of a deglycosylated form of the egg-white glycoprotein avidin and of its complex with biotin have been determined to 2.6 and 3.0 A, respectively. The structures reveal the amino acid residues critical for stabilization of the tetrameric assembly and for the exceptionally tight binding of biotin. Each monomer is an eight-stranded antiparallel beta-barrel, remarkably similar to that of the genetically distinct bacterial analog streptavidin. As in streptavidin, binding of biotin involves a highly stabilized network of polar and hydrophobic interactions. There are, however, some differences. The presence of additional hydrophobic and hydrophilic groups in the binding site of avidin (which are missing in streptavidin) may account for its higher affinity constant. Two amino acid substitutions are proposed to be responsible for its susceptibility to denaturation relative to streptavidin. Unexpectedly, a residual N-acetylglucosamine moiety was detected in the deglycosylated avidin monomer by difference Fourier synthesis.