Reversible unfolding and refolding behavior of a monomeric aldolase from Staphylococcus aureus.

Thermal and GdmCl-induced unfolding transitions of aldolase from Staphylococcus aureus are reversible under a variety of solvent conditions. Analysis of the transitions reveals that no partially folded intermediates can be detected under equilibrium conditions. The stability of the enzyme is very low with a delta G0 value of -9 +/- 2 kJ/mol at 20 degrees C. The kinetics of unfolding and refolding of aldolase are complex and comprise at least one fast and two slow reactions. This complexity arises from prolyl isomerization reactions in the unfolded chain, which are kinetically coupled to the actual folding reaction. Comparison with model calculations shows that at least two prolyl peptide bonds give rise to the observed slow folding reactions of aldolase and that all of the involved bonds are presumably in the trans conformation in the native state. The rate constant of the actual folding reaction is fast with a relaxation time of about 15 s at the midpoint of the folding transition at 15 degrees C. The data presented on the folding and stability of aldolase are comparable to the properties of much smaller proteins. This might be connected with the simple and highly repetitive tertiary structure pattern of the enzyme, which belongs to the group of alpha/beta barrel proteins.     

Low-n oligomers as therapeutic targets of Alzheimer's disease

The pathogenesis of Alzheimer's disease involves the progressive accumulation of amyloid β-protein (Aβ). Recent studies using synthetic Aβ peptides, a cell culture model, Aβ precursor protein transgenic mice models suggest that pre-fibrillar forms of Aβ are more deleterious than extracellular fibril forms. Recent findings obtained using synthetic Aβ peptides and human samples indicated that low-n oligomers (from dimers to octamers) may be proximate toxins for neuron and synapse. Here, we review the recent studies on the soluble oligomers, especially low-n oligomers in Alzheimer's disease.     

Crystal structure at 1.45-A resolution of the major allergen endo-beta-1,3-glucanase of banana as a molecular basis for the latex-fruit syndrome

Resolution of the crystal structure of the banana fruit endo-beta-1,3-glucanase by synchrotron X-ray diffraction at 1.45-A resolution revealed that the enzyme possesses the eightfold beta/alpha architecture typical for family 17 glycoside hydrolases. The electronegatively charged catalytic central cleft harbors the two glutamate residues (Glu94 and Glu236) acting as hydrogen donor and nucleophile residue, respectively. Modeling using a beta-1,3 linked glucan trisaccharide as a substrate confirmed that the enzyme readily accommodates a beta-1,3-glycosidic linkage in the slightly curved catalytic groove between the glucose units in positions -2 and -1 because of the particular orientation of residue Tyr33 delimiting subsite -2. The location of Phe177 in the proximity of subsite +1 suggested that the banana glucanase might also cleave beta-1,6-branched glucans. Enzymatic assays using pustulan as a substrate demonstrated that the banana glucanase can also cleave beta-1,6-glucans as was predicted from docking experiments. Similar to many other plant endo-beta-1,3-glucanases, the banana glucanase exhibits allergenic properties because of the occurrence of well-conserved IgE-binding epitopes on the surface of the enzyme. These epitopes might trigger some cross-reactions toward IgE antibodies and thus account for the IgE-binding cross-reactivity frequently reported in patients with the latex-fruit syndrome.     

Roles for the two N-terminal (β/α) modules in the folding of a (β/α)₈-barrel protein as studied by fragmentation analysis

The (β/α)₈‐barrel is one of the most abundant folds found in enzymes. To identify the independent folding units and the segment(s) that correspond to a minimum core structure within a (β/α)₈‐barrel protein, fragmentation experiments were performed with Escherichia coli phosphoribosylanthranilate isomerase, which has a single (β/α)₈‐barrel domain. Our previous studies indicated that the central four β/α segments comprise an independent folding unit; whereas, the role(s) of the first two β/α segments in folding had not been clarified prior to this report. Herein, we report the design and synthesis of a series of N‐terminally deleted fragments starting with (β/α)_1–5β₆ as the parent construct. Analytical gel filtration and urea‐induced equilibrium unfolding experiments indicated that deletions within the N‐terminal region, that is, within the first two β/α modules, resulted in reduced stability or aggregation of the remaining segments. The (β/α)_3–5β₆ segment appeared to fold into a stable structure and deletion of β₆ from (β/α)_3–5β₆ yielded (β/α)_3–5, which did not form native‐like secondary structures. However, urea‐induced unfolding of (β/α)_3–5, monitored by reduction of tryptophan fluorescence, indicated that the fragment contained a loosely packed hydrophobic core. Taken together, the results of our previous and present fragmentation experiments suggest the importance of the central (β/α)_3–4β₅ module in folding, which is a finding that is compatible with our simulated unfolding study performed previously. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

A synthetic peptide corresponding to a region of the human pericentriolar material 1 (PCM‐1) protein binds β‐amyloid (Aβ1‐42) oligomers

We have recently reported that a ~19‐kDa polypeptide, rPK‐4, is a protein kinase Cs inhibitor that is 89% homologous to the 1171–1323 amino acid region of the 228‐kDa human pericentriolar material‐1 (PCM‐1) protein (Chakravarthy et al. 2012). We have now discovered that rPK‐4 binds oligomeric amyloid‐β peptide (Aβ)_1‐42 with high affinity. Most importantly, a PCM‐1‐selective antibody co‐precipitated Aβ and amyloid β precursor protein (AβPP) from cerebral cortices and hippocampi from AD (Alzheimer's disease) transgenic mice that produce human AβPP and Aβ_1‐42, suggesting that PCM‐1 may interact with amyloid precursor protein/Aβ in vivo. We have identified rPK‐4′s Aβ‐binding domain using a set of overlapping synthetic peptides. We have found with ELISA, dot‐blot, and polyacrylamide gel electrophoresis techniques that a ~ 5 kDa synthetic peptide, amyloid binding peptide (ABP)‐p4‐5 binds Aβ_1‐42 at nM levels. Most importantly, ABP‐p4‐5, like rPK‐4, appears to preferentially bind Aβ_1‐42 oligomers, believed to be the toxic AD‐drivers. As expected from these observations, ABP‐p4‐5 prevented Aβ_1‐42 from killing human SH‐SY5Y neuroblastoma cells via apoptosis. These findings indicate that ABP‐p4‐5 is a possible candidate therapeutic for AD.     

Extracellular phosphorylation of the amyloid β-peptide promotes formation of toxic aggregates during the pathogenesis of Alzheimer's disease

Alzheimer's disease (AD) is the most common form of dementia and associated with progressive deposition of amyloid β-peptides (Aβ) in the brain. Aβ derives by sequential proteolytic processing of the amyloid precursor protein by β- and γ-secretases. Rare mutations that lead to amino-acid substitutions within or close to the Aβ domain promote the formation of neurotoxic Aβ assemblies and can cause early-onset AD. However, mechanisms that increase the aggregation of wild-type Aβ and cause the much more common sporadic forms of AD are largely unknown. Here, we show that extracellular Aβ undergoes phosphorylation by protein kinases at the cell surface and in cerebrospinal fluid of the human brain. Phosphorylation of serine residue 8 promotes formation of oligomeric Aβ assemblies that represent nuclei for fibrillization. Phosphorylated Aβ was detected in the brains of transgenic mice and human AD brains and showed increased toxicity in Drosophila models as compared with non-phosphorylated Aβ. Phosphorylation of Aβ could represent an important molecular mechanism in the pathogenesis of the most common sporadic form of AD.     

Spontaneous beta-barrel formation: an all-atom Monte Carlo study of Abeta16-22 oligomerization

Using all-atom Monte Carlo simulations with implicit water, combined with a cluster size analysis, we study the aggregation of Abeta(16) (-22), a peptide capable of forming amyloid fibrils. We consider a system of six initially randomly oriented Abeta(16) (-22) peptides, and investigate the thermodynamics and structural properties of aggregates formed by this system. The system is unaggregated without ordered secondary structure at high temperature, and forms beta-sheet rich aggregates at low temperature. At the crossover between these two regimes, we find that clusters of all sizes occur, whereas the beta-strand content is low. In one of several runs, we observe the spontaneous formation of a beta-barrel with six antiparallel strands. The beta-barrel stands out as the by far most long-lived aggregate seen in our simulations.     

A covalent S-F heterodimer of leucotoxin reveals molecular plasticity of beta-barrel pore-forming toxins

Staphylococcal leucotoxins, leucocidins, and gamma-hemolysins are bicomponent beta-barrel pore-forming toxins (beta-PFTs). Their production is associated with several clinical diseases. They have cytotoxic activity due to the synergistic action of a class S component and a class F component, which are secreted as water-soluble monomers and form hetero-oligomeric transmembrane pores, causing the lysis of susceptible cells. Structural information is currently available for the monomeric S and F proteins and the homoheptamer formed by the related alpha-hemolysin. These structures illustrate the start and end points in the mechanistic framework of beta-PFT assembly. Only limited structural data exist for the intermediate stages, including hetero-oligomeric complexes of leucotoxins. We investigated the protein-protein interactions responsible for maintaining the final bipartite molecular architecture and describe here the high-resolution crystal structure and low-resolution solution structure of a site-specific cross-linked heterodimer of gamma-hemolysin (HlgA T28C-HlgB N156C), which were solved by X-ray crystallography and small angle X-ray scattering, respectively. These structures reveal a molecular plasticity of beta-PFTs, which may facilitate the transition from membrane-bound monomers to heterodimers.     

Backbone makes a significant contribution to the electrostatics of alpha/beta-barrel proteins.

The electrostatic properties of seven alpha/beta-barrel enzymes selected from different evolutionary families were studied: triose phosphate isomerase, fructose-1,6-bisphosphate aldolase, pyruvate kinase, mandelate racemase, trimethylamine dehydrogenase, glycolate oxidase, and narbonin, a protein without any known enzymatic activity. The backbone of the alpha/beta-barrel has a distinct electrostatic field pattern, which is dipolar along the barrel axis. When the side chains are included in the calculations the general effect is to modulate the electrostatic pattern so that the electrostatic field is generally enhanced and is focused into a specific area near the active site. We use the electrostatic flux through a square surface near the active site to gauge the functionally relevant magnitude of the electrostatic field. The calculations reveal that in six out of the seven cases the backbone itself contributes greater than 45% of the total flux. The substantial electrostatic contribution of the backbone correlates with the known preference of alpha/beta-barrel enzymes for negatively charged substrates.     

The ensemble folding kinetics of the FBP28 WW domain revealed by an all-atom Monte Carlo simulation in a knowledge-based potential

In this work, we apply a detailed all‐atom model with a transferable knowledge‐based potential to study the folding kinetics of Formin‐Binding protein, FBP28, which is a canonical three‐stranded β‐sheet WW domain. Replica exchange Monte Carlo simulations starting from random coils find native‐like (Cα RMSD of 2.68 Å) lowest energy structure. We also study the folding kinetics of FBP28 WW domain by performing a large number of ab initio Monte Carlo folding simulations. Using these trajectories, we examine the order of formation of two β‐hairpins, the folding mechanism of each individual β‐hairpin, and transition state ensemble (TSE) of FBP28 WW domain and compare our results with experimental data and previous computational studies. To obtain detailed structural information on the folding dynamics viewed as an ensemble process, we perform a clustering analysis procedure based on graph theory. Further, a rigorous P_fold analysis is used to obtain representative samples of the TSEs showing good quantitative agreement between experimental and simulated Φ values. Our analysis shows that the turn structure between first and second β strands is a partially stable structural motif that gets formed before entering the TSE in FBP28 WW domain and there exist two major pathways for the folding of FBP28 WW domain, which differ in the order and mechanism of hairpin formation. Proteins 2011. © 2011 Wiley‐Liss, Inc.     

Solution structure and backbone dynamics of the DNA-binding domain of mouse Sox-5

The fold of the murine Sox-5 (mSox-5) HMG box in free solution has been determined by multidimensional NMR using (15)N-labeled protein and has been found to adopt the characteristic twisted L-shape made up of two wings: the major wing comprising helix 1 (F10--F25) and helix 2 (N32--A43), the minor wing comprising helix 3 (P51--Y67) in weak antiparallel association with the N-terminal extended segment. (15)N relaxation measurements show considerable mobility (reduced order parameter, S(2)) in the minor wing that increases toward the amino and carboxy termini of the chain. The mobility of residues C-terminal to Q62 is significantly greater than the equivalent residues of non-sequence-specific boxes, and these residues show a weaker association with the extended N-terminal segment than in non-sequence boxes. Comparison with previously determined structures of HMG boxes both in free solution and complexed with DNA shows close similarity in the packing of the hydrophobic cores and the relative disposition of the three helices. Only in hSRY/DNA does the arrangement of aromatic sidechains differ significantly from that of mSox-5, and only in rHMG1 box 1 bound to cisplatinated DNA does helix 1 have no kink. Helix 3 in mSox-5 is terminated by P68, a conserved residue in DNA sequence-specific HMG boxes, which results in the chain turning through approximately 90 degrees.     

The importance of surface loops for stabilizing an eightfold beta alpha barrel protein.

An important step in understanding how a protein folds is to determine those regions of the sequence that are critical to both its stability and its folding pathway. We chose phosphoribosyl anthranilate isomerase from Escherichia coli, which is a monomeric representative of the (beta alpha)8 barrel family of proteins, to construct a variant that carries an internal tandem duplication of the fifth beta alpha module. This (beta alpha)9 variant was enzymically active and therefore must have a wild-type (beta alpha)8 core. It had a choice a priori to fold to three different folding frames, which are distinguished by carrying the duplicated segment as an insert into one out of three different loops. Steady-state kinetic constants, the fluorescence properties of a crucial tryptophan residue, and limited proteolysis showed that the stable (beta alpha)9 variant carries the insertion between beta-strand 5 and alpha-helix 5. This preference can be explained by the important role of loops between alpha helices and beta strands in stabilizing the structure of the enzyme.     

The interaction of CK2alpha and CK2beta, the subunits of protein kinase CK2, requires CK2beta in a preformed conformation and is enthalpically driven

The protein kinase CK2 (former name: "casein kinase 2") predominantly occurs as a heterotetrameric holoenzyme composed of two catalytic chains (CK2alpha) and two noncatalytic subunits (CK2beta). The CK2beta subunits form a stable dimer to which the CK2alpha monomers are attached independently. In contrast to the cyclins in the case of the cyclin-dependent kinases CK2beta is no on-switch of CK2alpha; rather the formation of the CK2 holoenzyme is accompanied with an overall change of the enzyme's profile including a modulation of the substrate specificity, an increase of the thermostability, and an allocation of docking sites for membranes and other proteins. In this study we used C-terminal deletion variants of human CK2alpha and CK2beta that were enzymologically fully competent and in particular able to form a heterotetrameric holoenzyme. With differential scanning calorimetry (DSC) we confirmed the strong thermostabilization effect of CK2alpha on CK2beta with an upshift of the CK2alpha melting temperature of more than 9 degrees . Using isothermal titration calorimetry (ITC) we measured a dissociation constant of 12.6 nM. This high affinity between CK2alpha and CK2beta is mainly caused by enthalpic rather than entropic contributions. Finally, we determined a crystal structure of the CK2beta construct to 2.8 A resolution and revealed by structural comparisons with the CK2 holoenzyme structure that the CK2beta conformation is largely conserved upon association with CK2alpha, whereas the latter undergoes significant structural adaptations of its backbone.     

The adipokine SAA3 is induced by interleukin-1beta in mouse adipocytes

Serum amyloid A (SAA) 3 has been characterized as an inflammatory adipocyte-secreted acute-phase reactant. In the current study, regulation of SAA3 by the proinflammatory and insulin resistance-inducing cytokine interleukin (IL)-1beta was determined in 3T3-L1 and brown adipocytes. Interestingly, SAA3 mRNA and protein synthesis were dramatically increased by IL-1beta in a time-dependent fashion with maximal induction after 24 h. Furthermore, IL-1beta significantly induced SAA3 mRNA expression dose-dependently with maximal 36.4-fold upregulation seen at 2 ng/ml effector. Moreover, IL-1beta-induced SAA3 expression was mediated by nuclear factor-kappaB and janus kinase 2. Taken together, our data show a potent upregulation of SAA3 by IL-1beta.     

Effect of pH on the aggregate formation of a non-amyloid component (1-13).

The formation of aggregates including amyloid fibrils in the peptide fragment of non-amyloid-beta component (NAC(1-13)) was investigated under a variety of solution conditions. Two types of sample preparation method from neutral and acidic conditions were examined. Electron microscopy observation showed amorphous aggregates in the sample at pH 4.5 adjusted from the neutral condition. The CD and HPLC quantitative analyses indicated that the formation of the amorphous aggregate did not accompany a conformational conversion from a random coil in the sample solution. The analyses of pKa values determined by pH titration experiments in NMR spectroscopy indicated that the protonation of the carboxyl group of the N-terminal glutamic acid triggers the aggregation of NAC(1-13). On the other hand, electron microscopy observation showed that the samples at pH 2.2 and 4.5 adjusted from an initial pH of 2.2 form fibrils. A beta-structure was detected by CD spectroscopy in the 1 mM NAC(1-13) at pH 2.2 immediately after preparation. The CD analyses of samples at different concentrations and temperatures indicated that 1 mM NAC(1-13) immediately after preparation at pH 2.2 was oligomerized. The quantity of the beta-structure was increased depending on the Incubation time. The results strongly suggested that the beta-conformational oligomers play a critical role for the fibril nucleus.     

Employing a superior BACE1 cleavage sequence to probe cellular APP processing

The involvement of beta-secretase (BACE1; beta-site APP-cleaving enzyme) in producing the beta-amyloid component of plaques found in the brains of Alzheimer's patients, has fueled a major research effort to characterize this protease. Here, we describe work toward understanding the substrate specificity of BACE1 that began by considering the natural APP substrate and its Swedish mutant, APPSw, and proceeded on to include oxidized insulin B chain and ubiquitin substrates. From these findings, and the study of additional synthetic peptides, we determined that a decapeptide derived from APP in which the P3-P2' sequence, ...VKM--DA..., was replaced by ...ISY--EV... (-- = beta site of cleavage), yielded a substrate that was cleaved by BACE1 seven times faster than the corresponding APPSw peptide, SEVNL--DAEFR. The expanded peptide, GLTNIKTEEISEISY--EVEFRWKK, was cleaved an additional seven times faster than its decapeptide counterpart (boldface), and provides a substrate allowing assay of BACE1 at picomolar concentrations. Several APP mutants reflecting these beta-site amino acid changes were prepared as the basis for cellular assays. The APPISYEV mutant proved to be a cellular substrate that was superior to APPSw. The assay based on APPISYEV is highly specific for measuring BACE1 activity in cells; its homolog, BACE2, barely cleaved APPISYEV at the beta-site. Insertion of the optimized ISY--EV motif at either the beta-site (Asp1) or beta'-site (Glu11) directs the rate of cellular processing of APP at these two accessible sites. Thus, we have identified optimal BACE1 substrates that will be useful to elucidate the cellular enzymatic actions of BACE1, and for design of inhibitors that might be of therapeutic benefit in Alzheimer's disease.     

BetaPix-a enhances the activity of phospholipase Cgamma1 by binding SH3 domain in breast cancer

Phospholipase C-gamma1 (PLCgamma1) plays a critical role in cell growth and proliferation by generating the second messengers, diacylglycerol and 1, 4, 5-inositol triphosphate. To investigate the roles of Src homology domain 2 and domain 3 of PLCgamma1 in PLCgamma1-mediated cell signaling, we characterized some proteins binding to these domains in the MCF7 and MDA-MB-231 breast cancer cell lines. Of the several proteins that bind to glutathione-S-transferase-SH2/SH2/SH3, we identified an 85 kDa protein that binds to the SH3 domain of PLCgamma1 as the guanine nucleotide exchange factor, p21-activated protein kinase-interacting exchange factor-a (betaPix-a). BetaPix-a co-immunoprecipitated with PLCgamma1 in breast cancer tissues extracts and in MCF7 and MDA-MB-231 cell extracts. In addition, PDGF-stimulated PLCgamma1 activity was elevated in betaPix-a-overexpressing NIH3T3 cells. Our results suggest that betaPix-a binds to the Src homology domain 3 of PLCgamma1 and promotes tumor growth in breast cancer by enhancing the activity PLCgamma1.