Beta Residues Research Articles

Molecular interactions of resveratrol with Aβ 42 peptide and fibril during in-vitro Aβ 42 aggregation

Amyloid aggregates are responsible for the development of oxidative stress and neurotoxicity in the brain. Several amino acid residues of amyloid beta (Aβ) 42 establish different molecular interactions to form and stabilize these aggregates, which can be targeted to prevent the aggregation. Resveratrol (Res) has inhibitory properties against Aβ 42 aggregation but studies elucidating its interactions with different residues of Aβ 42 aggregates are scarce. In the present study, we have discerned the molecular interactions of Res with different amino acid residues of Aβ 42 peptide and fibril during in-vitro Aβ 42 aggregation. Inhibitory properties of Res against amyloid aggregation were established through ANS and Thioflavin-T fluorescence assay, congo red assay and CD spectroscopy. The molecular interactions were established through molecular docking and 100 ns molecular dynamics simulations. The hydrophobic regions of Aβ 42 peptide, responsible for the formation of aggregates, were better protected in the presence of Res as indicated by ANS assay. Th-T and congo red assay suggested that Res prevented the formation of cross β-sheet structures. CD spectra analysis revealed that in the presence of Res, the secondary structure content was significantly decreased. MD simulation analysis revealed that Res formed strong molecular interactions with hydrophobic and secondary structure forming amino acid residues, which are involved in the amyloid aggregation and stabilization of aggregates. In conclusion, these interactions might have led to the decline in secondary structure content, formation of unordered nontoxic aggregates and prevention of the formation of sufuranyl free radical.

Real-Time 1H NMR reveals position and sequence dependences of amino acid isomerization in amyloid beta fragments in situ

Amino acid isomers and their biologically relevant functions have been found in a variety of peptides and proteins in a living system. It is challenging, however, to specify how such isomerization proceeds at each amino acid independently in situ. In this work, the amino acid isomerization was quantified simultaneously at multiple residues in real time, by using solution-state 1H NMR in situ. The ability of NMR to distinguish isomerization product and reactant was demonstrated at different positions of aspartyl (Asp) residues in amyloid beta (Aβ) 1–40 fragments, as well as the glutamyl (Glu) residue. This enabled us to compare the isomerization of Asp1, Asp7, and Asp23, and Glu3 systematically in various Aβ fragments, in terms of the kinetics of isomerization in real time. It was found that Asp1 and Asp7 were prone to convert to iso-aspartyl (isoAsp) form, although Asp23 and Glu3 showed limited reactivity. The analysis demonstrated the role of histidine (His) in regulating Asp isomerization; His6 and His13/14 showed an inhibitory effect on the progress of isomerization at Asp1 and Asp7, respectively, by forming Asp-His complex that serves to slow down the conversion from Asp to isoAsp.

The antibody response to SARS-CoV-2 Beta underscores the antigenic distance to other variants

SummaryAlpha-B.1.1.7, Beta-B.1.351, Gamma-P.1, and Delta-B.1.617.2 variants of SARS-CoV-2 express multiple mutations in the spike protein (S). These may alter the antigenic structure of S, causing escape from natural or vaccine-induced immunity. Beta is particularly difficult to neutralize using serum induced by early pandemic SARS-CoV-2 strains and is most antigenically separated from Delta. To understand this, we generated 674 mAbs from Beta-infected individuals and performed a detailed structure-function analysis of the 27 most potent mAbs: one binding the spike N-terminal domain (NTD), the rest the receptor-binding domain (RBD). Two of these RBD-binding mAbs recognize a neutralizing epitope conserved between SARS-CoV-1 and -2, while 18 target mutated residues in Beta: K417N, E484K, and N501Y. There is a major response to N501Y, including a public IgVH4-39 sequence, with E484K and K417N also targeted. Recognition of these key residues underscores why serum from Beta cases poorly neutralizes early pandemic and Delta viruses.

A Double Mutant Cycle Involving the Charged Residues of Amyloid Beta

A critical difference between the fibril structures of Amyloid beta 40 (Aβ40) and its more toxic variant Abeta42 (Aβ42), associated with Alzheimer's disease, lies in the nature of a specific salt bridge involving Lysine-28 (K28). In Aβ42, it has a salt bridge with the C-terminal negative charge, while in Aβ40 it has the bridge with Aspartate-23 (D23). Interestingly, Aβ40 oligomers, which are aggregation intermediates known to be more toxic than the fibrils, do not have the D23-K28 salt bridge. So we ask whether transient interactions between K28 and the C-terminal of Aβ40, make the oligomers similar to the more toxic Aβ42. We use the powerful technique of ‘double mutant cycle’ to probe if K28 and the terminal COO- interact directly or indirectly during any stage of the aggregation process, and how that may influence the aggregation and toxicity of the peptide. We acetylate K28 and amidate the C-terminal, separately and also in the same mutant, and probe the aggregation kinetics, final solubility, secondary structure, membrane affinity and toxicity of the 4 variants. We find that the K28 acetylation has a very strong effect on the solubility, while amidation of the C-terminal has a mild but opposite effect. While K28 acetylation hardly affects the membrane affinity, amidation increases it sharply. However, the secondary structure of the amidated Abeta40 differs from the wild-type in the fibrillary state. Interestingly, both C-terminal amidation and K28-acetylation decrease the toxicity of the peptide considerably. Our results show that the K28 and the terminal COO- have independent but strong effects on the properties of the peptide. Moreover, the results prove the futility of focusing on reagents that just reduce aggregation propensity, since the toxicity of the peptide can vary independently.

Molecular docking analysis of phytoconstituent from Momordica charantia with Guanylate Cyclase catalytic domain.

Soluble guanylate cyclase (sGC) is a type of lyase enzyme with profoundly increasing importance in treatments of cardiovascular and neurodegenerative disorders. Modulation of sGC activity demonstrated beneficial effects against Parkinson's disease by reducing glutamate excitotoxicity. It is of interest to evaluate the pharmacological activity of Momordica charantia phytoconstituent (DGalacturonic acid) and ODQ with catalytic domain of sGC enzyme, using Autodock version 4.2 programs. Docking results revealed the binding ability of ODQ at the allosteric sites of sGC. D-galacturonic acid also shows binding interaction at the same allosteric sites in the catalytic domain of sGC like ODQ. Results show that both the ligands have efficient binding to THR 474 amino acid residue of beta 1 subunit of the enzyme. The drug likeliness score further implies the suitability of D-Galacturonic acid as a drug-like molecule. The binding property of ODQ and D-Galacturonic acid with the catalytic domain help to inhibit sGC activity having pharmacological effects. Moreover, ODQ interaction with heme site of sGC is already known while its interaction with the catalytic domain is shown in this report.

Effect of GNP functionalisation and multiple N-methylation of β-amyloid residue (32-37) on Gram-positive bacterium.

In the previous report, the authors showed the gold nanoparticle (GNP) functionalised multiple N-methylated fragments of the residue (32-37) of beta (β)-amyloid protein (1-42), CGGIGLMVG and CGGGGGIGLMVG toward disruption of β-amyloid (1-42), the predominant component of senile plaques. Herein the in vitro antimicrobial activities of both normal and multiple N-methylated sequences of CGGIGLMVG and CGGGGGIGLMVG were screened and it was found that all the eight sequences including four (non-functionalised with GNP) to possess activity against both Gram-positive [Staphylococcus aureus (ATCC 43300) and Enterococcus faecalis (ATCC 5129)] and Gram-negative [Escherichia coli (ATCC 35218), Pseudomonas aeruginosa (ATCC 27853) and Klebsiella pneumoniae (ATCC 700603)] bacteria. Among them, N-methylated sequences CGGIGLMVG and CGGGGGIGLMVG shown remarkable activity against Gram-positive bacteria.

Spreading of amyloid-β peptides via neuritic cell-to-cell transfer is dependent on insufficient cellular clearance

The spreading of pathology through neuronal pathways is likely to be the cause of the progressive cognitive loss observed in Alzheimer's disease (AD) and other neurodegenerative diseases. We have recently shown the propagation of AD pathology via cell-to-cell transfer of oligomeric amyloid beta (Aβ) residues 1–42 (oAβ1–42) using our donor–acceptor 3-D co-culture model. We now show that different Aβ-isoforms (fluorescently labeled 1–42, 3(pE)-40, 1–40 and 11–42 oligomers) can transfer from one cell to another. Thus, transfer is not restricted to a specific Aβ-isoform. Although different Aβ isoforms can transfer, differences in the capacity to clear and/or degrade these aggregated isoforms result in vast differences in the net amounts ending up in the receiving cells and the net remaining Aβ can cause seeding and pathology in the receiving cells. This insufficient clearance and/or degradation by cells creates sizable intracellular accumulations of the aggregation-prone Aβ1–42 isoform, which further promotes cell-to-cell transfer; thus, oAβ1–42 is a potentially toxic isoform. Furthermore, cell-to-cell transfer is shown to be an early event that is seemingly independent of later appearances of cellular toxicity. This phenomenon could explain how seeds for the AD pathology could pass on to new brain areas and gradually induce AD pathology, even before the first cell starts to deteriorate, and how cell-to-cell transfer can act together with the factors that influence cellular clearance and/or degradation in the development of AD.

Magic angle spinning NMR analysis of beta2-microglobulin amyloid fibrils in two distinct morphologies.

Beta(2)-microglobulin (beta(2)m) is the major structural component of amyloid fibrils deposited in a condition known as dialysis-related amyloidosis. Despite numerous studies that have elucidated important aspects of the fibril formation process in vitro, and a magic angle spinning (MAS) NMR study of the fibrils formed by a small peptide fragment, structural details of beta(2)m fibrils formed by the full-length 99-residue protein are largely unknown. Here, we present a site-specific MAS NMR analysis of fibrils formed by the full-length beta(2)m protein and compare spectra of fibrils prepared under two different conditions. Specifically, long straight (LS) fibrils are formed at pH 2.5, while a very different morphology denoted as worm-like (WL) fibrils is observed in preparations at pH 3.6. High-resolution MAS NMR spectra have allowed us to obtain (13)C and (15)N resonance assignments for 64 residues of beta(2)m in LS fibrils, including part of the highly mobile N-terminus. Approximately 25 residues did not yield observable signals. Chemical shift analysis of the sequentially assigned residues indicates that these fibrils contain an extensive beta-sheet core organized in a non-native manner, with a trans-P32 conformation. In contrast, WL fibrils exhibit more extensive dynamics and appear to have a smaller beta-sheet core than LS fibrils, although both cores seem to share some common elements. Our results suggest that the distinct macroscopic morphological features observed for the two types of fibrils result from variations in structure and dynamics at the molecular level.

Two Distinct Mechanisms of Inactivation of the Class Ic Ribonucleotide Reductase fromChlamydia trachomatisby Hydroxyurea: Implications for the Protein Gating of Intersubunit Electron Transfer

Catalysis by a class I ribonucleotide reductase (RNR) begins when a cysteine (C) residue in the alpha(2) subunit is oxidized to a thiyl radical (C(*)) by a cofactor approximately 35 A away in the beta(2) subunit. In a class Ia or Ib RNR, a stable tyrosyl radical (Y(*)) is the C oxidant, whereas a Mn(IV)/Fe(III) cluster serves this function in the class Ic enzyme from Chlamydia trachomatis (Ct). It is thought that, in either case, a chain of Y residues spanning the two subunits mediates C oxidation by forming transient "pathway" Y(*)s in a multistep electron transfer (ET) process that is "gated" by the protein so that it occurs only in the ready holoenzyme complex. The drug hydroxyurea (HU) inactivates both Ia/b and Ic beta(2) subunits by reducing their C oxidants. Reduction of the stable cofactor Y(*) (Y122(*)) in Escherichia coli class Ia beta(2) is faster in the presence of alpha(2) and a substrate (CDP), leading to speculation that HU might intercept a transient ET pathway Y(*) under these turnover conditions. Here we show that this mechanism is one of two that are operant in HU inactivation of the Ct enzyme. HU reacts with the Mn(IV)/Fe(III) cofactor to give two distinct products: the previously described homogeneous Mn(III)/Fe(III)-beta(2) complex, which forms only under turnover conditions (in the presence of alpha(2) and the substrate), and a distinct, diamagnetic Mn/Fe cluster, which forms approximately 900-fold less rapidly as a second phase in the reaction under turnover conditions and as the sole outcome in the reaction of Mn(IV)/Fe(III)-beta(2) only. Formation of Mn(III)/Fe(III)-beta(2) also requires (i) either Y338, the subunit-interfacial ET pathway residue of beta(2), or Y222, the surface residue that relays the "extra electron" to the Mn(IV)/Fe(IV) intermediate during activation of beta(2) but is not part of the catalytic ET pathway, and (ii) W51, the cofactor-proximal residue required for efficient ET between either Y222 or Y338 and the cofactor. The combined requirements for the catalytic subunit, the substrate, and, most importantly, a functional surface-to-cofactor electron relay system imply that HU effects the Mn(IV)/Fe(III) --> Mn(III)/Fe(III) reduction by intercepting a Y(*) that forms when the ready holoenzyme complex is assembled, the ET gate is opened, and the Mn(IV) oxidizes either Y222 or Y338.

γ-Amino Butyric Acid Type A Receptor Mutations at β2N265 Alter Etomidate Efficacy While Preserving Basal and Agonist-dependent Activity

Etomidate acts at gamma-Aminobutyric acid type A (GABAA) receptors containing beta2 or beta3, but not beta1 subunits. Mutations at beta residue 265 (Ser in beta1; Asn in beta2 or beta3) profoundly affect etomidate sensitivity. Whether these mutations alter etomidate binding remains uncertain. Heterologously expressed alpha1beta2gamma2L GABAA receptors and receptors with beta2(N265S) or beta2(N265M) mutations were studied electrophysiologically in both Xenopus oocytes and HEK293 cells. Experiments quantified the impact of beta2N265 mutations or substituting beta1 for beta2 on basal channel activation, GABA EC50, maximal GABA efficacy, etomidate-induced leftward shift in GABA responses, etomidate direct activation, and rapid macrocurrent kinetics. Results were analyzed in the context of an established allosteric co-agonist mechanism. Mutations produced only small changes in basal channel activity, GABA EC50, maximal GABA efficacy, and macrocurrent kinetics. Relative to wild-type, beta2(N265S) reduced etomidate enhancement of apparent GABA affinity six-fold, and it reduced etomidate direct activation efficacy 14-fold. beta2(N265M) totally eliminated both etomidate modulation of GABA responses and direct channel activation. Mechanism-based analysis showed that the function of both mutants remains consistent with the allosteric co-agonist model and that beta2(N265S) reduced etomidate allosteric efficacy five-fold, whereas etomidate-binding affinity dropped threefold. Experiments swapping beta2 subunits for beta1 indicated that etomidate efficacy is reduced 34-fold, whereas binding affinity drops less than two-fold. Mutations at beta2N265 profoundly alter etomidate sensitivity with only small changes in basal and GABA-dependent channel activity. Mutations at the beta2N265 residue or replacement of beta2 with beta1 influence etomidate efficacy much more than binding to inactive receptors.

The VE‐cadherin binding domain of fibrinogen induces endothelial barrier permeability and enhances transendothelial migration of malignant breast epithelial cells

Fibrin deposition and exudation of plasma fibrinogen (Fg) have long been recognized as hallmarks of inflammation, cardiovascular disease and neoplasia. The Fg-beta(15-42) domain binds to the endothelial cell adhesion molecule, VE-cadherin, promoting endothelial cell proliferation, angiogenesis and leukocyte diapedesis. Furthermore, spontaneous blood-borne and lymphatic metastasis of some types of tumor emboli requires plasma fibrin(ogen); however, the molecular mechanisms by which this occurs are poorly understood. We sought to determine whether Fg-beta(15-42) and VE-cadherin binding interactions promote endothelial barrier permeability and breast cancer cell transendothelial migration (TEM) using transwell insert culture systems. Synthetic peptides containing/missing residues beta(15-17) critical for Fg-beta(15-42) binding to VE-cadherin, and antibodies that bind to Fg-beta(15-21) (T2G1) and VE-cadherin (BV9) were used to induce or inhibit Fg-mediated permeability and TEM. Fg induced dose-dependent permeability of human umbilical vein and microvascular endothelial but not epithelial cell barriers. Maximal Fg-induced endothelial permeability required Fg-beta(15-42) and VE-cadherin-binding interactions involving Fg-beta(15-17). Fg-induced TEM of malignant MDA-MB-231 and MCF-7 breast cancer cells also required Fg-beta(15-42) and VE-cadherin binding; however, such TEM was independent of E-cadherin or estrogen receptor expression. In contrast, Fg did not induce TEM of nonmalignant MCF-10A breast epithelial cells. Fg-induced endothelial permeability was retained in the presence of MDA-MB-231 but inhibited in the presence of MCF-10A cells. It is intriguing to speculate that loss of Fg-beta(15-42) binding by premalignant breast epithelial cells serves as a molecular switch to induce a highly aggressive, metastatic breast cancer phenotype. Hence, Fg-beta(15-42) represents a potential molecular target for therapeutic intervention of breast cancer metastasis.

Contributions of the individual hydrophobic clefts of the Escherichia coli sliding clamp to clamp loading, DNA replication and clamp recycling

The homodimeric Escherichia coli β sliding clamp contains two hydrophobic clefts with which proteins involved in DNA replication, repair and damage tolerance interact. Deletion of the C-terminal five residues of β (βC) disrupted both clefts, severely impairing interactions of the clamp with the DnaX clamp loader, as well as the replicative DNA polymerase, Pol III. In order to determine whether both clefts were required for loading clamp onto DNA, stimulation of Pol III replication and removal of clamp from DNA after replication was complete, we developed a method for purification of heterodimeric clamp proteins comprised of one wild-type subunit (β+), and one βC subunit (β+/βC). The β+/βC heterodimer interacted normally with the DnaX clamp loader, and was loaded onto DNA slightly more efficiently than was β+. Moreover, β+/βC interacted normally with Pol III, and stimulated replication to the same extent as did β+. Finally, β+/βC was severely impaired for unloading from DNA using either DnaX or the δ subunit of DnaX. Taken together, these findings indicate that a single cleft in the β clamp is sufficient for both loading and stimulation of Pol III replication, but both clefts are required for unloading clamp from DNA after replication is completed.

Helicity of alpha(404-451) and beta(394-445) tubulin C-terminal recombinant peptides.

We have investigated the solution conformation of the functionally relevant C-terminal extremes of alpha- and beta-tubulin, employing the model recombinant peptides RL52alpha3 and RL33beta6, which correspond to the amino acid sequences 404-451(end) and 394-445(end) of the main vertebrate isotypes of alpha- and beta-tubulin, respectively, and synthetic peptides with the alpha-tubulin(430-443) and beta-tubulin(412-431) internal sequences. Alpha(404-451) and beta(394-445) are monomeric in neutral aqueous solution (as indicated by sedimentation equilibrium), and have circular dichroism (CD) spectra characteristic of nearly disordered conformation, consistent with low scores in peptide helicity prediction. Limited proteolysis of beta(394-445) with subtilisin, instead of giving extensive degradation, resulted in main cleavages at positions Thr409-Glu410 and Tyr422-Gln423-Gln424, defining the proteolysis resistant segment 410-422, which corresponds to the central part of the predicted beta-tubulin C-terminal helix. Both recombinant peptides inhibited microtubule assembly, probably due to sequestration of the microtubule stabilizing associated proteins. Trifluoroethanol (TFE)-induced markedly helical CD spectra in alpha(404-451) and beta(394-445). A substantial part of the helicity of beta(394-445) was found to be in the CD spectrum of the shorter peptide beta(412-431) with TFE. Two-dimensional 1H-NMR parameters (nonsequential nuclear Overhauser effects (NOE) and conformational C alphaH shifts) in 30% TFE permitted to conclude that about 25% of alpha(404-451) and 40% of beta(394-451) form well-defined helices encompassing residues 418-432 and 408-431, respectively, flanked by disordered N- and C-segments. The side chains of beta(394-451) residues Leu418, Val419, Ser420, Tyr422, Tyr425, and Gln426 are well defined in structure calculations from the NOE distance constraints. The apolar faces of the helix in both alpha and beta chains share a characteristic sequence of conserved residues Ala,Met(+4),Leu(+7),Tyr(+11). The helical segment of alpha(404-451) is the same as that described in the electron crystallographic model structure of alphabeta-tubulin, while in beta(394-451) it extends for nine residues more, supporting the possibility of a functional coil --> helix transition at the C-terminus of beta-tubulin. These peptides may be employed to construct model complexes with microtubule associated protein binding sites.

Exploration of Backbone Space in Foldamers Containing α‐ and β‐Amino Acid Residues: Developing Protease‐Resistant Oligomers that Bind Tightly to the BH3‐Recognition Cleft of Bcl‐xL

Protein-protein interactions play crucial roles in cell-signaling events and are often implicated in human disease. Molecules that bind tightly to functional protein-surface sites and show high stability to degradative enzymes could be valuable pharmacological tools for dissection of cell-signaling networks and might ultimately lead to therapeutic agents. We recently described oligomers containing both alpha- and beta-amino acid residues that bind tightly to the BH3 recognition site of the anti-apoptotic protein Bcl-x(L). The oligomers with highest affinity had a nine-residue N-terminal segment with a 1:1 alpha:beta residue repeat and a six-residue C-terminal segment containing exclusively proteinogenic alpha-residues. The N-terminal portions of such (alpha/beta+alpha)-peptides are highly resistant to proteolysis, but the C-terminal alpha-segments are susceptible. This study emerged from efforts to modify the alpha-segment in an (alpha/beta+alpha)-peptide in a way that would diminish proteolytic degradation but retain high affinity for Bcl-x(L). Some of the oligomers reported here could prove useful in certain biological applications, particularly those for which extended incubation in a biological milieu is required.

RETRACTED: Mechanism of nucleotide incorporation in DNA polymerase β

DNA polymerases play a central role in the mechanisms of DNA replication and repair. Here, we report mechanisms of the beta-polymerase catalyzed phosphoryl transfer reactions corresponding to correct and incorrect nucleotide incorporations in the DNA. Based on energy minimizations, molecular dynamics simulations, and free energy calculations of solvated ternary complexes of pol beta and by employing a mixed quantum mechanics molecular mechanics Hamiltonian, we have uncovered the identities of transient intermediates in the phosphoryl transfer pathways. Our study has revealed that an intriguing Grotthuss hopping mechanism of proton transfer involving water and three conserved aspartate residues in pol beta's active site mediates the phosphoryl transfer in the correct as well as misincorporation of nucleotides. The significance of this catalytic step in serving as a kinetic check point of polymerase fidelity may be unique to DNA polymerase beta, and is discussed in relation to other known mechanisms of DNA polymerases.

Mitochondrial ATP Synthase Residue βArginine-408, Which Interacts with the Inhibitory Site of Regulatory Protein IF1, Is Essential for the Function of the Enzyme

Mitochondrial ATP synthase (F(1)F(0)-ATPase) is regulated by an intrinsic ATPase inhibitor protein, IF(1). We previously found that six residues of the yeast IF(1) (Phe17, Arg20, Glu21, Arg22, Glu25, and Phe28) form an ATPase inhibitory site [Ichikawa, N. and Ogura, C. (2003) J. Bioenerg. Biomembr. 35, 399-407]. In the crystal structure of the F(1)/IF(1) complex [Cabezón, E. et al. (2003) Nat. Struct. Biol. 10, 744-750], the core residues of the inhibitory site interact with Arg408, Arg412 and Glu454 of the beta-subunit of F(1). In the present study, we examined the roles of the three beta residues by means of site-directed mutagenesis. A total of six yeast mutants were constructed: R408I, R408T, R412I, R412T, E454Q, and E454V. The betaArg412 and betaGlu454 mutants (R412I, R412T, E454Q, and E454V) could grow on a nonfermentable lactate medium, but the betaArg408 mutants (R408I and R408T) could not. The ATPase activity of isolated mitochondria was decreased in R412I, R412T, E454Q, and E454V mutant cells, and undetectable in R408I and R408T cells. The subunits of F(1) (alpha, beta, and gamma) were detected in mitochondria from each mutant on immunoblotting, and the F(1)F(0) complex was isolated from them. These results indicate that betaArg408 is essential not for assembly of the F(1)F(0) complex but for the catalytic activity of the enzyme. In the crystal structure of F(1), betaArg408 binds to alphaGlu399 in the alpha(DP)/beta(DP) pair and seems to be important for formation of the closed alpha(DP)/beta(DP) conformation. IF(1) seems to disrupt this alpha(DP)Glu399/beta(DP)Arg408 interaction by binding to beta(DP)Arg408, and to interfere with the change from the open alpha(DP)/beta(DP) conformation to the closed conformation that is required for catalysis by F(1)F(0)-ATPase.

Crystal Structure of Isoaspartyl Aminopeptidase in Complex with l-Aspartate

The crystal structure of Escherichia coli isoaspartyl aminopeptidase/asparaginase (EcAIII), an enzyme belonging to the N-terminal nucleophile (Ntn)-hydrolases family, has been determined at 1.9-A resolution for a complex obtained by cocrystallization with l-aspartate, which is a product of both enzymatic reactions catalyzed by EcAIII. The enzyme is a dimer of heterodimers, (alphabeta)(2). The (alphabeta) heterodimer, which arises by autoproteolytic cleavage of the immature protein, exhibits an alphabetabetaalpha-sandwich fold, typical for Ntn-hydrolases. The asymmetric unit contains one copy of the EcAIII.Asp complex, with clearly visible l-aspartate ligands, one bound in each of the two active sites of the enzyme. The l-aspartate ligand is located near Thr(179), the N-terminal residue of subunit beta liberated in the autoproteolytic event. Structural comparisons with the free form of EcAIII reveal that there are no major rearrangements of the active site upon aspartate binding. Although the ligand binding mode is similar to that observed in an l-aspartate complex of the related enzyme human aspartylglucosaminidase, the architecture of the EcAIII active site sheds light on the question of substrate specificity and explains why EcAIII is not able to hydrolyze glycosylated asparagine substrates.

Structural Analysis of Dispersin B, a Biofilm-releasing Glycoside Hydrolase from the Periodontopathogen Actinobacillus actinomycetemcomitans

Bacteria in a biofilm are enmeshed in a self-synthesized extracellular polysaccharide matrix that holds the bacteria together in a mass and firmly attaches the bacterial mass to the underlying surface. A major component of the extracellular polysaccharide matrix in several phylogenetically diverse bacteria is PGA, a linear polymer of N-acetylglucosamine residues in beta(1,6)-linkage. PGA is produced by the Gram-negative periodontopathogen Actinobacillus actinomycetemcomitans as well as by the Gram-positive device-associated pathogen Staphylococcus epidermidis. We recently reported that A.actinomycetemcomitans produces a soluble glycoside hydrolase named dispersin B, which degrades PGA. Here, we present the crystal structure of dispersin B at 2.0A in complex with a glycerol and an acetate ion at the active site. The enzyme crystallizes in the orthorhombic space group C222(1) with cell dimensions a=41.02A, b=86.13A, c=185.77A. The core of the enzyme consists a (beta/alpha)(8) barrel topology similar to other beta-hexosaminidases but significant differences exist in the arrangement of loops hovering in the vicinity of the active site. The location and interactions of the glycerol and acetate moieties in conjunction with the sequence analysis suggest that dispersin B cleaves beta(1,6)-linked N-acetylglucosamine polymer using a catalytic machinery similar to other family 20 hexosaminidases which cleave beta(1,4)-linked N-acetylglucosamine residues.

Alpha,beta hybrid peptides: a polypeptide helix with a central segment containing two consecutive beta-amino acid residues.

Conformational studies on the synthetic 11-aa peptide t-butoxycarbonyl (Boc)-Val-Ala-Phe-alpha-aminoisobutyric acid (Aib)-(R)-beta3-homovaline (betaVal)-(S)-beta3-homophenylalanine (betaPhe)-Aib-Val-Ala-Phe-Aib-methyl ester (OMe) (peptide 1; betaVal and betaPhe are beta amino acids generated by homologation of the corresponding l-residues) establish that insertion of two consecutive beta residues into a polypeptide helix can be accomplished without significant structural distortion. Crystal-structure analysis reveals a continuous helical conformation encompassing the segment of residues 2-10 of peptide 1. At the site of insertion of the betabeta segment, helical hydrogen-bonded rings are expanded. A C15 hydrogen bond for the alphabetabeta segment and two C14 hydrogen bonds for the alphaalphabeta or betaalphaalpha segments have been characterized. The following conformational angles were determined from the crystal structure for the beta residues: betaVal-5 (= -126 degrees, = 76 degrees, and psi = -124) and betaPhe-6 (=-88 degrees, = 80 degrees, and psi =-118). The N terminus of the peptide is partially unfolded in crystals. The 500-MHz 1H-NMR studies establish a continuous helix over the entire length of the peptide in CDCl3 solution, as evidenced by diagnostic nuclear Overhauser effects. The presence of seven intramolecular hydrogen bonds is also established by using solvent dependence of NH chemical shifts.

Extracellular Signal-regulated Kinase Activated by Epidermal Growth Factor and Cell Adhesion Interacts with and Phosphorylates Vinexin

Extracellular signal-regulated kinase 1/2 (ERK1/2) is activated by various extracellular stimuli including growth factors and cytokines and plays a pivotal role in regulating cell proliferation and differentiation by phosphorylating nuclear transcription factors. Recently, it was reported that activated ERK1/2 also concentrates at adhesion sites and regulates cell spreading and migration. Vinexin is a focal adhesion protein regulating both cell spreading and growth factor signaling. We show here that vinexin was directly phosphorylated by ERK1/2 upon stimulation with growth factors. ERK1/2 phosphorylated the linker region of vinexin between the second and third SH3 domains. Site-directed mutagenesis revealed that ERK2 mainly phosphorylated the serine 189 residue of vinexin beta. Furthermore, vinexin beta interacted with ERK1/2 both in vitro and in vivo. Vinexin interacted with the active but not inactive form of ERK1/2. A putative DEF (docking for ERK FXFP) domain located in the linker region of vinexin was required for the interaction with ERK1/2 and efficient phosphorylation of vinexin beta by ERK2. Finally, we showed that cell adhesion to fibronectin also induced the association of vinexin beta with ERK2 and the phosphorylation of vinexin beta. Furthermore, vinexin and ERK were co-localized to the periphery of cells during cell spreading on fibronectin. Together, these results suggest that vinexin is a novel substrate of ERK2 and may play roles in ERK-dependent cell regulation during cell spreading as well as in growth factor-induced responses.