Phosphorylation of glucose by a guanosine-5′-triphosphate (GTP)-dependent glucokinase in Fibrobacter succinogenes subsp. succinogenes S85

Cell extracts of Fibrobacter succinogenes subsp. succinogenes S85 phosphorylated glucose with a GTP-dependent glucokinase. The enzyme showed little activity with ATP (12% of that with GTP). Of other phosphate donors tested, only dGTP and ITP gave high glucokinase activities. Dialyzed extracts required Mg⁺² and K⁺ for maximal activity. In potassium phosphate buffer, glucokinase showed maximum activity at pH 7.5 with glucose-6-phosphate dehydrogenase as the coupling enzyme. In this assay, glucokinase was active with glucose (100%), 2-deoxy-d-glucose (40%), and mannose (20%). Partially purified glucokinase had a molecular weight of 82,000 and a pl of 4.82. Double-reciprocal plots of substrate concentration versus velocity were linear and the enzyme had apparent K_m values of 55 M for glucose and 72 M for GTP. Dialyzed cell extracts of Fibrobacter intestinalis C1A also contained a GTP-dependent glucokinase that showed little activity with ATP. Potassium also stimulated the activity of this enzyme. These results suggest that this unusual glucokinase may be characteristic of the genus Fibrobacter.Abbreviations CHES cyclohexylaminoethanesulfonic acid - GK glucokinase - PEP phosphoenolpyruvate Published with the approval of the Director of the North Dakota Agricultural Experiment Station as journal article no. 2186     

Crystal Structure of Calmodulin Binding Domain of Orai1 in Complex with Ca2+?Calmodulin Displays a Unique Binding Mode

Orai1 is a plasma membrane protein that in its tetrameric form is responsible for calcium influx from the extracellular environment into the cytosol in response to interaction with the Ca²⁺-depletion sensor STIM1. This is followed by a fast Ca²⁺·calmodulin (CaM)-dependent inhibition, resulting from CaM binding to an Orai1 region called the calmodulin binding domain (CMBD). The interaction between Orai1 and CaM at the atomic level remains unknown. Here, we report the crystal structure of a CaM·Orai1-CMBD complex showing one CMBD bound to the C-terminal lobe of CaM, differing from other CaM-target protein complexes, in which both N- and C-terminal lobes of CaM (CaM-N and CaM-C) are involved in target binding. Orai1-CMBD binds CaM-C mainly through hydrophobic interactions, primarily involving residue Trp⁷⁶ of Orai1-CMBD, which interacts with the hydrophobic pocket of CaM-C. However, NMR data, isothermal titration calorimetry data, and pulldown assays indicated that CaM-N and CaM-C both can bind Orai1-CMBD, with CaM-N having ∼4 times weaker affinity than CaM-C. Pulldown assays of a Orai1-CMBD(W76E) mutant, gel filtration chromatography data, and NOE signals indicated that CaM-N and CaM-C can each bind one Orai1-CMBD. Thus our studies support an unusual, extended 1:2 binding mode of CaM to Orai1-CMBDs, and quantify the affinity of Orai1 for CaM. We propose a two-step mechanism for CaM-dependent Orai1 inactivation initiated by binding of the C-lobe of CaM to the CMBD of one Orai1 followed by the binding of the N-lobe of CaM to the CMBD of a neighboring Orai1.     

Characterization of a Hexameric Exo-Acting GH51 α-l-Arabinofuranosidase from the Mesophilic Bacillus subtilis

α-l-Arabinofuranosidases (α-l-Abfases, EC 3.2.1.55) display a broad specificity against distinct glycosyl moieties in branched hemicellulose and recent studies have demonstrated their synergistic use with cellulases and xylanases for biotechnological processes involving plant biomass degradation. In this study, we examined the structural organization of the arabinofuranosidase (GH51 family) from the mesophilic Bacillus subtilis (AbfA) and its implications on function and stability. The recombinant AbfA showed to be active over a broad temperature range with the maximum activity between 35 and 50 °C, which is desirable for industrial applications. Functional studies demonstrated that AbfA preferentially cleaves debranched or linear arabinan and is an exo-acting enzyme producing arabinose from arabinoheptaose. The enzyme has a canonical circular dichroism spectrum of α/β proteins and exhibits a hexameric quaternary structure in solution, as expected for GH51 members. Thermal denaturation experiments indicated a melting temperature of 53.5 °C, which is in agreement with the temperature–activity curves. The mechanisms associated with the unfolding process were investigated through molecular dynamics simulations evidencing an important contribution of the quaternary arrangement in the stabilization of the β-sandwich accessory domain and other regions involved in the formation of the catalytic interface of hexameric Abfases belonging to GH51 family.     

Modular Organization of α-Toxins from Scorpion Venom Mirrors Domain Structure of Their Targets,Sodium Channels

To gain success in the evolutionary “arms race,” venomous animals such as scorpions produce diverse neurotoxins selected to hit targets in the nervous system of prey. Scorpion α-toxins affect insect and/or mammalian voltage-gated sodium channels (Na_vs) and thereby modify the excitability of muscle and nerve cells. Although more than 100 α-toxins are known and a number of them have been studied into detail, the molecular mechanism of their interaction with Na_vs is still poorly understood. Here, we employ extensive molecular dynamics simulations and spatial mapping of hydrophobic/hydrophilic properties distributed over the molecular surface of α-toxins. It is revealed that despite the small size and relatively rigid structure, these toxins possess modular organization from structural, functional, and evolutionary perspectives. The more conserved and rigid “core module” is supplemented with the “specificity module” (SM) that is comparatively flexible and variable and determines the taxon (mammal versus insect) specificity of α-toxin activity. We further show that SMs in mammal toxins are more flexible and hydrophilic than in insect toxins. Concomitant sequence-based analysis of the extracellular loops of Na_vs suggests that α-toxins recognize the channels using both modules. We propose that the core module binds to the voltage-sensing domain IV, whereas the more versatile SM interacts with the pore domain in repeat I of Na_vs. These findings corroborate and expand the hypothesis on different functional epitopes of toxins that has been reported previously. In effect, we propose that the modular structure in toxins evolved to match the domain architecture of Na_vs.     

Carbohydrate and Protein-based Inhibitors of Porcine Pancreatic α-Amylase: Structure Analysis and Comparison of Their Binding Characteristics

The crystal structures of porcine pancreatic α-amylase isozyme II (PPA II) in its free form and complexed with the trestatin A derived pseudo-octasaccharide V-1532 have been determined using Patterson search techniques at resolutions of 2.3 and 2.2 Å, respectively. Seven rings of the competitive inhibitor V-1532 could be detected in the active site region as well as two maltose units in secondary binding sites on the surface.V-1532 occupies the five central sugar binding subsites similar to the PPA/acarbose structure. A sixth ring exists at the reducing end, connecting two symmetry related PPA molecules. The seventh moiety, a 6-hydroxymethylconduritol ring, is located at the non-reducing end. The electron density for this ring is relatively weak, indicating considerable disorder.This study shows that PPA is able to accommodate more than five rings in the active site region, but that additional rings would increase the binding affinity only slightly, which is in accordance with kinetic experiments.A comparison of the structures of free PPA, PPA/V-1532 and PPA/Tendamistat shows the characteristic conformational changes that accompany inhibitor binding and distinguish pseudo-oligosaccharide inhibitors from proteinaceous inhibitors. Although both classes of inhibitors block the sugar binding subsites in the active site region, the extreme specificity and binding affinity of the proteinaceous inhibitors is probably due to an intricate interaction pattern involving areas further away from the catalytic center.     

Elucidation of the Molecular Basis for Arabinoxylan-Debranching Activity of a Thermostable Family GH62 α-l-Arabinofuranosidase from Streptomyces thermoviolaceus

Xylan-debranching enzymes facilitate the complete hydrolysis of xylan and can be used to alter xylan chemistry. Here, the family GH62 α-l-arabinofuranosidase from Streptomyces thermoviolaceus (SthAbf62A) was shown to have a half-life of 60 min at 60°C and the ability to cleave α-1,3 l-arabinofuranose (l-Araf) from singly substituted xylopyranosyl (Xylp) backbone residues in wheat arabinoxylan; low levels of activity on arabinan as well as 4-nitrophenyl α-l-arabinofuranoside were also detected. After selective removal of α-1,3 l-Araf substituents from disubstituted Xylp residues present in wheat arabinoxylan, SthAbf62A could also cleave the remaining α-1,2 l-Araf substituents, confirming the ability of SthAbf62A to remove α-l-Araf residues that are (1→2) and (1→3) linked to monosubstituted β-d-Xylp sugars. Three-dimensional structures of SthAbf62A and its complex with xylotetraose and l-arabinose confirmed a five-bladed β-propeller fold and revealed a molecular Velcro in blade V between the β1 and β21 strands, a disulfide bond between Cys27 and Cys297, and a calcium ion coordinated in the central channel of the fold. The enzyme-arabinose complex structure further revealed a narrow and seemingly rigid l-arabinose binding pocket situated at the center of one side of the β propeller, which stabilized the arabinofuranosyl substituent through several hydrogen-bonding and hydrophobic interactions. The predicted catalytic amino acids were oriented toward this binding pocket, and the catalytic essentiality of Asp53 and Glu213 was confirmed by site-specific mutagenesis. Complex structures with xylotetraose revealed a shallow cleft for xylan backbone binding that is open at both ends and comprises multiple binding subsites above and flanking the l-arabinose binding pocket.     

Inhibition of αIIbβ3 Ligand Binding by an αIIb Peptide that Clasps the Hybrid Domain to the βI Domain of β3

Agonist-stimulated platelet activation triggers conformational changes of integrin αIIbβ3, allowing fibrinogen binding and platelet aggregation. We have previously shown that an octapeptide, ^p1YMESRADR⁸, corresponding to amino acids 313–320 of the β-ribbon extending from the β-propeller domain of αIIb, acts as a potent inhibitor of platelet aggregation. Here we have performed in silico modelling analysis of the interaction of this peptide with αIIbβ3 in its bent and closed (not swing-out) conformation and show that the peptide is able to act as a substitute for the β-ribbon by forming a clasp restraining the β3 hybrid and βI domains in a closed conformation. The involvement of species-specific residues of the β3 hybrid domain (E356 and K384) and the β1 domain (E297) as well as an intrapeptide bond (^pE315-^pR317) were confirmed as important for this interaction by mutagenesis studies of αIIbβ3 expressed in CHO cells and native or substituted peptide inhibitory studies on platelet functions. Furthermore, NMR data corroborate the above results. Our findings provide insight into the important functional role of the αIIb β-ribbon in preventing integrin αIIbβ3 head piece opening, and highlight a potential new therapeutic approach to prevent integrin ligand binding.     

The Interaction of Integrin αIIbβ3 with Fibrin Occurs through Multiple Binding Sites in the αIIb β-Propeller Domain

The currently available antithrombotic agents target the interaction of platelet integrin α_IIbβ₃ (GPIIb-IIIa) with fibrinogen during platelet aggregation. Platelets also bind fibrin formed early during thrombus growth. It was proposed that inhibition of platelet-fibrin interactions may be a necessary and important property of α_IIbβ₃ antagonists; however, the mechanisms by which α_IIbβ₃ binds fibrin are uncertain. We have previously identified the γ370–381 sequence (P3) in the γC domain of fibrinogen as the fibrin-specific binding site for α_IIbβ₃ involved in platelet adhesion and platelet-mediated fibrin clot retraction. In the present study, we have demonstrated that P3 can bind to several discontinuous segments within the α_IIb β-propeller domain of α_IIbβ₃ enriched with negatively charged and aromatic residues. By screening peptide libraries spanning the sequence of the α_IIb β-propeller, several sequences were identified as candidate contact sites for P3. Synthetic peptides duplicating these segments inhibited platelet adhesion and clot retraction but not platelet aggregation, supporting the role of these regions in fibrin recognition. Mutant α_IIbβ₃ receptors in which residues identified as critical for P3 binding were substituted for homologous residues in the I-less integrin α_Mβ₂ exhibited reduced cell adhesion and clot retraction. These residues are different from those that are involved in the coordination of the fibrinogen γ404–411 sequence and from auxiliary sites implicated in binding of soluble fibrinogen. These results map the binding of fibrin to multiple sites in the α_IIb β-propeller and further indicate that recognition specificity of α_IIbβ₃ for fibrin differs from that for soluble fibrinogen.     

Crystal Structure and Characterization of the Glycoside Hydrolase Family 62 α-l-Arabinofuranosidase from Streptomyces coelicolor

α-l-Arabinofuranosidase, which belongs to the glycoside hydrolase family 62 (GH62), hydrolyzes arabinoxylan but not arabinan or arabinogalactan. The crystal structures of several α-l-arabinofuranosidases have been determined, although the structures, catalytic mechanisms, and substrate specificities of GH62 enzymes remain unclear. To evaluate the substrate specificity of a GH62 enzyme, we determined the crystal structure of α-l-arabinofuranosidase, which comprises a carbohydrate-binding module family 13 domain at its N terminus and a catalytic domain at its C terminus, from Streptomyces coelicolor. The catalytic domain was a five-bladed β-propeller consisting of five radially oriented anti-parallel β-sheets. Sugar complex structures with l-arabinose, xylotriose, and xylohexaose revealed five subsites in the catalytic cleft and an l-arabinose-binding pocket at the bottom of the cleft. The entire structure of this GH62 family enzyme was very similar to that of glycoside hydrolase 43 family enzymes, and the catalytically important acidic residues found in family 43 enzymes were conserved in GH62. Mutagenesis studies revealed that Asp²⁰² and Glu³⁶¹ were catalytic residues, and Trp²⁷⁰, Tyr⁴⁶¹, and Asn⁴⁶² were involved in the substrate-binding site for discriminating the substrate structures. In particular, hydrogen bonding between Asn⁴⁶² and xylose at the nonreducing end subsite +2 was important for the higher activity of substituted arabinofuranosyl residues than that for terminal arabinofuranoses.     

An Activating Mutation Reveals a Second Binding Mode of the Integrin α2 I Domain to the GFOGER Motif in Collagens

The GFOGER motif in collagens (O denotes hydroxyproline) represents a high-affinity binding site for all collagen-binding integrins. Other GxOGER motifs require integrin activation for maximal binding. The E318W mutant of the integrin α2β1 I domain displays a relaxed collagen specificity, typical of an active state. E318W binds more strongly than the wild-type α2 I domain to GMOGER, and forms a 2:1 complex with a homotrimeric, collagen-like, GFOGER peptide. Crystal structure analysis of this complex reveals two E318W I domains, A and B, bound to a single triple helix. The E318W I domains are virtually identical to the collagen-bound wild-type I domain, suggesting that the E318W mutation activates the I domain by destabilising the unligated conformation. E318W I domain A interacts with two collagen chains similarly to wild-type I domain (high-affinity mode). E318W I domain B makes favourable interactions with only one collagen chain (low-affinity mode). This observation suggests that single GxOGER motifs in the heterotrimeric collagens V and IX may support binding of activated integrins.     

Flow Cytometry for the Analysis of α-Dystroglycan Glycosylation in Fibroblasts from Patients with Dystroglycanopathies

α-dystroglycan (α-DG) is a peripheral membrane protein that is an integral component of the dystrophin-glycoprotein complex. In an inherited subset of muscular dystrophies known as dystroglycanopathies, α-DG has reduced glycosylation which results in lower affinity binding to several extracellular matrix proteins including laminins. The glycosylation status of α-DG is normally assessed by the binding of the α-DG antibody IIH6 to a specific glycan epitope on α-DG involved in laminin binding. Immunocytochemistry and immunoblotting are two of the most widely used methods to detect the amount of α-DG glycosylation in muscle. While the interpretation of the presence or absence of the epitope on muscle using these techniques is straightforward, the assessment of a mild defect can be challenging. In this study, flow cytometry was used to compare the amount of IIH6-reactive glycans in fibroblasts from dystroglycanopathy patients with defects in genes known to cause α-DG hypoglycosylation to the amount in fibroblasts from healthy and pathological control subjects. A total of twenty one dystroglycanopathy patient fibroblasts were assessed, as well as fibroblasts from three healthy controls and seven pathological controls. Control fibroblasts have clearly detectable amounts of IIH6-reactive glycans, and there is a significant difference in the amount of this glycosylation, as measured by the mean fluorescence intensity of an antibody recognising the epitope and the percentage of cells positive for the epitope, between these controls and dystroglycanopathy patient fibroblasts (p<0.0001 for both). Our results indicate that the amount of α-DG glycosylation in patient fibroblasts is comparable to that in patient skeletal muscle. This method could complement existing immunohistochemical assays in skeletal muscle as it is quantitative and simple to perform, and could be used when a muscle biopsy is not available. This test could also be used to assess the pathogenicity of variants of unknown significance in genes involved in dystroglycanopathies.     

Characterization of the Catalytic and Nucleotide Binding Properties of the α-Kinase Domain of Dictyostelium Myosin-II Heavy Chain Kinase A

The α-kinases are a widely expressed family of serine/threonine protein kinases that exhibit no sequence identity with conventional eukaryotic protein kinases. In this report, we provide new information on the catalytic properties of the α-kinase domain of Dictyostelium myosin-II heavy chain kinase-A (termed A-CAT). Crystallization of A-CAT in the presence of MgATP yielded structures with AMP or adenosine in the catalytic cleft together with a phosphorylated Asp-766 residue. The results show that the β- and α-phosphoryl groups are transferred either directly or indirectly to the catalytically essential Asp-766. Biochemical assays confirmed that A-CAT hydrolyzed ATP, ADP, and AMP with k_cat values of 1.9, 0.6, and 0.32 min⁻¹, respectively, and showed that A-CAT can use ADP to phosphorylate peptides and proteins. Binding assays using fluorescent 2′/3′-O-(N-methylanthraniloyl) analogs of ATP and ADP yielded K_d values for ATP, ADP, AMP, and adenosine of 20 ± 3, 60 ± 20, 160 ± 60, and 45 ± 15 μm, respectively. Site-directed mutagenesis showed that Glu-713, Leu-716, and Lys-645, all of which interact with the adenine base, were critical for nucleotide binding. Mutation of the highly conserved Gln-758, which chelates a nucleotide-associated Mg²⁺ ion, eliminated catalytic activity, whereas loss of the highly conserved Lys-722 and Arg-592 decreased k_cat values for kinase and ATPase activities by 3–6-fold. Mutation of Asp-663 impaired kinase activity to a much greater extent than ATPase, indicating a specific role in peptide substrate binding, whereas mutation of Gln-768 doubled ATPase activity, suggesting that it may act to exclude water from the active site.     

Comparative Analysis of MPTP Neurotoxicity in Mice with a Constitutive Knockout of the α-Synuclein Gene

Molecular Biology - Aggregated forms of α-synuclein are core components of pathohistological inclusions known as Lewy bodies in substantia nigra (SN) neurons of patients with Parkinson’s...     

Degradation of Granular Starch by the Bacterium Microbacterium aurum Strain B8.A Involves a Modular α-Amylase Enzyme System with FNIII and CBM25 Domains

The bacterium Microbacterium aurum strain B8.A, originally isolated from a potato plant wastewater facility, is able to degrade different types of starch granules. Here we report the characterization of an unusually large, multidomain M. aurum B8.A α-amylase enzyme (MaAmyA). MaAmyA is a 1,417-amino-acid (aa) protein with a predicted molecular mass of 148 kDa. Sequence analysis of MaAmyA showed that its catalytic core is a family GH13_32 α-amylase with the typical ABC domain structure, followed by a fibronectin (FNIII) domain, two carbohydrate binding modules (CBM25), and another three FNIII domains. Recombinant expression and purification yielded an enzyme with the ability to degrade wheat and potato starch granules by introducing pores. Characterization of various truncated mutants of MaAmyA revealed a direct relationship between the presence of CBM25 domains and the ability of MaAmyA to form pores in starch granules, while the FNIII domains most likely function as stable linkers. At the C terminus, MaAmyA carries a 300-aa domain which is uniquely associated with large multidomain amylases; its function remains to be elucidated. We concluded that M. aurum B8.A employs a multidomain enzyme system to initiate degradation of starch granules via pore formation.     

Orthosteric Binding of ρ-Da1a,a Natural Peptide of Snake Venom Interacting Selectively with the α1A-Adrenoceptor

ρ-Da1a is a three-finger fold toxin from green mamba venom that is highly selective for the α_1A-adrenoceptor. This toxin has atypical pharmacological properties, including incomplete inhibition of ³H-prazosin or ¹²⁵I-HEAT binding and insurmountable antagonist action. We aimed to clarify its mode of action at the α_1A-adrenoceptor. The affinity (pKi 9.26) and selectivity of ρ-Da1a for the α_1A-adrenoceptor were confirmed by comparing binding to human adrenoceptors expressed in eukaryotic cells. Equilibrium and kinetic binding experiments were used to demonstrate that ρ-Da1a, prazosin and HEAT compete at the α_1A-adrenoceptor. ρ-Da1a did not affect the dissociation kinetics of ³H-prazosin or ¹²⁵I-HEAT, and the IC₅₀ of ρ-Da1a, determined by competition experiments, increased linearly with the concentration of radioligands used, while the residual binding by ρ-Da1a remained stable. The effect of ρ-Da1a on agonist-stimulated Ca²⁺ release was insurmountable in the presence of phenethylamine- or imidazoline-type agonists. Ten mutations in the orthosteric binding pocket of the α_1A-adrenoceptor were evaluated for alterations in ρ-Da1a affinity. The D106^3.32A and the S188^5.42A/S192^5.46A receptor mutations reduced toxin affinity moderately (6 and 7.6 times, respectively), while the F86^2.64A, F288^6.51A and F312^7.39A mutations diminished it dramatically by 18- to 93-fold. In addition, residue F86^2.64 was identified as a key interaction point for ¹²⁵I-HEAT, as the variant F86^2.64A induced a 23-fold reduction in HEAT affinity. Unlike the M1 muscarinic acetylcholine receptor toxin MT7, ρ-Da1a interacts with the human α_1A-adrenoceptor orthosteric pocket and shares receptor interaction points with antagonist (F86^2.64, F288^6.51 and F312^7.39) and agonist (F288^6.51 and F312^7.39) ligands. Its selectivity for the α_1A-adrenoceptor may result, at least partly, from its interaction with the residue F86^2.64, which appears to be important also for HEAT binding.     

Danio rerio αE-catenin Is a Monomeric F-actin Binding Protein with Distinct Properties from Mus musculus αE-catenin

It is unknown whether homologs of the cadherin·catenin complex have conserved structures and functions across the Metazoa. Mammalian αE-catenin is an allosterically regulated actin-binding protein that binds the cadherin·β-catenin complex as a monomer and whose dimerization potentiates F-actin association. We tested whether these functional properties are conserved in another vertebrate, the zebrafish Danio rerio. Here we show, despite 90% sequence identity, that Danio rerio and Mus musculus αE-catenin have striking functional differences. We demonstrate that D. rerio αE-catenin is monomeric by size exclusion chromatography, native PAGE, and small angle x-ray scattering. D. rerio αE-catenin binds F-actin in cosedimentation assays as a monomer and as an α/β-catenin heterodimer complex. D. rerio αE-catenin also bundles F-actin, as shown by negative stained transmission electron microscopy, and does not inhibit Arp2/3 complex-mediated actin nucleation in bulk polymerization assays. Thus, core properties of α-catenin function, F-actin and β-catenin binding, are conserved between mouse and zebrafish. We speculate that unique regulatory properties have evolved to match specific developmental requirements.     

NMR Structures of a Nonapeptide from DNA Binding Domain of Human Polymerase-α Determined by Iterative Complete-Relaxation-Matrix Approach

Abstract

Nuclear magnetic resonance structures of a nonapeptide, ERFKCPCPT, selected from the DNA binding domain of human polymerase-a, were determined by complete relaxation matrix analysis of transverse NOE data. The structures exhibit a type III turn with residues KCPC, and the remaining residues exhibit non-ordered structures. The turn was confirmed by α, N (i,i+3) connectivity, a low temperature coefficient of NH chemical shift (?3.1 × 1O^?3) of the fourth residue, ³J_NHα coupling constants, and characteristic CD peaks at 228 and 200 nm. Furthermore, ø and ψ dihedral angles for the i + 1, and i + 2 residues of the tum are found to be?80 and?41 and?60 and?40 degrees. The first proline residue is trans- while the second exists in both eis- and trans- configurations, with trans- being more than 80% populated. The trans-configuration was established from C5α-P6α correlation and ø and ψ angles of the proline. The five-membered proline ring is in DOWN puckered (C-β-exo/C-γ-endo) conformation. The structure of the peptide reveals that the two cysteine thiols are?5 A° apart and appropriately positioned to covalently bind cis-diamminedichloroplatinum(II), a widely used anti-cancer drug.     

Analysis of the Cytoprotective Role of α-Crystallins in Cell Survival and Implication of the αA-Crystallin C-Terminal Extension Domain in Preventing Bax-Induced Apoptosis

α-Crystallins, initially described as the major structural proteins of the lens, belong to the small heat shock protein family. Apart from their function as chaperones, α-crystallins are involved in the regulation of intracellular apoptotic signals. αA- and αB-crystallins have been shown to interfere with the mitochondrial apoptotic pathway triggering Bax pro-apoptotic activity and downstream activation of effector caspases. Differential regulation of α-crystallins has been observed in several eye diseases such as age-related macular degeneration and stress-induced and inherited retinal degenerations. Although the function of α-crystallins in healthy and diseased retina remains poorly understood, their altered expression in pathological conditions argue in favor of a role in cellular defensive response. In the Rpe65^−/− mouse model of Leber''s congenital amaurosis, we previously observed decreased expression of αA- and αB-crystallins during disease progression, which was correlated with Bax pro-death activity and photoreceptor apoptosis. In the present study, we demonstrated that α-crystallins interacted with pro-apoptotic Bax and displayed cytoprotective action against Bax-triggered apoptosis, as assessed by TUNEL and caspase assays. We further observed in staurosporine-treated photoreceptor-like 661W cells stably overexpressing αA- or αB-crystallin that Bax-dependent apoptosis and caspase activation were inhibited. Finally, we reported that the C-terminal extension domain of αA-crystallin was sufficient to provide protection against Bax-triggered apoptosis. Altogether, these data suggest that α-crystallins interfere with Bax-induced apoptosis in several cell types, including the cone-derived 661W cells. They further suggest that αA-crystallin-derived peptides might be sufficient to promote cytoprotective action in response to apoptotic cell death.     

The Structure of Integrin α1I Domain in Complex with a Collagen-mimetic Peptide

We have determined the structure of the human integrin α1I domain bound to a triple-helical collagen peptide. The structure of the α1I-peptide complex was investigated using data from NMR, small angle x-ray scattering, and size exclusion chromatography that were used to generate and validate a model of the complex using the data-driven docking program, HADDOCK (High Ambiguity Driven Biomolecular Docking). The structure revealed that the α1I domain undergoes a major conformational change upon binding of the collagen peptide. This involves a large movement in the C-terminal helix of the αI domain that has been suggested to be the mechanism by which signals are propagated in the intact integrin receptor. The structure suggests a basis for the different binding selectivity observed for the α1I and α2I domains. Mutational data identify residues that contribute to the conformational change observed. Furthermore, small angle x-ray scattering data suggest that at low collagen peptide concentrations the complex exists in equilibrium between a 1:1 and 2:1 α1I-peptide complex.