Dissociation of Bimolecular ��IIb��3-Fibrinogen Complex under a Constant Tensile Force

The regulated ability of integrin αIIbβ3 to bind fibrinogen plays a crucial role in platelet aggregation, adhesion, and hemostasis. Employing an optical-trap-based electronic force clamp, we studied the thermodynamics and kinetics of αIIbβ3-fibrinogen bond formation and dissociation under constant unbinding forces, mimicking the forces of physiologic blood shear on a thrombus. The distribution of bond lifetimes was bimodal, indicating that the αIIbβ3-fibrinogen complex exists in two bound states with different mechanical stability. The αIIbβ3 antagonist, abciximab, inhibited binding without affecting the unbinding kinetics, whereas Mn²⁺ biased the αIIbβ3-fibrinogen complex to the strong bound state with reduced off-rate. The average bond lifetimes decreased exponentially with increasing pulling force from ∼5 pN to 50 pN, suggesting that in this force range the αIIbβ3-fibrinogen interactions are classical slip bonds. We found no evidence for catch bonds, which is consistent with the known lack of shear-enhanced platelet adhesion on fibrinogen-coated surfaces. Taken together, these data provide important quantitative and qualitative characteristics of αIIbβ3-fibrinogen binding and unbinding that underlie the dynamics of platelet adhesion and aggregation in blood flow.     

The Clamp Loader Assembles the �� Clamp onto Either a 3�� or 5�� Primer Terminus: THE UNDERLYING BASIS FAVORING 3�� LOADING*

Clamp loaders assemble sliding clamps onto 3′ primed sites for DNA polymerases. Clamp loaders are thought to be specific for a 3′ primed site, and unable to bind a 5′ site. We demonstrate here that the Escherichia coli γ complex clamp loader can load the β clamp onto a 5′ primed site, although with at least 20-fold reduced efficiency relative to loading at a 3′ primed site. Preferential clamp loading at a 3′ site does not appear to be due to DNA binding, as the clamp loader forms an avid complex with β at a 5′ site. Preferential loading at a 3′ versus a 5′ site occurs at the ATP hydrolysis step, needed to close the ring around DNA. We also address DNA structural features that are recognized for preferential loading at a 3′ site. Although the single-stranded template strand extends in opposite directions from 3′ and 5′ primed sites, thus making it a favorite candidate for distinguishing between 3′ and 5′ sites, the single-strand polarity at a primed template junction does not determine 3′ site selection for clamp loading. Instead, we find that clamp loader recognition of a 3′ site lies in the duplex portion of the primed site, not the single-strand portion. We present evidence that the β clamp facilitates its own loading specificity for a 3′ primed site. Implications to eukaryotic clamp loader complexes are proposed.     

Drosophila RecQ4 Has a 3��-5�� DNA Helicase Activity That Is Essential for Viability

Members of the RecQ family of proteins are highly conserved DNA helicases that have important functions in the maintenance of genomic stability. Deficiencies in RecQ4 have been linked to human diseases including Rothmund-Thomson, RAPADILINO, and Baller-Gerold syndromes, all of which are characterized by developmental defects, tumor propensity, and genetic instability. However, there are conflicting results shown in the literature regarding the DNA helicase activity of RecQ4. We report here the expression of Drosophila melanogaster RecQ4 with a baculoviral vector and its purification to near homogeneity. The purified protein has a DNA-dependent ATPase activity and is a 3′-5′ DNA helicase dependent on hydrolysis of ATP. The presence of 5′-adenylyl-β,γ-imidodiphosphate (AMPPNP), a nonhydrolyzable ATP analog, promotes stable complex formation between RecQ4 and single-stranded DNA. Drosophila RecQ4 can also anneal complementary single strands; this activity was reduced in the presence of AMPPNP, possibly because of the stable protein-DNA complex formed under such conditions. A point mutation of the highly conserved lysine residue in the helicase domain, although retaining the wild type level of annealing activity, inactivated ATPase and helicase activities and eliminated stable complex formation. These results suggest that the helicase domain alone is responsible for the DNA unwinding action of the Drosophila enzyme. We generated a null recq4 mutant that is homozygous lethal, which we used to test the genetic function of the helicase-dead mutant in flies. Complementation tests showed that the helicase-dead mutant recq4 transgenes are incapable of rescuing the null mutation, demonstrating that the helicase activity has an essential biological function.     

DNA stabilization at the Bacillus subtilis PolX core��a binding model to coordinate polymerase, AP-endonuclease and 3��-5�� exonuclease activities

Family X DNA polymerases (PolXs) are involved in DNA repair. Their binding to gapped DNAs relies on two conserved helix-hairpin-helix motifs, one located at the 8-kDa domain and the other at the fingers subdomain. Bacterial/archaeal PolXs have a specifically conserved third helix-hairpin-helix motif (GFGxK) at the fingers subdomain whose putative role in DNA binding had not been established. Here, mutagenesis at the corresponding residues of Bacillus subtilis PolX (PolXBs), Gly130, Gly132 and Lys134 produced enzymes with altered DNA binding properties affecting the three enzymatic activities of the protein: polymerization, located at the PolX core, 3′-5′ exonucleolysis and apurinic/apyrimidinic (AP)-endonucleolysis, placed at the so-called polymerase and histidinol phosphatase domain. Furthermore, we have changed Lys192 of PolXBs, a residue moderately conserved in the palm subdomain of bacterial PolXs and immediately preceding two catalytic aspartates of the polymerization reaction. The results point to a function of residue Lys192 in guaranteeing the right orientation of the DNA substrates at the polymerization and histidinol phosphatase active sites. The results presented here and the recently solved structures of other bacterial PolX ternary complexes lead us to propose a structural model to account for the appropriate coordination of the different catalytic activities of bacterial PolXs.     

Reconstitution of a Frizzled8��Wnt3a��LRP6 Signaling Complex Reveals Multiple Wnt and Dkk1 Binding Sites on LRP6

Wnt/β-catenin signaling is initiated at the cell surface by association of secreted Wnt with its receptors Frizzled (Fz) and low density lipoprotein receptor-related protein 5/6 (LRP5/6). The study of these molecular interactions has been a significant technical challenge because the proteins have been inaccessible in sufficient purity and quantity. In this report we describe insect cell expression and purification of soluble mouse Fz8 cysteine-rich domain and human LRP6 extracellular domain and show that they inhibit Wnt/β-catenin signaling in cellular assays. We determine the binding affinities of Wnts and Dickkopf 1 (Dkk1) to the relevant co-receptors and reconstitute in vitro the Fz8 CRD·Wnt3a·LRP6 signaling complex. Using purified fragments of LRP6, we further show that Wnt3a binds to a region including only the third and fourth β-propeller domains of LRP6 (E3E4). Surprisingly, we find that Wnt9b binds to a different part of the LRP6 extracellular domain, E1E2, and we demonstrate that Wnt3a and Wnt9b can bind to LRP6 simultaneously. Dkk1 binds to both E1E2 and E3E4 fragments and competes with both Wnt3a and Wnt9b for binding to LRP6. The existence of multiple, independent Wnt binding sites on the LRP6 co-receptor suggests new possibilities for the architecture of Wnt signaling complexes and a model for broad-spectrum inhibition of Wnt/β-catenin signaling by Dkk1.     

Phosphoinositide 3-Kinase p110�� Regulates Integrin ��IIb��3 Avidity and the Cellular Transmission of Contractile Forces

Phosphoinositide (PI) 3-kinase (PI3K) signaling processes play an important role in regulating the adhesive function of integrin α_IIbβ₃, necessary for platelet spreading and sustained platelet aggregation. PI3K inhibitors are effective at reducing platelet aggregation and thrombus formation in vivo and as a consequence are currently being evaluated as novel antithrombotic agents. PI3K regulation of integrin α_IIbβ₃ activation (affinity modulation) primarily occurs downstream of G_i-coupled and tyrosine kinase-linked receptors linked to the activation of Rap1b, AKT, and phospholipase C. In the present study, we demonstrate an important role for PI3Ks in regulating the avidity (strength of adhesion) of high affinity integrin α_IIbβ₃ bonds, necessary for the cellular transmission of contractile forces. Using knock-out mouse models and isoform-selective PI3K inhibitors, we demonstrate that the Type Ia p110β isoform plays a major role in regulating thrombin-stimulated fibrin clot retraction in vitro. Reduced clot retraction induced by PI3K inhibitors was not associated with defects in integrin α_IIbβ₃ activation, actin polymerization, or actomyosin contractility but was associated with a defect in integrin α_IIbβ₃ association with the contractile cytoskeleton. Analysis of integrin α_IIbβ₃ adhesion contacts using total internal reflection fluorescence microscopy revealed an important role for PI3Ks in regulating the stability of high affinity integrin α_IIbβ₃ bonds. These studies demonstrate an important role for PI3K p110β in regulating the avidity of high affinity integrin α_IIbβ₃ receptors, necessary for the cellular transmission of contractile forces. These findings may provide new insight into the potential antithrombotic properties of PI3K p110β inhibitors.     

The Novel S527F Mutation in the Integrin ��3 Chain Induces a High Affinity ��IIb��3 Receptor by Hindering Adoption of the Bent Conformation

Three heterozygous mutations were identified in the genes encoding platelet integrin receptor αIIbβ3 in a patient with an ill defined platelet disorder: one in the β3 gene (S527F) and two in the αIIb gene (R512W and L841M). Five stable Chinese hamster ovary cell lines were constructed expressing recombinant αIIbβ3 receptors bearing the individual R512W, L841M, or S527F mutation; both the R512W and L841M mutations; or all three mutations. All receptors were expressed on the cell surface, and mutations R512W and L841M had no effect on integrin function. Interestingly, the β3 S527F mutation produced a constitutively active receptor. Indeed, both fibrinogen and the ligand-mimetic antibody PAC-1 bound to non-activated αIIbβ3 receptors carrying the S527F mutation, indicating that the conformation of this receptor was altered and corresponded to the high affinity ligand binding state. In addition, the conformational change induced by S527F was evident from basal anti-ligand-induced binding site antibody binding to the receptor. A molecular model bearing this mutation was constructed based on the crystal structure of αIIbβ3 and revealed that the S527F mutation, situated in the third integrin epidermal growth factor-like (I-EGF3) domain, hindered the αIIbβ3 receptor from adopting a wild type-like bent conformation. Movement of I-EGF3 into a cleft in the bent conformation may be hampered both by steric hindrance between Phe⁵²⁷ in β3 and the calf-1 domain in αIIb and by decreased flexibility between I-EGF2 and I-EGF3.The platelet receptor αIIbβ3 belongs to the family of integrin receptors that consist of noncovalently linked α/β-heterodimers. They are cell-surface receptors that play a role in cell-cell and cell-matrix interactions. Under resting conditions, integrin receptors adopt the low affinity conformation and do not interact with their ligands. Inside-out signaling turns the receptor into a high affinity conformation capable of ligand binding. Ligand binding itself induces additional conformational changes resulting in exposure of neoantigenic sites called ligand-induced binding sites (LIBS)³ and generates in turn outside-in signaling, which triggers a range of downstream signals (1, 2).Integrin αIIbβ3 is expressed on platelets and megakaryocytes. In flowing blood under resting conditions, αIIbβ3 does not interact with its ligand fibrinogen. When a blood vessel is damaged, platelets adhere at sites of vascular injury and become activated. As a consequence, αIIbβ3 adopts the high affinity conformation and binds fibrinogen. This results in platelet aggregation and thrombus formation, which eventually will stop the bleeding (3).The topology of integrins comprises an extracellular, globular, N-terminal ligand-binding head domain (the β-propeller domain in the αIIb chain and the βI domain in the β3 chain) standing on two long legs or stalks (consisting of thigh, calf-1, and calf-2 domains in the αIIb chain and hybrid, plexin/semaphorin/integrin (PSI), four integrin endothelial growth factor-like (I-EGF), and β-tail domains in the β3 chain), followed by transmembrane and cytoplasmic domains (1, 2). X-ray crystal structures of the extracellular domain of non-activated αVβ3 revealed that the legs are severely bent, putting the head domain next to the membrane-proximal portions of the legs (4, 5). The bending occurs between I-EGF1 and I-EGF2 in the β-subunit and between the thigh and calf-1 domains in the α-subunit. This bent conformation represents the low affinity state of the receptor. The high affinity state of the receptor is induced by activation and is associated with a large-scale conformational rearrangement in which the integrin extends with a switchblade-like motion (2). Recently, the crystal structure of the entire extracellular domain of αIIbβ3 in its low affinity conformation was resolved and revealed that this integrin also adopts the bent conformation under resting conditions (6). Structural rearrangements in αIIbβ3 between the bent and extended conformations are similar to what has been reported for other integrins (7).We report here that the S527F mutation in the I-EGF3 region of the β3 polypeptide chain of the αIIbβ3 receptor induces a constitutively active receptor adopting an extended high affinity conformation. This was evidenced by spontaneous PAC-1, fibrinogen, and anti-LIBS antibody binding. These data were further corroborated by modeling the replacement of Ser⁵²⁷ with Phe in the crystal structure of the extracellular domain of αIIbβ3. In this model, the S527F mutation decreases the flexibility of I-EGF3 and appears to prevent movement of the lower β-leg into the cleft between the upper β-leg and the lower α-leg. As a consequence, formation of the bent conformation of the non-activated receptor is hampered.     

CP12 from Chlamydomonas reinhardtii, a Permanent Specific ��Chaperone-like�� Protein of Glyceraldehyde-3-phosphate Dehydrogenase

A new role is reported for CP12, a highly unfolded and flexible protein, mainly known for its redox function with A₄ glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Both reduced and oxidized CP12 can prevent the in vitro thermal inactivation and aggregation of GAPDH from Chlamydomonas reinhardtii. This mechanism is thus not redox-dependent. The protection is specific to CP12, because other proteins, such as bovine serum albumin, thioredoxin, and a general chaperone, Hsp33, do not fully prevent denaturation of GAPDH. Furthermore, CP12 acts as a specific chaperone, since it does not protect other proteins, such as catalase, alcohol dehydrogenase, or lysozyme. The interaction between CP12 and GAPDH is necessary to prevent the aggregation and inactivation, since the mutant C66S that does not form any complex with GAPDH cannot accomplish this protection. Unlike the C66S mutant, the C23S mutant that lacks the N-terminal bridge is partially able to protect and to slow down the inactivation and aggregation. Tryptic digestion coupled to mass spectrometry confirmed that the S-loop of GAPDH is the interaction site with CP12. Thus, CP12 not only has a redox function but also behaves as a specific “chaperone-like protein” for GAPDH, although a stable and not transitory interaction is observed. This new function of CP12 may explain why it is also present in complexes involving A₂B₂ GAPDHs that possess a regulatory C-terminal extension (GapB subunit) and therefore do not require CP12 to be redox-regulated.CP12 is a small 8.2-kDa protein present in the chloroplasts of most photosynthetic organisms, including cyanobacteria (1, 2), higher plants (3), the diatom Asterionella formosa (4, 5), and green (1) and red algae (6). It allows the formation of a supramolecular complex between phosphoribulokinase (EC 2.7.1.19) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH),³ two key enzymes of the Calvin cycle pathway, and was recently shown to interact with fructose bisphosphate aldolase, another enzyme of the Calvin cycle pathway (7). The phosphoribulokinase·GAPDH·CP12 complex has been extensively studied in Chlamydomonas reinhardtii (8, 9) and in Arabidopsis thaliana (10, 11). In the green alga C. reinhardtii, the interaction between CP12 and GAPDH is strong (8). GAPDH may exist as a homotetramer composed of four GapA subunits (A₄) in higher plants, cyanobacteria, and green and red algae (6, 12), but in higher plants, it can also exist as a heterotetramer (A₂B₂), composed of two subunits, GapA and GapB (13, 14). GapB, up to now, has exclusively been found in Streptophyta, but recently two prasinophycean green algae, Ostreococcus tauri and Ostreococcus lucimarinus, were also shown to possess a GapB gene, whereas CP12 is missing (15). The GapB subunit is similar to the GapA subunit but has a C-terminal extension containing two redox-regulated cysteine residues (16). Thus, although the A₄ GAPDHs lack these regulatory cysteine residues (13, 14, 17–20), they are also redox-regulated through its interaction with CP12, since the C terminus of this small protein resembles the C-terminal extension of the GapB subunit. The regulatory cysteine residues for GapA are thus supplied by CP12, as is well documented in the literature (1, 8, 11, 16).CP12 belongs to the family of intrinsically unstructured proteins (IUPs) (21–26). The amino acid composition of these proteins causes them to have no or few secondary structures. Their total or partial lack of structure and their high flexibility allow them to be molecular adaptors (27, 28). They are often able to bind to several partners and are involved in most cellular functions (29, 30). Recently, some IUPs have been described in photosynthetic organisms (31, 32).There are many functional categories of IUPs (22, 33). They can be, for instance, involved in permanent binding and have (i) a scavenger role, neutralizing or storing small ligands; (ii) an assembler role by forming complexes; and (iii) an effector role by modulating the activity of a partner molecule (33). These functions are not exclusive; thus, CP12 can form a stable complex with GAPDH, regulating its redox properties (8, 34, 35), and can also bind a metal ion (36, 37). IUPs can also bind transiently to partners, and some of them have been found to possess a chaperone activity (31, 38). This chaperone function was first shown for α-synuclein (39) and for α-casein (40), which are fully disordered. The amino acid composition of IUPs is less hydrophobic than those of soluble proteins; hence, they lack hydrophobic cores and do not become insoluble when heated. Since CP12 belongs to this family, we tested if it was resistant to heat treatment and finally, since it is tightly bound to GAPDH, if it could prevent aggregation of its partner, GAPDH, an enzyme well known for its tendency to aggregate (41–44) and consequently a substrate commonly used in chaperone studies (45, 46).Unlike chaperones, which form transient, dynamic complexes with their protein substrates through hydrophobic interactions (47, 48), CP12 forms a stable complex with GAPDH. The interaction involves the C-terminal part of the protein and the presence of negatively charged residues on CP12 (35). However, only a site-directed mutagenesis has been performed to characterize the interaction site on GAPDH. Although the mutation could have an indirect effect, the residue Arg-197 was shown to be a good candidate for the interaction site (49).In this report, we accordingly used proteolysis experiments coupled with mass spectrometry to detect which regions of GAPDH are protected by its association with CP12. To conclude, the aim of this report was to characterize a chaperone function of CP12 that had never been described before and to map the interaction site on GAPDH using an approach that does not involve site-directed mutagenesis.     

In Vitro Characterization of a Recombinant Blh Protein from an Uncultured Marine Bacterium as a ��-Carotene 15,15��-Dioxygenase

Codon optimization was used to synthesize the blh gene from the uncultured marine bacterium 66A03 for expression in Escherichia coli. The expressed enzyme cleaved β-carotene at its central double bond (15,15′) to yield two molecules of all-trans-retinal. The molecular mass of the native purified enzyme was ∼64 kDa as a dimer of 32-kDa subunits. The K_m, k_cat, and k_cat/K_m values for β-carotene as substrate were 37 μm, 3.6 min⁻¹, and 97 mm⁻¹ min⁻¹, respectively. The enzyme exhibited the highest activity for β-carotene, followed by β-cryptoxanthin, β-apo-4′-carotenal, α-carotene, and γ-carotene in decreasing order, but not for β-apo-8′-carotenal, β-apo-12′-carotenal, lutein, zeaxanthin, or lycopene, suggesting that the presence of one unsubstituted β-ionone ring in a substrate with a molecular weight greater than C₃₅ seems to be essential for enzyme activity. The oxygen atom of retinal originated not from water but from molecular oxygen, suggesting that the enzyme was a β-carotene 15,15′-dioxygenase. Although the Blh protein and β-carotene 15,15′-monooxygenases catalyzed the same biochemical reaction, the Blh protein was unrelated to the mammalian β-carotene 15,15′-monooxygenases as assessed by their different properties, including DNA and amino acid sequences, molecular weight, form of association, reaction mechanism, kinetic properties, and substrate specificity. This is the first report of in vitro characterization of a bacterial β-carotene-cleaving enzyme.Vitamin A (retinol) is a fat-soluble vitamin and important for human health. In vivo, the cleavage of β-carotene to retinal is an important step of vitamin A synthesis. The cleavage can proceed via two different biochemical pathways (1, 2). The major pathway is a central cleavage catalyzed by mammalian β-carotene 15,15′-monooxygenases (EC 1.14.99.36). β-Carotene is cleaved by the enzyme symmetrically into two molecules of all-trans-retinal, and retinal is then converted to vitamin A in vivo (3–5). The second pathway is an eccentric cleavage that occurs at double bonds other than the central 15,15′-double bond of β-carotene to produce β-apo-carotenals with different chain lengths, which are catalyzed by carotenoid oxygenases from mammals, plants, and cyanobacteria (6). These β-apo-carotenals are degraded to one molecule of retinal, which is subsequently converted to vitamin A in vivo (2).β-Carotene 15,15′-monooxygenase was first isolated as a cytosolic enzyme by identifying the product of β-carotene cleavage as retinal (7). The characterization of the enzyme and the reaction pathway from β-carotene to retinal were also investigated (4, 8). The enzyme activity has been found in mammalian intestinal mucosa, jejunum enterocytes, liver, lung, kidney, and brain (5, 9, 10). Molecular cloning, expression, and characterization of β-carotene 15,15′-monooxygenase have been reported from various species, including chickens (11), fruit flies (12), humans (13), mice (14), and zebra fishes (15).Other proteins thought to convert β-carotene to retinal include bacterioopsin-related protein (Brp) and bacteriorhodopsin-related protein-like homolog protein (Blh) (16). Brp protein is expressed from the bop gene cluster, which encodes the structural protein bacterioopsin, consisting of at least three genes as follows: bop (bacterioopsin), brp (bacteriorhodopsin-related protein), and bat (bacterioopsin activator) (17). brp genes were reported in Haloarcula marismortui (18), Halobacterium sp. NRC-1 (19), Halobacterium halobium (17), Haloquadratum walsbyi, and Salinibacter ruber (20). Blh protein is expressed from the proteorhodopsin gene cluster, which contains proteorhodopsin, crtE (geranylgeranyl-diphosphate synthase), crtI (phytoene dehydrogenase), crtB (phytoene synthase), crtY (lycopene cyclase), idi (isopentenyl diphosphate isomerase), and blh gene (21). Sources of blh genes were previously reported in Halobacterium sp. NRC-1 (19), Haloarcula marismortui (18), Halobacterium salinarum (22), uncultured marine bacterium 66A03 (16), and uncultured marine bacterium HF10 49E08 (21). β-Carotene biosynthetic genes crtE, crtB, crtI, crtY, ispA, and idi encode the enzymes necessary for the synthesis of β-carotene from isopentenyl diphosphate, and the Idi, IspA, CrtE, CrtB, CrtI, and CrtY proteins have been characterized in vitro (23–28). Blh protein has been proposed to catalyze or regulate the conversion of β-carotene to retinal (29, 30), but there is no direct proof of the enzymatic activity.In this study, we used codon optimization to synthesize the blh gene from the uncultured marine bacterium 66A03 for expression in Escherichia coli, and we performed a detailed biochemical and enzymological characterization of the expressed Blh protein. In addition, the properties of the enzyme were compared with those of mammalian β-carotene 15,15′-monooxygenases.