Selective regulation of IP3-receptor-mediated Ca2+ signaling and apoptosis by the BH4 domain of Bcl-2 versus Bcl-Xl

Antiapoptotic B-cell lymphoma 2 (Bcl-2) targets the inositol 1,4,5-trisphosphate receptor (IP(3)R) via its BH4 domain, thereby suppressing IP(3)R Ca(2+)-flux properties and protecting against Ca(2+)-dependent apoptosis. Here, we directly compared IP(3)R inhibition by BH4-Bcl-2 and BH4-Bcl-Xl. In contrast to BH4-Bcl-2, BH4-Bcl-Xl neither bound the modulatory domain of IP(3)R nor inhibited IP(3)-induced Ca(2+) release (IICR) in permeabilized and intact cells. We identified a critical residue in BH4-Bcl-2 (Lys17) not conserved in BH4-Bcl-Xl (Asp11). Changing Lys17 into Asp in BH4-Bcl-2 completely abolished its IP(3)R-binding and -inhibitory properties, whereas changing Asp11 into Lys in BH4-Bcl-Xl induced IP(3)R binding and inhibition. This difference in IP(3)R regulation between BH4-Bcl-2 and BH4-Bcl-Xl controls their antiapoptotic action. Although both BH4-Bcl-2 and BH4-Bcl-Xl had antiapoptotic activity, BH4-Bcl-2 was more potent than BH4-Bcl-Xl. The effect of BH4-Bcl-2, but not of BH4-Bcl-Xl, depended on its binding to IP(3)Rs. In agreement with the IP(3)R-binding properties, the antiapoptotic activity of BH4-Bcl-2 and BH4-Bcl-Xl was modulated by the Lys/Asp substitutions. Changing Lys17 into Asp in full-length Bcl-2 significantly decreased its binding to the IP(3)R, its ability to inhibit IICR and its protection against apoptotic stimuli. A single amino-acid difference between BH4-Bcl-2 and BH4-Bcl-Xl therefore underlies differential regulation of IP(3)Rs and Ca(2+)-driven apoptosis by these functional domains. Mutating this residue affects the function of Bcl-2 in Ca(2+) signaling and apoptosis.     

Recombinant human uteroglobin/CC10 inhibits the adhesion and migration of primary human endothelial cells via specific and saturable binding to fibronectin

Uteroglobin (UG) or Clara Cell 10 kDa protein (CC10) is a small, stable, epithelial secretory anti-inflammatory protein. Uteroglobin has been shown to inhibit neointimal formation in vivo after balloon angioplasty through an unknown mechanism. An interaction between UG and plasma fibronectin (Fn) has been demonstrated in mice. Since Fn plays a key role in endothelial cell (EC) migration and angiogenesis, we investigated whether recombinant human UG (rhUG) affects EC migration via Fn binding. In this report, we show a saturable binding of rhUG to Fn depending on Fn conformation and that rhUG is covalently cross-linked to Fn by transglutaminase (TGase). Additionally, our study highlights that rhUG can also bind to exogenously added or self-secreted Fn on the membrane of human primary microvascular endothelial cells (HMVEC), although these complexes are weakly associated with the plasmalemma. Upon the interaction with Fn in solid phase, rhUG strongly inhibits HMVEC attachment on Fn, but not on other ECM proteins. Consequently, rhUG also inhibits cell migration in a dose dependent fashion (I.C.50 = 65 nM) and hinders the "wound healing" in vitro. The small size, stability and human tolerability of rhUG suggest that rhUG in slow-release form or genetically delivered could be used in humans to modulate cell/Fn interactions in the context of tumor microenvironment or in the context of inflammation and fibrosis.     

Activation of Ca2+-independent membrane-damaging activity in Lys49-phospholipase A2 promoted by amphiphilic molecules

Association of class-II phospholipase A(2) (PLA(2)) with aggregated phospholipid substrate results in elevated levels of the Ca(2+)-dependent hydrolytic activity. The Asp49 residue participates in coordination of the Ca(2+) ion cofactor, however, in Lys49-PLA(2) homologues (Lys49-PLA(2)s), substitution of the Asp49 by Lys results in loss of Ca(2+) binding and lack of detectable phospholipid hydrolysis. Nevertheless, Lys49-PLA(2)s cause Ca(2+)-independent damage of liposome membranes. Bothropstoxin-I is a homodimeric Lys49-PLA(2) from the venom of Bothrops jararacussu, and in fluorescent marker release and dynamic light scattering experiments with DPPC liposomes we demonstrate activation of the Ca(2+)-independent membrane damaging activity by approximately 4 molecules of sodium dodecyl sulphate (SDS) per protein monomer. Activation is accompanied by significant changes in the intrinsic tryptophan fluorescence emission (ITFE) and near UV circular dichroism (UVCD) spectra of the protein. Subsequent binding of 7-10 SDS molecules results in further alterations in the ITFE and far UVCD spectra. Reduction in the rate of N-bromosuccinimide modification of Trp77 at the dimer interface suggests that initial binding of SDS to this region accompanies the activation of the membrane damaging activity. 1-anilinonaphthalene-8-sulphonic acid binding studies indicate that subsequent SDS binding to the active site is concomitant with the second structural transition. These results provide insights in the structural basis of amphiphile/protein coupling in class-II PLA(2)s.     

A pH-induced dissociation of the dimeric form of a lysine 49-phospholipase A2 abolishes Ca2+-independent membrane damaging activity

The hydrolysis of phospholipids by class II phospholipase A2 (PLA2) involves a Ca2+ ion cofactor bound to the Asp49 residue in the active site region. In the lysine 49 phospholipase A2 homologues (Lys49-PLA2), the Asp49 residue is substituted by Lys, and consequently the Lys49-PLA2s show no Ca2+ binding and no detectable phospholipid hydrolysis. Nevertheless, the Lys49-PLA2s demonstrate membrane damaging activity by an incompletely understood Ca2+-independent mechanism of action. Using a combination of steady-state and time-resolved fluorescence techniques, we have examined the effect of pH on the monomer-dimer equilibrium of bothropstoxin I (BthTX-I), a Lys49-PLA2 from the venom of Bothrops jararacussu which contains a single Trp77 residue located at the dimer interface. At pH 5.0, we observe a decreased quantum yield, a decreased rotational correlation time, and an increased bimolecular quenching rate constant with iodide. These results are consistent with a pH-induced dissociation of the BthTX-I dimer, with the consequent exposure of the Trp77 residue to aqueous solvent. In the presence of liposomes, membrane damaging activity is observed only under conditions in which the dimeric form of the BthTX-I is favored. These results demonstrate that the dimeric form of the protein is essential for the initiation of the Ca2+-independent membrane damaging activity.     

Roles of conserved P domain residues and Mg2+ in ATP binding in the ground and Ca2+-activated states of sarcoplasmic reticulum Ca2+-ATPase

Residues in conserved motifs (625)TGD, (676)FARXXPXXK, and (701)TGDGVND in domain P of sarcoplasmic reticulum Ca(2+)-ATPase, as well as in motifs (601)DPPR and (359)NQR(/K)MSV in the hinge segments connecting domains N and P, were examined by mutagenesis to assess their roles in nucleotide and Mg(2+) binding and stabilization of the Ca(2+)-activated transition state for phosphoryl transfer. In the absence of Mg(2+), mutations removing the charges of domain P residues Asp(627), Lys(684), Asp(703), and Asp(707) increased the affinity for ATP and 2',3'-O-(2,4,6-trinitrophenyl)-8-azidoadenosine 5'-triphosphate. These mutations, as well as Gly(626)--> Ala, were inhibitory for ATP binding in the presence of Mg(2+) and for tight binding of the beta,gamma-bidentate chromium(III) complex of ATP. The hinge mutations had pronounced, but variable, effects on ATP binding only in the presence of Mg(2+). The data demonstrate an unfavorable electrostatic environment for binding of negatively charged nucleotide in domain P and show that Mg(2+) is required to anchor the phosphoryl group of ATP at the phosphorylation site. Mutants Gly(626) --> Ala, Lys(684) --> Met, Asp(703) --> Ala/Ser/Cys, and mutants with alteration to Asp(707) exhibited very slow or negligible phosphorylation, making it possible to measure ATP binding in the pseudo-transition state attained in the presence of both Mg(2+) and Ca(2+). Under these conditions, ATP binding was almost completely blocked in Gly(626) --> Ala and occurred with 12- and 7-fold reduced affinities in Asp(703) --> Ala and Asp(707) --> Cys, respectively, relative to the situation in the presence of Mg(2+) without Ca(2+), whereas in Lys(684) --> Met and Asp(707) --> Ser/Asn the affinity was enhanced 14- and 3-5-fold, respectively. Hence, Gly(626) and Asp(703) seem particularly critical for mediating entry into the transition state for phosphoryl transfer upon Ca(2+) binding at the transport sites.     

Functional consequences of alterations to amino acids located in the catalytic center (isoleucine 348 to threonine 357) and nucleotide-binding domain of the Ca2+-ATPase of sarcoplasmic reticulum

The sequence of 10 amino acids (ICSDKTGTLT357) at the site of phosphorylation of the rabbit fast twitch muscle Ca2+-ATPase is highly conserved in the family of cation-transporting ATPases. We changed each of the residues flanking Asp351, Lys352, and Thr353 to an amino acid differing in size or polarity and assayed the mutant for Ca2+ transport activity and autophosphorylation with ATP or P1. We found that conservative changes (Ile----Leu, Thr----Ser, Gly----Ala) or the alteration of Cys349 to alanine did not destroy Ca2+ transport activity or phosphoenzyme formation, whereas nonconservative changes (Ile----Thr, Leu----Ser) did disrupt function. These results indicate that very conservative changes in the amino acids flanking Asp351, Lys352, and Thr353 can be accommodated. A number of mutations were also introduced into amino acids predicted to be involved in nucleotide binding, in particular those in the conserved sequences KGAPE519, RDAGIRVIMITGDNK629, and KK713. Our results indicate that amino acids KGAPE519, Arg615, Gly618, Arg620, and Lys712-Lys713 are not essential for nucleotide binding, although changes to Lys515 diminished Ca2+ transport activity but not phosphoenzyme formation. Changes of Gly626 and Asp627 abolished phosphoenzyme formation with both ATP and Pi, indicating that these residues may contribute to the conformation of the catalytic center.     

Secreted phospholipase A2 inhibitors are also potent blockers of binding to the M-type receptor

Mammalian secreted phospholipases A(2) (sPLA(2)s) constitute a family of structurally related enzymes that are likely to play numerous biological roles because of their phospholipid hydrolyzing activity and binding to soluble and membrane-bound proteins, including the M-type receptor. Over the past decade, a number of competitive inhibitors have been developed against the inflammatory-type human group IIA (hGIIA) sPLA(2) with the aim of specifically blocking its catalytic activity and pathophysiological functions. The fact that many of these inhibitors, including the indole analogue Me-Indoxam, inhibit several other sPLA(2)s that bind to the M-type receptor prompted us to investigate the impact of Me-Indoxam and other inhibitors on the sPLA(2)-receptor interaction. By using a Ca(2+) loop mutant derived from a venom sPLA(2) which is insensitive to hGIIA inhibitors but still binds to the M-type receptor, we demonstrate that Me-Indoxam dramatically decreases the affinity of various sPLA(2)s for the receptor, yet an sPLA(2)-Me-Indoxam-receptor complex can form at very high sPLA(2) concentrations. Me-Indoxam inhibits the binding of iodinated mouse sPLA(2)s to the mouse M-type receptor expressed on live cells but also enhances binding of sPLA(2) to phospholipids. Because Me-Indoxam and other competitive inhibitors protrude out of the sPLA(2) catalytic groove, it is likely that the inhibitors interfere with the sPLA(2)-receptor interaction by steric hindrance and to different extents that depend on the type of sPLA(2) and inhibitor. Our finding suggests that the various anti-inflammatory therapeutic effects of sPLA(2) inhibitors may be due not only to inhibition of enzymatic activity but also to modulation of binding of sPLA(2) to the M-type receptor or other as yet unknown protein targets.     

Mechanism of the Ca2+-induced enhancement of the intrinsic factor VIIa activity

The intrinsic activity of coagulation factor VIIa (FVIIa) is dependent on Ca(2+) binding to a loop (residues 210-220) in the protease domain. Structural analysis revealed that Ca(2+) may enhance the activity by attenuating electrostatic repulsion of Glu(296) and/or by facilitating interactions between the loop and Lys(161) in the N-terminal tail. In support of the first mechanism, the mutations E296V and D212N resulted in similar, about 2-fold, enhancements of the amidolytic activity. Moreover, mutation of the Lys(161)-interactive residue Asp(217) or Asp(219) to Ala reduced the amidolytic activity by 40-50%, whereas the K161A mutation resulted in 80% reduction. Hence one of these Asp residues in the Ca(2+)-binding loop appears to suffice for some residual interaction with Lys(161), whereas the more severe effect upon replacement of Lys(161) is due to abrogation of the interaction with the N-terminal tail. However, Ca(2+) attenuation of the repulsion between Asp(212) and Glu(296) keeps the activity above that of apoFVIIa. Altogether, our data suggest that repulsion involving Asp(212) in the Ca(2+)-binding loop suppresses FVIIa activity and that optimal activity requires a favorable interaction between the Ca(2+)-binding loop and the N-terminal tail. Crystal structures of tissue factor-bound FVIIa(D212N) and FVIIa(V158D/E296V/M298Q) revealed altered hydrogen bond networks, resembling those in factor Xa and thrombin, after introduction of the D212N and E296V mutations plausibly responsible for tethering the N-terminal tail to the activation domain. The charge repulsion between the Ca(2+)-binding loop and the activation domain appeared to be either relieved by charge removal and new hydrogen bonds (D212N) or abolished (E296V). We propose that Ca(2+) stimulates the intrinsic FVIIa activity by a combination of charge neutralization and loop stabilization.     

Essential Ca(2+)-independent role of the group IVA cytosolic phospholipase A(2) C2 domain for interfacial activity

The cytosolic Group IVA phospholipase A2 (GIVAPLA2) translocates to intracellular membranes to catalyze the release of lysophospholipids and arachidonic acid. GIVAPLA2 translocation and subsequent activity is regulated by its Ca2+-dependent phospholipid binding C2 domain. Phosphatidylinositol 4,5-bisphosphate (PI-4,5-P2) also binds with high affinity and specificity to GIVAPLA2, facilitating membrane binding and activity. Herein, we demonstrate that GIVAPLA2 possessed full activity in the absence of Ca2+ when PI-4,5-P2 or phosphatidylinositol 3,4,5-trisphosphate were present. A point mutant, D43N, that is unable to bind Ca2+ also had full activity in the presence of PI-4,5-P2. However, when GIVAPLA2 was expressed without its Ca2+-binding C2 domain (DeltaC2), there was no interfacial activity. GIVAPLA2 and DeltaC2 both had activity on monomeric lysophospholipids. DeltaC2, but not the C2 domain alone, binds to phosphoinositides (PIPns) in the same manner as the full-length GIVAPLA2, confirming the location of the PIPn binding site as the GIVAPLA2 catalytic domain. Moreover, proposed PIPn-binding residues in the catalytic domain (Lys488, Lys541, Lys543, and Lys544) were confirmed to be essential for PI-4,5-P2-dependent activity increases. Exploiting the effects of PI-4,5-P2, we have discovered that the C2 domain plays a critical role in the interfacial activity of GIVAPLA2 above and beyond its Ca2+-dependent phospholipid binding.     

Single amino acid substitutions in one Ca2+ binding site of uvomorulin abolish the adhesive function

We show that a synthetic peptide corresponding to the sequence of one putative Ca2+ binding motif of the cell adhesion molecule uvomorulin is able to complex Ca2+. This function is abolished if the first Asp in the peptide is replaced by Lys. Accordingly, we expressed in L cells mutant uvomorulin with a replacement of Asp to Lys or Ala. Mutant protein was resistant to Ca2+/trypsin under mild conditions but became susceptible at or near the site of replacement at higher concentrations, leaving the remaining Ca2+ binding domains protected. Remarkably, in cell aggregation assays both mutant uvomorulins failed to mediate cell adhesiveness, demonstrating that a single amino acid substitution in one Ca2+ binding site inactivates the adhesive function.     

Cyclic peptide models of the Ca2+-binding loop of alpha-lactalbumin

A series of cyclic peptides with different linkers were designed and synthesized to model the elbow-type Ca2+-binding loop of alpha-lactalbumin (LA). All amino acids of the Ca2+-binding loop are strikingly well conserved among LAs of different species with the sequence Lys79-Phe-Leu-Asp82-Asp-Asp-Leu-Thr- Asp87-Asp88, where three carboxylates of Asp82, Asp87, and Asp88 and the amide carbonyl oxygen atoms of Lys79 and Asp84 participate in Ca2+ binding. Alanine-containing models were also prepared for monitoring the role of the binding (82, 87-88) and nonbinding Asp residues (83-84) in coordinating the cation. The structural features of synthetic peptides and their Ca2+-binding properties were investigated in solution by circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy. In water, the CD curves show a strong negative band below 200 nm as a sign of the presence of unfolded conformers. In TFE, all cyclic peptides were found to have a CD spectrum, reflecting the presence of folded (turn) conformers. The effect of Ca2+ was dependent on the structure and concentration of the model and the Ca2+ to peptide ratio (r(cat)). A surprising time dependence of the FTIR spectra of Ca2+ complexes of the Ala-containing peptides was observed. The shape of the broad amide I band showed no more change after approximately 60 min. Contrary to this, the deprotonation of the side chain COOH group(s) and formation of the final coordination sphere of Ca2+ took more time. Infrared spectra showed that in the Ca2+ complex of model comprising the binding Asp residues of LA, the cation is coordinated to the COO- groups of all three Asps, while in the complex of model comprising nonbinding Asp residues of LA, the two neighboring Asp side chains form a bridged Ca2+-binding system.     

Ca2+ activation of the cPLA2 C2 domain: ordered binding of two Ca2+ ions with positive cooperativity

During Ca(2+) activation, the Ca(2+)-binding sites of C2 domains typically bind multiple Ca(2+) ions in close proximity. These binding events exhibit positive cooperativity, despite the strong charge repulsion between the adjacent divalent cations. Using both experimental and computational approaches, the present study probes the detailed mechanisms of Ca(2+) activation and positive cooperativity for the C2 domain of cytosolic phospholipase A(2), which binds two Ca(2+) ions in sites I and II, separated by only 4.1 A. First, each of the five coordinating side chains in the Ca(2+)-binding cleft is individually mutated and the effect on Ca(2+)-binding affinity and cooperativity is measured. The results identify Asp 43 as the major contributor to Ca(2+) affinity, while the two coordinating side chains that provide bridging coordination to both Ca(2+) ions, Asp 43 and Asp 40, are observed to make the largest contributions to positive cooperativity. Electrostatic calculations reveal that Asp 43 possesses the highest pseudo-pK(a) of the coordinating acidic residues, as well as the highest general cation affinity, due to its relatively buried location within 3.5 A of seven protein oxygens with full or partial negative charges. These calculations therefore explain the greater importance of Asp 43 in defining the Ca(2+) affinity. Overall, the experimental and computational results support an activation model in which the first Ca(2+) ion binds usually to site I, thereby preordering both bridging side chains Asp 40 and 43, and partially or fully deprotonating the three coordinating Asp residues. This initial binding event prepares the conformation and protonation state of the remaining site for Ca(2+) binding, enabling the second Ca(2+) ion to bind with higher affinity than the first as required for positive cooperativity.     

Rat brain cortex mitochondria release group II secretory phospholipase A(2) under reduced membrane potential

Activation of brain mitochondrial phospholipase(s) A(2) (PLA(2)) might contribute to cell damage and be involved in neurodegeneration. Despite the potential importance of the phenomenon, the number, identities, and properties of these enzymes are still unknown. Here, we demonstrate that isolated mitochondria from rat brain cortex, incubated in the absence of respiratory substrates, release a Ca(2+)-dependent PLA(2) having biochemical properties characteristic to secreted PLA(2) (sPLA(2)) and immunoreacting with the antibody raised against recombinant type IIA sPLA(2) (sPLA(2)-IIA). Under identical conditions, no release of fumarase in the extramitochondrial medium was observed. The release of sPLA(2) from mitochondria decreases when mitochondria are incubated in the presence of respiratory substrates such as ADP, malate, and pyruvate, which causes an increase of transmembrane potential determined by cytofluorimetric analysis using DiOC(6)(3) as a probe. The treatment of mitochondria with the uncoupler carbonyl cyanide 3-chlorophenylhydrazone slightly enhances sPLA(2) release. The increase of sPLA(2) specific activity after removal of mitochondrial outer membrane indicates that the enzyme is associated with mitoplasts. The mitochondrial localization of the enzyme has been confirmed by electron microscopy in U-251 astrocytoma cells and by confocal laser microscopy in the same cells and in PC-12 cells, where the structurally similar isoform type V-sPLA(2) has mainly nuclear localization. In addition to sPLA(2), mitochondria contain another phospholipase A(2) that is Ca(2+)-independent and sensitive to bromoenol lactone, associated with the outer mitochondrial membrane. We hypothesize that, under reduced respiratory rate, brain mitochondria release sPLA(2)-IIA that might contribute to cell damage.     

Identification of group X secretory phospholipase A(2) as a natural ligand for mouse phospholipase A(2) receptor

Phospholipase A(2) receptor (PLA(2)R) mediates various biological responses elicited by group IB secretory phospholipase A(2) (sPLA(2)-IB). The recently cloned group X sPLA(2) (sPLA(2)-X) possesses several structural features characteristic of sPLA(2)-IB. Here, we detected a specific binding site of sPLA(2)-X in mouse osteoblastic MC3T3-E(1) cells. Cross-linking experiments demonstrated its molecular weight (180 kDa) to be similar to that of PLA(2)R. In fact, sPLA(2)-X was found to bind the recombinant PLA(2)R expressed in COS-7 cells, and its specific binding detected in mouse lung membranes was abolished by the deficiency of PLA(2)R. These findings demonstrate sPLA(2)-X to be one of the high-affinity ligands for mouse PLA(2)R.     

Identification of vascular endothelial growth factor receptor-binding protein in the venom of eastern cottonmouth. A new role of snake venom myotoxic Lys49-phospholipase A2

Vascular endothelial growth factor (VEGF165) and its receptor KDR (kinase insert domain-containing receptor) are central regulators of blood vessel formation. We herein report a KDR-binding protein we have isolated in the venom of eastern cottonmouth (Agkistrodon piscivorus piscivorus). Sequence analysis revealed the isolated KDR-binding protein (designated KDR-bp) is identical to Lys49-phosholipase A2 (Lys49PLA2), an inactive PLA2 homologue with strong myotoxicity, in which Lys49 substitutes Asp49, a key residue for binding the essential cofactor Ca2+. KDR-bp binds to the extracellular domain of KDR with subnanomolar affinity. KDR-bp also binds to a lesser extent with Flt-1 and IgG but not to other receptors with similar immunoglobulin-like domain structures such as platelet-derived growth factor receptor alpha. The interaction between KDR-bp and KDR was blocked by VEGF165, and KDR-bp specifically inhibited VEGF165-stimulated endothelial cell proliferation, indicating KDR-bp is an antagonistic ligand for KDR. Lys49PLA2s from another snake venom were found to exhibit similar receptor binding properties to KDR-bp. This is the first report to demonstrate that an exogenous factor antagonizes VEGF and its receptor system. Our observation offers further insight into the as yet unknown molecular mechanism of myotoxic activity of snake venom Lys49PLA2s. Furthermore, KDR-bp would make a valuable tool for studying the structure and function of KDR, such as that expressed on skeletal muscle cells.     

High-affinity insulin binding: insulin interacts with two receptor ligand binding sites

The interaction of insulin with its receptor is complex. Kinetic and equilibrium binding studies suggest coexistence of high- and low-affinity binding sites or negative cooperativity. These phenomena and high-affinity interactions are dependent on the dimeric structure of the receptor. Structure-function studies of insulin analogs suggest insulin has two receptor binding sites, implying a bivalent interaction with the receptor. Alanine scanning studies of the secreted recombinant receptor implicate the L1 domain and a C-terminal peptide of the receptor alpha subunit as components of one ligand binding site. Functional studies suggest that the first and second type III fibronectin repeats of the receptor contain a second ligand binding site. We have used structure-directed alanine scanning mutagenesis to identify determinants in these domains involved in ligand interactions. cDNAs encoding alanine mutants of the holo-receptor were transiently expressed in 293 cells, and the binding properties of the expressed receptor were determined. Alanine mutations of Lys(484), Leu(552), Asp(591), Ile(602), Lys(616), Asp(620), and Pro(621) compromised affinities for insulin 2-5-fold. With the exception of Asp(620), none of these mutations compromised the affinity of the recombinant secreted receptor for insulin, indicating that the perturbation of the interaction is at the site of mutation and not an indirect effect on the interaction with the binding site of the secreted receptor. These residues thus form part of a novel ligand binding site of the insulin receptor. Complementation experiments demonstrate that insulin interacts in trans with both receptor binding sites to generate high-affinity interactions.     

The structural integrity exerted by N-terminal pyroglutamate is crucial for the cytotoxicity of frog ribonuclease from Rana pipiens

Onconase, a cytotoxic ribonuclease from Rana pipiens, possesses pyroglutamate (Pyr) at the N-terminus and has a substrate preference for uridine–guanine (UG). To identify residues responsible for onconase’s cytotoxicity, we cloned the rpr gene from genomic DNA and expressed it in Escherichia coli BL21(DE3). The recombinant onconase with Met at the N-terminus had reduced thermostability, catalytic activity and antigenicity. Therefore, we developed two methods to produce onconase without Met. One relied on the endogeneous E.coli methionine aminopeptidase and the other relied on the cleavage of a pelB signal peptide. The Pyr1 substitutional variants maintained similar secondary structures to wild-type onconase, but with less thermostability and specific catalytic activity for the innate substrate UG. However, the non-specific catalytic activity for total RNAs varied depending on the relaxation of base specificity. Pyr1 promoted the structural integrity by forming a hydrogen bond network through Lys9 in α1 and Val96 in β6, and participated in catalytic activity by hydrogen bonds to Lys9 and P₁ catalytic phosphate. Residues Thr35 and Asp67 determined B₁ base specificity, and Glu91 determined B₂ base specificity. The cytotoxicity of onconase is largely determined by structural integrity and specific catalytic activity for UG through Pyr1, rather than non-specific activity for total RNAs.     

Roles of catalytic domain residues in interfacial binding and activation of group IV cytosolic phospholipase A2

Group IV cytosolic phospholipase A(2) (cPLA(2)) has been shown to play a critical role in eicosanoid biosynthesis. cPLA(2) is composed of the C2 domain that mediates the Ca(2+)-dependent interfacial binding of protein and the catalytic domain. To elucidate the mechanism of interfacial activation of cPLA(2), we measured the effects of mutations of selected ionic and hydrophobic residues in the catalytic domain on the enzyme activity and the membrane binding of cPLA(2). Mutations of anionic residues located on (Glu(419) and Glu(420)) or near (Asp(436), Asp(438), Asp(439), and Asp(440)) the active site lid enhanced the affinity for cPLA(2) for anionic membranes, implying that the electrostatic repulsion between these residues and the anionic membrane surface might trigger the opening of the active site. This notion is further supported by a biphasic dependence of cPLA(2) activity on the anionic lipid composition of the vesicles. Mutations of a cluster of cationic residues (Lys(541), Lys(543), Lys(544), and Arg(488)), while significantly enhancing the activity of enzyme, abrogated the specific activation effect by phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). These data, in conjunction with cell activity of cPLA(2) and mutants transfected into HEK293 cells, suggest that the cationic residues form a specific binding site for PtdIns(4,5)P(2) and that the specific PtdIns(4,5)P(2) binding is involved in cellular activation of cPLA(2). Also, three hydrophobic residues at the rim of the active site (Ile(399), Leu(400), and Leu(552)) were shown to partially penetrate the membrane, thereby promoting membrane binding and activation of cPLA(2). Based on these results, we propose an interfacial activation mechanism for cPLA(2) which involves the removal of the active site lid by nonspecific electrostatic repulsion, the interdomain hinge movement induced by specific PtdIns(4,5)P(2) binding, and the partial membrane penetration by catalytic domain hydrophobic residues.