Mechanism of Inducible Nitric-oxide Synthase Dimerization Inhibition by Novel Pyrimidine Imidazoles

Overproduction of nitric oxide (NO) by inducible nitric-oxide synthase (iNOS) has been etiologically linked to several inflammatory, immunological, and neurodegenerative diseases. As dimerization of NOS is required for its activity, several dimerization inhibitors, including pyrimidine imidazoles, are being evaluated for therapeutic inhibition of iNOS. However, the precise mechanism of their action is still unclear. Here, we examined the mechanism of iNOS inhibition by a pyrimidine imidazole core compound and its derivative (PID), having low cellular toxicity and high affinity for iNOS, using rapid stopped-flow kinetic, gel filtration, and spectrophotometric analysis. PID bound to iNOS heme to generate an irreversible PID-iNOS monomer complex that could not be converted to active dimers by tetrahydrobiopterin (H₄B) and l-arginine (Arg). We utilized the iNOS oxygenase domain (iNOSoxy) and two monomeric mutants whose dimerization could be induced (K82AiNOSoxy) or not induced (D92AiNOSoxy) with H₄B to elucidate the kinetics of PID binding to the iNOS monomer and dimer. We observed that the apparent PID affinity for the monomer was 11 times higher than the dimer. PID binding rate was also sensitive to H₄B and Arg site occupancy. PID could also interact with nascent iNOS monomers in iNOS-synthesizing RAW cells, to prevent their post-translational dimerization, and it also caused irreversible monomerization of active iNOS dimers thereby accomplishing complete physiological inhibition of iNOS. Thus, our study establishes PID as a versatile iNOS inhibitor and therefore a potential in vivo tool for examining the causal role of iNOS in diseases associated with its overexpression as well as therapeutic control of such diseases.     

Control of nitric oxide synthase dimer assembly by a heme-NO-dependent mechanism

Homodimer formation is a key step that follows heme incorporation during assembly of an active inducible nitric oxide synthase (iNOS). In cells, heme incorporation into iNOS becomes limited due to interaction between self-generated NO and cellular heme [Albakri, Q., and Stuehr, D. J. (1996) J. Biol. Chem. 271, 5414-5421]. Here we investigated if NO can regulate at points downstream in the process by inhibiting dimerization of heme-containing iNOS monomer. Heme-containing monomers were generated by treating iNOS dimer or iNOS oxygenase domain dimer (iNOSoxy) with urea. Both monomers dimerized when incubated with Arg and 6R-tetrahydrobiopterin (H4B), as shown previously [Abu-Soud, H. M., Loftus, M., and Stuehr, D. J. (1995) Biochemistry 34, 11167-11175]. The NO-releasing drug S-nitrosyl-N-acetyl-D,L-penicillamine (SNAP; 0-0.5 mM) inhibited dimerization of iNOS monomer in a dose- and time-dependent manner, without causing heme release. SNAP-pretreated monomer also did not dimerize in response to H4B plus Arg. SNAP converted Arg- and H4B-free iNOS dimer into monomer that could not redimerize, but had no effect on iNOS dimer preincubated with Arg and H4B. Anaerobic spectral analysis showed that NO from SNAP bound to the ferric heme of iNOSoxy monomer or dimer. Adding imidazole as an alternative heme ligand prevented SNAP from inhibiting iNOS monomer dimerization. We conclude that NO and related species can block iNOS dimerization at points downstream from heme incorporation. The damage to heme-containing monomer results from a reaction with the protein and appears irreversible. Although dimeric structure alone does not protect, it does enable Arg and H4B to bind and protect. Inhibition appears mediated by NO coordinating to the ferric heme iron of the monomer.     

Role of regulatory F-domain in hepatocyte nuclear factor-4alpha ligand specificity

The F-domain of rat HNF-4alpha1 has a crucial impact on the ligand binding affinity, ligand specificity and secondary structure of HNF-4alpha. (i) Fluorescent binding assays indicate that wild-type, full-length HNF-4alpha (amino acids 1-455) has high affinity (Kd=0.06-12 nm) for long chain fatty acyl-CoAs (LCFA-CoA) and low affinity (Kd=58-296 nm) for unesterified long chain fatty acids (LCFAs). LCFA-CoA binding was due to close molecular interaction as shown by fluorescence resonance energy transfer (FRET) from full-length HNF-4alpha tryptophan (FRET donor) to bound cis-parinaroyl-CoA (FRET acceptor), which yielded an intermolecular distance of 33 A, although no FRET to cis-parinaric acid was detected. (ii) Deleting the N-terminal A-D-domains, comprising the AF1 and DNA binding functions, only slightly affected affinities for LCFA-CoAs (Kd=0.9-4 nm) and LCFAs (Kd=93-581 nm). (iii) Further deletion of the F-domain robustly reduced affinities for LCFA-CoA and reversed ligand specificity (i.e. high affinity for LCFAs (Kd=1.5-32 nm) and low affinity for LCFA-CoAs (Kd=54-302 nm)). No FRET from HNF-4alpha-E (amino acids 132-370) tryptophan (FRET donor) to bound cis-parinaroyl-CoA (FRET acceptor) was detected, whereas an intermolecular distance of 28 A was calculated from FRET between HNF-4alpha-E and cis-parinaric acid. (iv) Circular dichroism showed that LCFA-CoA, but not LCFA, altered the secondary structure of HNF-4alpha only when the F-domain was present. (v) cis-Parinaric acid bound to HNF-4alpha with intact F-domain was readily displaceable by S-hexadecyl-CoA, a nonhydrolyzable thioether analogue of LCFA-CoAs. Truncation of the F-domain significantly decreased cis-parinaric acid displacement. Hence, the C-terminal F-domain of HNF-4alpha regulated ligand affinity, ligand specificity, and ligand-induced conformational change of HNF-4alpha. Thus, characteristics of F-domain-truncated mutants may not reflect the properties of full-length HNF-4alpha.     

Characterization of the metal ion-binding domains from rat alpha- and beta-parvalbumins

We have examined the metal ion-binding domains from rat alpha and beta parvalbumin. We find that the CD-EF fragments differ markedly in their tendency to self-associate. Whereas Ca(2+)-free alpha CD-EF is monomeric, the Ca(2+)-free beta peptide dimerizes weakly (K(2) = 2400 +/- 200 M(-1)). In buffer containing 1.0 mM Ca(2+), the apparent dimerization constant for beta CD-EF (191,000 +/- 29,000 M(-1)) is more than 50 times that of alpha (3400 +/- 200 M(-1)). Alpha CD-EF binds two Ca(2+) with positive cooperativity. Titration calorimetry data afford binding constants of 3.7(0.1) x 10(3) M(-1) and 8.6(0.2) x 10(4) M(-1). Beta CD-EF also binds two Ca(2+) cooperatively but with lower affinity. Equilibrium dialysis yields Adair constants of 4.2(0.1) x 10(3) and 6.1(0.2) x 10(3) M(-1). Significantly, the difference in Ca(2+) affinity is substantially smaller than that observed for the full-length proteins-suggesting that the AB domain can modulate divalent ion affinity. Analysis of beta calorimetry data requires explicit consideration of the self-association behavior. Data collected at low CD-EF concentration are consistent with preferential occupation of the EF site, dimerization of singly bound monomers, and cooperative filling of the CD sites. At higher concentrations, apo-protein dimerization can apparently precede cooperative occupation of the EF sites. In the presence of Ca(2+), alpha CD-EF exhibits higher thermal stability, consistent with its higher Ca(2+) affinity. However, the beta melting temperature shows greater concentration dependence, consistent with its greater tendency to dimerize. Neither fragment exhibits a sigmoidal melting curve in the Ca(2+)-free state, suggesting that the apo-peptides are disordered.     

Characterization of Calmodulin-Free Murine Inducible Nitric-Oxide Synthase

Nitric-Oxide Synthase (NOS), that produces the biological signal molecule Nitric-Oxide (NO), exists in three different isoforms called, neuronal (nNOS), endothelial (eNOS) and inducible (iNOS). All NOS isoforms require post-translational interaction with the calcium-binding protein, calmodulin (CaM) for manifesting their catalytic activity. However, CaM has been suggested to control the translational assembly of the enzyme as well, particularly in helping its inducible isoform, iNOS assume a stable, heme-replete, dimeric and active form. Expression of recombinant murine iNOS in E.coli in the absence of CaM has been previously shown to give extremely poor yield of the enzyme which was claimed to be absolutely heme-free, devoid of flavins, completely monomeric and catalytically inactive when compared to the heme-replete, active, dimeric iNOS, generated through co-expression with CaM. In contrast, we found that although iNOS expressed without CaM does produce significantly low amounts of the CaM-free enzyme, the iNOS thus produced, is not completely devoid of heme and is neither entirely monomeric nor absolutely bereft of catalytic activity as reported before. In fact, iNOS synthesized in the absence of CaM undergoes compromised heme incorporation resulting in extremely poor dimerization and activity compared to its counterpart co-expressed with CaM. Moreover, such CaM-free iNOS has similar flavin content and reductase activity as iNOS co-expressed with CaM, suggesting that CaM may not be as much required for the functional assembly of the iNOS reductase domain as its oxygenase domain. LC-MS/MS-based peptide mapping of the CaM-free iNOS confirmed that it had the same full-length sequence as the CaM-replete iNOS. Isothermal calorimetric measurements also revealed high affinity for CaM binding in the CaM-free iNOS and thus the possible presence of a CaM-binding domain. Thus CaM is essential but not indispensible for the assembly of iNOS and such CaM-free iNOS may help in elucidating the role of CaM on iNOS catalysis.     

Dimeric fragment of the insulin receptor alpha-subunit binds insulin with full holoreceptor affinity

The insulin receptor (IR) is a dimeric receptor, and its activation is thought to involve cross-linking between monomers initiated by binding of a single insulin molecule to separate epitopes on each monomer. We have previously shown that a minimized insulin receptor consisting of the first three domains of the human IR fused to 16 amino acids from the C-terminal of the alpha-subunit was monomeric and bound insulin with nanomolar affinity (Kristensen, C., Wiberg, F. C., Sch?ffer, L., and Andersen, A. S. (1998) J. Biol. Chem. 273, 17780-17786). To investigate the insulin binding properties of dimerized alpha-subunits, we have reintroduced the domains containing alpha-alpha disulfide bonds into this minireceptor. When inserting either the first fibronectin type III domain or the full-length sequence of exon 10, the receptor fragments were predominantly secreted as disulfide-linked dimers that both had nanomolar affinity for insulin, similar to the affinity found for the minireceptor. However, when both these domains were included we obtained a soluble dimeric receptor that bound insulin with 1000-fold higher affinity (4-8 pm) similar to what was obtained for the solubilized holoreceptor (14-24 pm). Moreover, dissociation of labeled insulin from this receptor was accelerated in the presence of unlabeled insulin, demonstrating another characteristic feature of the holoreceptor. This is the first direct demonstration showing that the alpha-subunit of IR contains all the epitopes required for binding insulin with full holoreceptor affinity.     

Binding stoichiometry and kinetics of the interaction of a human anthrax toxin receptor, CMG2, with protective antigen

The protective antigen (PA) moiety of anthrax toxin binds to cellular receptors and mediates entry of the two enzymatic moieties of the toxin into the cytosol. Two PA receptors, anthrax toxin receptor (ATR)/tumor endothelial marker 8 (TEM8) and capillary morphogenesis protein 2 (CMG2), have been identified. We expressed and purified the von Willebrand A (VWA) domain of CMG2 and examined its interactions with monomeric and heptameric forms of PA. Monomeric PA bound a stoichiometric equivalent of CMG2, whereas the heptameric prepore form bound 7 eq. The Kd of the VWA domain-PA interaction is 170 pm when liganded by Mg2+, reflecting a 1000-fold tighter interaction than most VWA domains with their endogenous ligands. The dissociation rate constant is extremely slow, indicating a 30-h lifetime for the CMG2.PA monomer complex. CMG2 metal ion-dependent adhesion site (MIDAS) was studied kinetically and thermodynamically. The association rate constant (approximately 10(5) m(-1) s(-1)) is virtually identical in the presence or absence of Mg2+ or Ca2+ , but the dissociation rate of metal ion liganded complex is up to 4 orders of magnitude slower than metal ion free complex. Residual affinity (Kd approximately 960 nm) in the absence of divalent metal ions allowed the free energy for the contribution of the metal ion to be calculated as 5 kcal mol(-1), demonstrating that the metal ion-dependent adhesion site is directly coordinated by CMG2 and PA in the binding interface. The high affinity of the VWA domain for PA supports its potency in neutralizing anthrax toxin, demonstrating its potential utility as a novel therapeutic for anthrax.     

1-(1,3-Benzodioxol-5-ylmethyl)-3-[4-(1H-imidazol-1-yl)phenoxy]-piperidine analogs as potent and selective inhibitors of nitric oxide formation

A new series of 1-(1,3-benzodioxol-5-ylmethyl)-3-[4-(1H-Imidazol-1-yl)phenoxy]-piperidine analogs were designed and identified as potent and selective inhibitors of NO formation based both on the crystal structure of a murine iNOS Delta114 monomer domain/ inhibitor complex and inhibition of the NO formation in human A172 cell assays. Compound 12S showed high potency and high iNOS selectivity versus nNOS and eNOS.     

Phospholipid binding properties of human placental anticoagulant protein-I, a member of the lipocortin family

Human placental anticoagulant protein-I (PAP-I) is a member of the lipocortin/calpactin/annexin family of Ca2+-dependent phospholipid binding proteins. PAP-I was labeled with fluorescein 5-isothiocyanate (1 mol/mol); this derivative had anticoagulant activity identical to the unlabeled protein and could be used to measure Ca2+-dependent binding to phospholipid vesicles through changes in fluorescence quenching. At 1.2 mM Ca2+, 0.50 M ionic strength, pH 7.4, 25 degrees C, fluorescein-labeled PAP-I bound to phospholipid vesicles containing 80% phosphatidylcholine, 20% phosphatidylserine with a Kd of 1.2 +/- 0.2 nM (mean +/- S.D.). At an ionic strength of 0.15 M, the Kd decreased to less than 0.1 nM. Prothrombin and factor Xa both competed with fluorescein-labeled PAP-I for binding to anionic phospholipid vesicles, but with affinities at least 1000-fold weaker than PAP-I. PAP-I bound only weakly (Kd greater than 2 x 10(-5) M) to neutral or anionic phospholipid monomers, and this binding was not calcium-dependent. These results show that the affinity of PAP-I for anionic phospholipid surfaces is sufficient to explain its potency as an in vitro anticoagulant.     

Oxalomalate affects the inducible nitric oxide synthase expression and activity

Inducible nitric oxide synthase (iNOS) is an homodimeric enzyme which produces large amounts of nitric oxide (NO) in response to inflammatory stimuli. Several factors affect the synthesis and catalytic activity of iNOS. Particularly, dimerization of NOS monomers is promoted by heme, whereas an intracellular depletion of heme and/or L-arginine considerably decreases NOS resistance to proteolysis. In this study, we found that oxalomalate (OMA, oxalomalic acid, alpha-hydroxy-beta-oxalosuccinic acid), an inhibitor of both aconitase and NADP-dependent isocitrate dehydrogenase, inhibited nitrite production and iNOS protein expression in lipopolysaccharide (LPS)-activated J774 macrophages, without affecting iNOS mRNA content. Furthermore, injection of OMA precursors to LPS-stimulated rats also decreased nitrite production and iNOS expression in isolated peritoneal macrophages. Interestingly, alpha-ketoglutarate or succinyl-CoA administration reversed OMA effect on NO production, thus correlating NO biosynthesis with the anabolic capacity of Krebs cycle. When protein synthesis was blocked by cycloheximide in LPS-activated J774 cells treated with OMA, iNOS protein levels, evaluated by Western blot analysis and (35)S-metabolic labelling, were decreased, suggesting that OMA reduces iNOS biosynthesis and induces an increase in the degradation rate of iNOS protein. Moreover, we showed that OMA inhibits the activity of the iNOS from lung of LPS-treated rats by enzymatic assay. Our results, demonstrating that OMA acts regulating synthesis, catalytic activity and degradation of iNOS, suggest that this compound might have a potential role in reducing the NO overproduction occurring in some pathological conditions.     

Interaction of hemopexin with Sn-protoporphyrin IX, an inhibitor of heme oxygenase. Role for hemopexin in hepatic uptake of Sn-protoporphyrin IX and induction of mRNA for heme oxygenase

Sn-protoporphyrin IX (SnPP), an inhibitor of heme oxygenase and a potential therapeutic agent for neonatal hyperbilirubinemia, is bound tightly by hemopexin. The apparent dissociation constant (Kd) at pH 7.4 is 0.25 +/- 0.15 microM, but estimation of the Kd for the SnPP-hemopexin complex is hampered by the fact that at physiological pH SnPP exists as monomers and dimers, both of which are bound by hemopexin. SnPP is readily displaced from hemopexin by heme (Kd less than 1 pM). The hemopexin-SnPP interaction, like that of heme-hemopexin, is dependent on the histidine residues of hemopexin. However, as expected from the differences in the coordination chemistries of tin and iron, the stability of the histidyl-metalloporphyrin complex is lower for SnPP-hemopexin than for mesoheme-hemopexin. Nevertheless, when SnPP binds to hemopexin, certain of the ligand-induced changes in the conformation of hemopexin which increase the affinity of the protein for its receptor are produced. Binding of SnPP produces the conformational change in hemopexin which protects the hinge region of hemopexin from proteolysis, but SnPP does not produce the characteristic increase in the ellipticity of hemopexin at 231 nm that heme does. Competition experiments confirmed that human serum albumin (apparent Kd = 4 +/- 2 microM) has a significantly lower affinity for SnPP than does hemopexin. Appreciable amounts of SnPP (up to 35% in adults and 20% in neonates) would be bound by hemopexin in the circulation, and the remainder of SnPP would be associated with albumin due to the latter's high concentration in serum. Essentially no non-protein-bound SnPP is present. Importantly, SnPP-hemopexin binds to the hemopexin receptor on mouse hepatoma cells with an affinity comparable to that of heme-hemopexin and treatment of the hepatoma cells with SnPP-hemopexin causes a rapid increase in the steady state level of heme oxygenase messenger RNA. These results show that hemopexin participates in the transport of SnPP to heme oxygenase and in its regulation by SnPP.     

Baculovirus-directed expression of the human insulin receptor and an insulin-binding ectodomain

In this report we describe the use of the baculovirus expression system to overproduce the human insulin holoreceptor (HIR) and a truncated, secretory version of the HIR cDNA (HIRsec) consisting of the alpha subunit and the extracellular portion of the beta subunit (beta'). Sf9 cells infected with the full-length HIR viruses synthesize recombinant HIR (rHIR) with an insulin-binding alpha subunit of apparent Mr = 110,000 and a beta subunit of apparent Mr = 80,000. Uncleaved alpha beta proreceptor accumulates in infected cells. Both of these forms assemble into higher order disulfide-linked dimers or heterotetramers of apparent Mr greater than 350,000. Insulin-binding activity in cells infected with rHIR viruses is present predominantly on the extracellular aspect of the plasma membrane (greater than 80%). Insulin binding to the full-length rHIR occurs with typical complex kinetics with Kd1 = 0.5-1 x 10(-9) M and Kd2 = 10(-7) M and receptors are present in large amounts in infected cells (1 x 10(6) receptors/cell; 1-2 mg HIR/10(9) cells). The full-length rHIR undergoes insulin-dependent autophosphorylation; half-maximal activation of beta subunit autophosphorylation occurs at 1-2 x 10(-8) M. The alpha beta proreceptor also becomes phosphorylated in vitro. Analysis of tryptic phosphopeptides derived from in vitro autophosphorylated beta subunit and alpha beta proreceptor reveals a pattern of phosphorylation that is indistinguishable from that of authentic placental HIR. Sf9 cells infected with rHIRsec viruses synthesize and secrete an (alpha beta')2 heterotetrameric complex having an insulin-binding alpha subunit of apparent Mr = 110,000 and a truncated beta' subunit of apparent Mr = 45,000 that lacks kinase activity. The rHIRsec complex purified from the conditioned medium of infected cells binds insulin with high affinity (Kd = 10(-9) M).     

3,4-Dihydro-1-isoquinolinamines: a novel class of nitric oxide synthase inhibitors with a range of isoform selectivity and potency

3-Phenyl-3.4-dihydro-1-isoquinolinamine is a weak inhibitor of iNOS and nNOS. Structural variation of 5a results in inhibitors with a range of potency and selectivity for the NOS enzymes, including a potent and very selective iNOS inhibitor 5j.     

Full-length myosin VI dimerizes and moves processively along actin filaments upon monomer clustering

Myosin VI is a reverse direction actin-based motor capable of taking large steps (30-36 nm) when dimerized. However, all dimeric myosin VI molecules so far examined have included non-native coiled-coil sequences, and reports on full-length myosin VI have failed to demonstrate the existence of dimers. Herein, we demonstrate that full-length myosin VI is capable of forming stable, processive dimers when monomers are clustered, which move up to 1-2 mum in approximately 30 nm, hand-over-hand steps. Furthermore, we present data consistent with the monomers being prevented from dimerizing unless they are held in close proximity and that dimerization is somewhat inhibited by the cargo binding tail. A model thus emerges that cargo binding likely clusters and initiates dimerization of full-length myosin VI molecules. Although this mechanism has not been previously described for members of the myosin superfamily, it is somewhat analogous to the proposed mechanism of dimerization for the kinesin Unc104.     

Binding of L-arginine and imidazole suggests heterogeneity of rat brain neuronal nitric oxide synthase

Nitric oxide synthase (NOS) is inhibited by imidazole, which binds to the heme in a low-spin complex absorbing at 428 nm. Conversion by L-arginine of this complex into a high-spin species absorbing at 395 nm is a common method to determine the binding parameters of Arg. However, both Arg-competitive and noncompetitive inhibition of NOS by imidazole has been reported, and optical studies with neuronal NOS provided no evidence for imidazole affecting Arg binding. We investigated the cause for these paradoxical observations with recombinant rat brain neuronal NOS. Imidazole bound to nNOS with a K(d)(app) of 50 microM; tetrahydrobiopterin (BH4) lowered the affinity of nNOS for imidazole 4-fold. The enzyme behaved heterogeneously with respect to Arg binding. Most of nNOS (65-80%) showed competition between Arg and imidazole. In the presence of BH4, a K(d)(Arg) of 1 microM could be estimated for this fraction, as well as apparent association and dissociation rate constants of 2.5 x 10(6) M(-1) x s(-1) and 2.5 s(-1). A second fraction of nNOS (20-30%) exhibited little or no competition. Consequently, Arg binding did not cause dissociation of the imidazole complex for this fraction, and complete generation of the high-spin state by Arg could not be achieved in the presence of imidazole. A third fraction (< or =10%) bound Arg with low affinity (K(d) 1-2 mM). Because of this heterogeneity, titration curves with Arg became almost uninterpretable. We propose that this heterogeneous response of nNOS toward Arg and imidazole is underlying the apparently conflicting results reported in the literature.     

The active form of tumor necrosis factor is a trimer

Natural human and recombinant human and murine tumor necrosis factors (TNF) were fractionated by gel filtration chromatography on Sephadex G-75. The active form of TNF was identified by its inhibitory activity in receptor binding assays with HeLa cells and was eluted as a protein of Mr approximately 55,000. Radioiodinated human and murine TNF were fractionated by gel filtration into a major peak of Mr approximately 55,000, corresponding to a trimer, and a minor peak of Mr approximately 17,000, corresponding to a monomer. Binding assays showed that the timer was at least 8-fold more active than the monomer. The human TNF partially dissociated into monomers upon addition of the nonionic detergent Triton X-100. Isolated monomers showed low binding affinity (KD = 70 nM) and reduced cytotoxicity, whereas trimers showed high binding affinity (KD = 90 pM) and cytotoxicity. When 125I-TNF was bound to cells, no release of monomer was detectable, suggesting that the trimer could directly bind to cellular receptors without dissociating into subunits. Further evidence for such binding was obtained by cross-linking 125I-TNF trimers with bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone. These trimers were bound to HeLa cells, could be dissociated from cellular receptors, and elicited a cytotoxic response. These results show that trimers, whether native or cross-linked, bind to receptors and are the biologically active form of TNF.     

Quantitative analysis of the effect of Acanthamoeba profilin on actin filament nucleation and elongation

The current view of the mechanism of action of Acanthamoeba profilin is that it binds to actin monomers, forming a complex that cannot polymerize [Tobacman, L. S., & Korn, E. D. (1982) J. Biol. Chem. 257, 4166-4170; Tseng, P., & Pollard, T. D. (1982) J. Cell Biol. 94, 213-218; Tobacman, L. S., Brenner, S. L., & Korn, E. D. (1983) J. Biol. Chem. 258, 8806-8812]. This simple model fails to predict two new experimental observations made with Acanthamoeba actin in 50 mM KC1, 1 mM MgCl2, and 1 mM EGTA. First, Acanthamoeba profilin inhibits elongation of actin filaments far more at the pointed end than at the barbed end. According, to the simple model, the Kd for the profilin-actin complex is less than 5 microM on the basis of observations at the pointed end and greater than 50 microM for the barbed end. Second, profilin inhibits nucleation more strongly than elongation. According to the simple model, the Kd for the profilin-actin complex is 60-140 microM on the basis of two assays of elongation but 2-10 microM on the basis of polymerization kinetics that reflect nucleation. These new findings can be explained by a new and more complex model for the mechanism of action that is related to a proposal of Tilney and co-workers [Tilney, L. G., Bonder, E. M., Coluccio, L. M., & Mooseker, M. S. (1983) J. Cell Biol. 97, 113-124]. In this model, profilin can bind both to actin monomers with a Kd of about 5 microM and to the barbed end of actin filaments with a Kd of about 50-100 microM. An actin monomer bound to profilin cannot participate in nucleation or add to the pointed end of an actin filament. It can add to the barbed end of a filament. When profilin is bound to the barbed end of a filament, actin monomers cannot bind to that end, but the terminal actin protomer can dissociate at the usual rate. This model includes two different Kd's--one for profilin bound to actin monomers and one for profilin bound to an actin molecule at the barbed end of a filament. The affinity for the end of the filament is lower by a factor of 10 than the affinity for the monomer, presumably due to the difference in the conformation of the two forms of actin or to steric constraints at the end of the filament.     

DNA-induced dimerization of the Escherichia coli rep helicase. Allosteric effects of single-stranded and duplex DNA.

The Escherichia coli Rep helicase is a stable monomer (Mr = 72,802) in the absence of DNA; however, binding of single-stranded (ss) or duplex (ds) DNA induces Rep monomers to dimerize. Furthermore, a chemically cross-linked Rep dimer retains both its DNA-dependent ATPase and helicase activities, suggesting that the functionally active Rep helicase is a dimer (Chao, K., and Lohman, T. M. (1991) J. Mol. Biol. 221, 1165-1181). Using a modified "double-filter" nitrocellulose filter binding assay, we have examined quantitatively the equilibrium binding of Rep to a series of ss-oligodeoxynucleotides, d(pN)n (8 less than or equal to n less than or equal to 20) and two 16-base pair duplex oligodeoxynucleotides, which are short enough so that only a single Rep monomer can bind to each oligonucleotide. This strategy has enabled us to examine the linkage between DNA binding and dimerization. We also present a statistical thermodynamic model to describe the DNA-induced Rep dimerization in the presence of ss- and/or ds-oligodeoxynucleotides. We observe quantitative agreement between this model and the experimental binding isotherms and have analyzed these isotherms to obtain the seven independent interaction constants that describe Rep-DNA binding and Rep dimerization. We find that Rep monomers (P) can bind either ss-DNA (S) or ds-DNA (D) to form PS or PD, respectively, which can then dimerize to form P2S or P2D. Furthermore, both protomers of the DNA-induced Rep dimer can bind DNA to form either P2S2, P2D2 or the mixed dimer species P2SD and ss- and ds-DNA compete for the same sites on the Rep protein. When bound to DNA, the Rep dimerization constants are approximately 1-2 x 10(8) M-1 (6 mM NaCl, pH 7.5, 4 degrees C), which are greater than the dimerization constant for free Rep monomers by at least 10(4)-fold. The Rep-ss-DNA interaction constants are independent of base composition and sequence, consistent with its role as a nonspecific DNA-binding protein. Allosteric effects are associated with ss- and ds-DNA binding to the half-saturated Rep dimers, i.e. the affinity of either ss- or ds-DNA to the free promoter of a half-saturated Rep dimer is clearly influenced by the conformation of DNA bound to the first protomer. These allosteric effects further support the proposal that the Rep dimer is functionally important and that the Rep-DNA species P2S2 and P2SD may serve as useful models for intermediates that occur during DNA unwinding.(ABSTRACT TRUNCATED AT 400 WORDS)     

Dimerization inhibitors of HIV-1 protease

By targeting the highly conserved antiparallel beta-sheet formed by the interdigitation of the N- and C-terminal strands of each monomer, dimerization inhibitors of HIV-1 protease may be useful to overcome the drug resistance observed with current active-site directed antiproteases. Sequestration of the monomer by the inhibitor (or disruption of the dimer interface) prevents the correct assembly of the inactive monomers to active enzyme. Strategies for the design of drugs targeting the dimer interface are described. Various dimerization inhibitors are reported including N- and C-terminal mimetics, lipopeptides and cross-linked interface peptides.