Is SH1-SH2-cross-linked myosin subfragment 1 a structural analog of the weakly-bound state of myosin?

Myosin subfragment 1 (S1) with SH1 (Cys(707)) and SH2 (Cys(697)) groups cross-linked by p-phenylenedimaleimide (pPDM-S1) is thought to be an analog of the weakly bound states of myosin bound to actin. The structural properties of pPDM-S1 were compared in this study to those of S1.ADP.BeF(x) and S1.ADP.AlF(4)(-), i.e., the established structural analogs of the myosin weakly bound states. To distinguish between the conformational effects of SH1-SH2 cross-linking and those due to their monofunctional modification, we used S1 with the SH1 and SH2 groups labeled with N-phenylmaleimide (NPM-S1) as a control in our experiments. The state of the nucleotide pocket was probed using a hydrophobic fluorescent dye, 3-[4-(3-phenyl-2-pyrazolin-1-yl)benzene-1-sulfonylamido]phen ylboronic acid (PPBA). Differential scanning calorimetry (DSC) was used to study the thermal stability of S1. By both methods the conformational state of pPDM-S1 was different from that of unmodified S1 in the S1.ADP.BeF(x) and S1.ADP.AlF(4)(-) complexes and closer to that of nucleotide-free S1. Moreover, BeF(x) and AlF(4)(-) binding failed to induce conformational changes in pPDM-S1 similar to those observed in unmodified S1. Surprisingly, when pPDM cross-linking was performed on S1.ADP.BeF(x) complex, ADP.BeF(x) protected to some extent the nucleotide pocket of S1 from the effects of pPDM modification. NPM-S1 behaved similarly to pPDM-S1 in our experiments. Overall, this work presents new evidence that the conformational state of pPDM-S1 is different from that of the weakly bound state analogs, S1.ADP.BeF(x) and S1.ADP.AlF(4)(-). The similar structural effects of pPDM cross-linking of SH1 and SH2 groups and their monofunctional labeling with NPM are ascribed to the inhibitory effects of these modifications on the flexibility/mobility of the SH1-SH2 helix.     

Mechanism of GTP hydrolysis in tubulin polymerization: characterization of the kinetic intermediate microtubule-GDP-Pi using phosphate analogues

Beryllium fluoride (BeF3-) has previously been shown to bind tightly to microtubules as a structural analogue of Pi and to mimic the GDP-Pi transient state in tubulin polymerization [Carlier, M.-F., Didry, D., Melki, R., Chabre, M., & Pantaloni, D. (1988) Biochemistry 27, 3555-3559]. The interaction of BeF3- with tubulin is analyzed here in greater detail. BeF3- binds to and dissociates from microtubule GDP subunits at very slow rates (k+ congruent to 100 M-1 s-1; k- congruent to 6 x 10(-4) s-1), suggesting that a slow conformation change of tubulin, linked to the stabilization of the microtubule structure, follows BeF3- binding. The possibility is evoked that BeF3- acts as a transition-state analogue in the GTPase reaction of tubulin. BeF3- does not bind to dimeric nor to oligomeric GDP-tubulin with high affinity. Substoichiometric binding of BeF3- to microtubules provides extensive stabilization of the structure. An original mechanistic model that accounts for the data is proposed. The kinetic parameters for microtubule elongation in the presence of GTP- and GDP-tubulin with and without BeF3- have been determined. Data support the following views: (i) Microtubules at steady state and in a regime of slow growth in the presence of GTP are stabilized by a cap of GDP-Pi subunits functionally similar to GDP-BeF3 subunits. (ii) In the presence of BeF3-, microtubules elongate from GDP-tubulin within the following sequence of reactions: initial nonproductive binding of GDP-tubulin to microtubule ends is followed by the binding of BeF3- and the associated conformation change allowing sustained elongation.     

Roles of asp126 and asp156 in the enzyme function of sphingomyelinase from Bacillus cereus.

To elucidate the roles of conserved Asp residues of Bacillus cereus sphingomyelinase (SMase) in the kinetic and binding properties of the enzyme toward various substrates and Mg2+, the kinetic data on mutant SMases (D126G and D156G) were compared with those of wild type (WT) enzyme. The stereoselectivity of the enzyme in the hydrolysis of monodispersed short-chain sphingomyelin (SM) analogs and the binding of Mg2+ to the enzyme were not affected by the replacement of Asp126 or Asp156. The pH-dependence curves of kinetic parameters (1/Km and kcat) for D156G-catalyzed hydrolysis of micellar SM mixed with Triton X-100 (1:10) and of micellar 2-hexadecanoylamino-4-nitrophenylphosphocholine (HNP) were similar in shape to those for WT enzyme-catalyzed hydrolysis. On the other hand, the curves for D126G lacked the transition observed for D156G and WT enzymes. Comparison of the values and the shape of pH-dependence curves of kinetic parameters indicated that Asp126 of WT SMase enhances the enzyme's catalytic activity toward both substrates and its binding of HNP but not SM. The deprotonation of Asp126 enhances the substrate binding and slightly suppresses the catalytic activity toward both substrates. Asp156 of WT SMase acts to decrease the binding of both substrates and the catalytic activity to HNP but not SM. From the present study and the predicted three-dimensional structure of B. cereus SMase, Asp126 was thought to be located close to the active site, and its ionization was shown to affect the catalytic activity and substrate binding.     

X-ray structures of the Dictyostelium discoideum myosin motor domain with six non-nucleotide analogs

The three-dimensional structures of the truncated myosin head from Dictyostelium discoideum myosin II complexed with dinitrophenylaminoethyl-, dinitrophenylaminopropyl-, o-nitrophenylaminoethyl-, m-nitrophenylaminoethyl-, p-nitrophenylaminoethyl-, and o-nitrophenyl-N-methyl-aminoethyl-diphosphate.beryllium fluoride have been determined to better than 2.3-A resolution. The structure of the protein and nucleotide binding pocket in these complexes is very similar to that of S1dC.ADP.BeF(x) (Fisher, A. J., Smith, C. A., Thoden, J., Smith, R., Sutoh, K., Holden, H. M., and Rayment, I. (1995) Biochemistry 34, 8960-8972). The position of the triphosphate-like moiety is essentially identical in all complexes. Furthermore, the alkyl-amino group plays the same role as the ribose by linking the triphosphate to the adenine binding pocket; however, none of the phenyl groups lie in the same position as adenine in S1dC.MgADP.BeF(x), even though several of these nucleotide analogs are functionally equivalent to ATP. Rather the former location of adenine is occupied by water in the nanolog complexes, and the phenyl groups are organized in a manner that attempts to optimize their hydrogen bonding interactions with this constellation of solvent molecules. A comparison of the kinetic and structural properties of the nanologs relative to ATP suggests that the ability of a substrate to sustain tension and to generate movement correlates with a well defined interaction with the active site water structure observed in S1dC.MgADP.BeF(x).     

Structure-activity relationship of sphingomyelin analogs with sphingomyelinase from Bacillus cereus

The aim of this study was to examine how structural properties of different sphingomyelin (SM) analogs affected their substrate properties with sphingomyelinase (SMase) from Bacillus cereus. Using molecular docking and dynamics simulations (for SMase-SM complex), we then attempted to explain the relationship between SM structure and enzyme activity. With both micellar and monolayer substrates, 3O-methylated SM was found not to be degraded by the SMase. 2N-methylated SM was a substrate, but was degraded at about half the rate of its 2NH-SM control. PhytoPSM was readily hydrolyzed by the enzyme. PSM lacking one methyl in the phosphocholine head group was a good substrate, but PSM lacking two or three methyls failed to act as substrates for SMase. Based on literature data, and our docking and MD simulations, we conclude that the 3O-methylated PSM fails to interact with Mg(2+) and Glu53 in the active site, thus preventing hydrolysis. Methylation of 2NH was not crucial for binding to the active site, but appeared to interfere with an induced fit activation of the SMase via interaction with Asp156. An OH on carbon 4 in the long-chain base of phytoPSM appeared not to interfere with the 3OH interacting with Mg(2+) and Glu53 in the active site, and thus did not interfere with catalysis. Removing two or three methyls from the PSM head group apparently increased the positive charge on the terminal N significantly, which most likely led to ionic interactions with Glu250 and Glu155 adjacent to the active site. This likely interaction could have misaligned the SM substrate and hindered proper catalysis.     

BeF(x) stops the chaperonin cycle of GroEL-GroES and generates a complex with double folding chambers

Coupling with ATP hydrolysis and cooperating with GroES, the double ring chaperonin GroEL assists the folding of other proteins. Here we report novel GroEL-GroES complexes formed in fluoroberyllate (BeF(x)) that can mimic the phosphate part of the enzyme-bound nucleotides. In ATP, BeF(x) stops the functional turnover of GroEL by preventing GroES release and produces a symmetric 1:2 GroEL-GroES complex in which both GroEL rings contain ADP.BeF(x) and an encapsulated substrate protein. In ADP, the substrate protein-loaded GroEL cannot bind GroES. In ADP plus BeF(x), however, it can bind GroES to form a stable 1:1 GroEL-GroES complex in which one of GroEL rings contains ADP.BeF(x) and an encapsulated substrate protein. This 1:1 GroEL-GroES complex is converted into the symmetric 1:2 GroEL-GroES complex when GroES is supplied in ATP plus BeF(x). Thus, BeF(x) stabilizes two GroEL-GroES complexes; one with a single folding chamber and the other with double folding chambers. These results shed light on the intermediate ADP.P(i) nucleotide states in the functional cycle of GroEL.     

Noncooperative stabilization effect of phalloidin on ADP.BeFx- and ADP.AlF4-actin filaments

Actin plays important roles in eukaryotic cell motility. During actin polymerization, the actin-bound ATP is hydrolyzed to ADP and P i. We carried out differential scanning calorimetry experiments to characterize the cooperativity of the stabilizing effect of phalloidin on actin filaments in their ADP.P i state. The ADP.P i state was mimicked by using ADP.BeF x or ADP.AlF 4. The results showed that the binding of the nucleotide analogues or phalloidin stabilized the actin filaments to a similar extent when added separately. Phalloidin binding to ADP.BeF x- or ADP.AlF 4-actin filaments further stabilized them, indicating that the mechanism by which phalloidin and the nucleotide analogues affect the filament structure was different. The results also showed that the stabilization effect of phalloidin binding to ADP.BeF x or ADP.AlF 4-bound actin filaments was not cooperative. Since the effect of phalloidin binding was cooperative in the absence of these nucleotide analogues, these results suggest that the binding of ADP.BeF x or ADP.AlF 4 to the actin modified the protomer-protomer interactions along the actin filaments.     

Activation of sphingomyelinase from Bacillus cereus by Zn2+ hitherto accepted as a strong inhibitor

Sphingomyelinase (SMase) from Bacillus cereus has been known to be activated by Mg2+, Mn2+, and Co2+, but strongly inhibited by Zn2+. In the present study, we investigated the effects of several kinds of metal ions on the catalytic activity of B. cereus SMase, and found that the activity was inhibited by Zn2+ at its higher concentrations or at higher pH values, but unexpectedly activated at lower Zn2+ concentrations or at lower pH values. This result indicates that SMase possesses at least two different binding sites for Zn2+ and that the Zn2+ binding to the high-affinity site can activate the enzyme, whereas the Zn2+ binding to the low-affinity site can inactivate it. We also found that the binding of substrate to the enzyme was independent of the Zn2+ binding to the high-affinity site, but was competitively inhibited by the Zn2+ binding to the low-affinity site. The binding affinity of the metal ions to the site for activating the enzyme was determined to be in the rank-order of Mg2+ = Co2+ < Mn2+ < Zn2+. It was also demonstrated that these four metal ions competed with each other for the same binding site on the enzyme molecule.     

Klebsiella pneumoniae nitrogenase: formation and stability of putative beryllium fluoride-ADP transition state complexes.

Incubation of the MoFe protein (Kp1) and Fe protein (Kp2), the component proteins of Klebsiella pneumoniae nitrogenase, with BeF(3)(-) and MgADP resulted in a progressive inhibition of nitrogenase activity. We have shown that at high Kp2 to Kp1 molar ratios this inhibition is due to the formation of an inactive complex with a stoichiometry corresponding to Kp1.{Kp2.(MgADP.BeFx)2}2. At lower Kp2:Kp1 ratios, an equilibrium between this 2:1 complex, the partially active 1:1 Kp1.Kp2.(MgADP. BeFx)2 complex, and active nitrogenase components was demonstrated. The inhibition was reversible since incubation of the 1:1 complex in the absence of MgADP and beryllium resulted in complete restoration of activity over 30 h. Under pseudo-first-order conditions with regard to nitrogenase components and MgADP, the kinetics of the rate of inhibition with increasing concentrations of BeF(3)(-) showed a square dependence on [BeF(3)(-)], consistent with the binding of two Be atoms by Kp2 in the complex. Analytical fplc gel filtration profiles of Kp1.Kp2 incubation mixtures at equilibrium resolved the 2:1 complex and the 1:1 complex from free Kp1. Deconvolution of the equilibrium profiles gave concentrations of the components allowing constants for their formation of 2.1 x 10(6) and 5.6 x 10(5) M(-1) to be calculated for the 1:1 and 2:1 complexes, respectively. When the active site concentration of the different species was taken into account, values for the two constants were the same, indicating the two binding sites for Kp2 are the same for Kp1 with one or both sites unoccupied. The value for K(1) we obtain from this study is comparable with the value derived from pre-steady-state studies of nitrogenase. Analysis of the elution profile obtained on gel filtration of a 1:1 ratio incubation mixture containing 20 microM nitrogenase components showed 97% of the Kp2 present initially to be complexed. These data provide the first unequivocal demonstration that Fe protein preparations which may contain up to 50% of a species of Fe protein defective in electron transfer is nevertheless fully competent in complex formation with MoFe protein.     

Glu-53 of Bacillus cereus sphingomyelinase acts as an indispensable ligand of Mg2+ essential for catalytic activity

Bacillus cereus sphingomyelinase (SMase) is an extracellular hemolysin classified into a group of Mg(2+)-dependent neutral SMases (nSMase). Sequence comparison of bacterial and eukaryotic Mg(2+)-dependent nSMases has shown that several amino acid residues, including Glu-53 of B. cereus SMase, are conserved, suggesting a catalytic mechanism common to these enzymes. Mutational analysis has revealed that hemolytic and SM-hydrolyzing activities are abolished by E53A and E53Q mutations. Only the E53D mutant enzyme partially retains these activities, however, a significant decrease in the apparent k(cat)/K(m) for SM hydrolysis is observed by this mutation. Mg(2+) activates the wild-type enzyme in a two-step manner, i.e., at least two binding sites for Mg(2+), high- and low-affinity, are present on the enzyme. The binding affinity of essential Mg(2+) for the high-affinity site is decreased by the mutation. In addition, the binding affinities of Mn(2+) and Co(2+) (substitutes for Mg(2+)) are also decreased. On the contrary, the inhibitory effects of Ca(2+), Cu(2+), and Zn(2+) on SM-hydrolyzing activity are not influenced by the mutation. The results indicate that Glu-53 of B. cereus SMase acts as a ligand for Mg(2+) and is involved in the high-affinity Mg(2+)-binding site, which is independent of the binding site for inhibitory metals.     

Structure–activity relationship of sphingomyelin analogs with sphingomyelinase from Bacillus cereus

The aim of this study was to examine how structural properties of different sphingomyelin (SM) analogs affected their substrate properties with sphingomyelinase (SMase) from Bacillus cereus. Using molecular docking and dynamics simulations (for SMase–SM complex), we then attempted to explain the relationship between SM structure and enzyme activity. With both micellar and monolayer substrates, 3O-methylated SM was found not to be degraded by the SMase. 2N-methylated SM was a substrate, but was degraded at about half the rate of its 2NH–SM control. PhytoPSM was readily hydrolyzed by the enzyme. PSM lacking one methyl in the phosphocholine head group was a good substrate, but PSM lacking two or three methyls failed to act as substrates for SMase. Based on literature data, and our docking and MD simulations, we conclude that the 3O-methylated PSM fails to interact with Mg^2 + and Glu53 in the active site, thus preventing hydrolysis. Methylation of 2NH was not crucial for binding to the active site, but appeared to interfere with an induced fit activation of the SMase via interaction with Asp156. An OH on carbon 4 in the long-chain base of phytoPSM appeared not to interfere with the 3OH interacting with Mg^2 + and Glu53 in the active site, and thus did not interfere with catalysis. Removing two or three methyls from the PSM head group apparently increased the positive charge on the terminal N significantly, which most likely led to ionic interactions with Glu250 and Glu155 adjacent to the active site. This likely interaction could have misaligned the SM substrate and hindered proper catalysis.     

Role of the beta 93-100 determinant loop sequence in receptor binding and biological activity of human luteinizing hormone and chorionic gonadotropin

The intercysteine loop sequence (93-100) in the beta-subunit has been postulated to be important for receptor binding and specificity in the glycoprotein hormones, LH and human CG (hCG). To demonstrate this directly, and to characterize the structural features essential for activity, we prepared a series of synthetic peptides and analogs incorporating this determinant loop region. Peptides were assayed for inhibition of labeled hCG binding to ovarian membrane receptors and stimulation of testosterone production in Leydig cells. Peptides with the native (93-100) sequence from hCG and hLH inhibited hCG binding half maximally at 2.18 and 2.62 x 10(-4) M, respectively, while the sequence from FSH was inactive. Isosteric substitution of Ala for Cys resulted in an inactive peptide, indicating that the (93-100) disulfide bridge is essential for activity. Optimal binding activity requires at least one net positive charge among the side chains, as shown by loss of activity in hybrid analogs with neutral or negative charges conferred by progressive replacement of Arg by Asp at 94 and 95 or by introduction of Asp at 96 and 97. Despite binding to receptors, the native sequence did not promote testosterone production at doses up to 10(-2) M. This contrasts with a second receptor binding sequence, beta (38-57) that activates testosterone production. There are differences between the (93-100) and (38-57) loop sequences in their chemical and physical properties, biological activity and antigenicity. While the cumulative evidence suggests that they associate with counterpart sites in alpha-subunit to form a topographical binding domain in the whole hormone, our results suggest that each sequence may contribute in different ways to activation of postreceptor events.     

Band-3 mediated uptake of beryllofluoride complexes by human erythrocytes.

Beryllium forms several multivalent fluoride complexes in aqueous solution; the relative concentration of each is governed by the relative concentrations of the constituent ions and pH. In 9Be NMR spectra the 9Be (spin = 3/2) and 19F (spin = 1/2) spin coupling gave rise to an overlapping resonance triplet, quartet, and quintet of BeF2, BeF3-, and BeF4(2-), respectively. The low frequency shift of the quartet (0.31 ppm) and the quintet (0.62 ppm) from the triplet correlated with an increase in the number of 19F-ions in each complex. 19F NMR spectra of the complexes showed that the spin-coupled quartet of each complex was progressively shifted to higher frequency with an increase in the number of F- ions in the complex. Using 9Be and 19F NMR, the multiple equilibrium mixture of complexes was found to shift substantially to favor the BeF3- and BeF4(2-) with a relative increase of NaF concentration. The association constants for BeF2, BeF3-, and BeF4(2-) at 25 degrees C were determined directly from the peak intensities of the spectra, and by a numerical fitting procedure for multiple spectra, and were 0.51 +/- 0.17 mM-2, 0.26 +/- 0.03 mM-1, and 1.0 x 10(-2) +/- 0.1 x 10(-2) mM-1, respectively. 19F NMR spectra of human erythrocytes to which Be2+ and F- were added showed separate resonances from the intracellular populations of the complexes and these were shifted to higher frequency from their extracellular counterparts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the aluminum and beryllium fluoride species which activate transducin. Analysis of the binding and dissociation kinetics.

Aluminofluoride and beryllofluoride complexes can activate the heterotrimeric G-proteins by binding next to GDP in the nucleotide site of their G alpha subunit and acting as analogs of the gamma-phosphate of a GTP. However, the exact structures of the activatory complexes in solution as well as those of the bound complexes in the nucleotide site are still disputed. We have studied, by monitoring the activation-dependent tryptophan fluorescence of transducin T alpha subunit, the pF (-log[F-]) and pH dependencies of the kinetics of activation and deactivation of T alpha GDP in the presence of NaF and aluminum or beryllium salts. Comparisons were made with the calculated pF and pH dependencies of the distribution of the metallofluoride complexes, in order to identify the activating species. We observed that the contribution of a magnesium-dependent mechanism of activation by fluoride (Antonny, B., Bigay, J., and Chabre, M. (1990) FEBS Lett. 268, 277-280) and effects due to slow equilibration kinetics between various aluminofluoride complexes could give rise to puzzling kinetics that had caused misinterpretations of previous results. Once corrected for these effects, our results suggest that with aluminum AlF3(OH)- is, rather than AlF4-, the main activating species and that the bound form of the complex is tetracoordinated GDP-AlF3. Deactivation kinetics depend on the free fluoride concentration in the medium, suggesting that the simple bimolecular scheme: T alpha GDP-AlF3 in equilibrium with T alpha GDP+AlF3(OH) does not fully describe the interaction. Fluorides in the bound complexes can also exchange with free F- ions in solution. With beryllium, two complexes are activatory: BeF3-.H2O and BeF2(OH)-.H2O. In the nucleotide site these give two tetracoordinated complexes, GDP.BeF3 and GDP.BeF2(OH), as shown by their different dissociation rates.     

Mn2+ and Mg2+ improved sphingomyelinase production by Lactobacillus rhamnosus FTDC 8313 and binding affinity to sphingomyelin for generation of ceramides

The present research aims to optimize the sphingomyelinase (SMase) activity produced by Lactobacillus rhamnosus FTDC 8313 using divalent metal ions via response surface methodology and to further study the effects of the divalent metal ions on SMase activity using molecular modeling approach. This study also aimed to assess the possibility of increasing ceramide levels in vitro on cultured keratinocytes upon treatment with the extracellular extract of the optimized L. rhamnosus FTDC 8313. Using a central composite design, an optimum point of SMase activity (6.54 mU ml^?1) was produced from a combination of 0.65% (w/v) MnSO₄ and 0.82% (w/v) MgSO₄. 3D response surface indicated that the altered availability of the two ions (Mn²⁺ and Mg²⁺) reduced their effects on SMase activity. In addition, the treatment of the HaCaT cells with optimized extracellular extract of L. rhamnosus FTDC8313 significantly increased (P < 0.05) the conversion of sphingomyelin to ceramide as compared to the control. Molecular docking demonstrated that the addition of Mn²⁺ and Mg²⁺ into the active site of SMase improved the binding affinity between the SMase and sphingomyelin based on its free energy of binding as well as the interaction distances between the important catalytic residues Glu53 and His296.     

Switched or not?: the structure of unphosphorylated CheY bound to the N terminus of FliM

Phosphorylation of Escherichia coli CheY increases its affinity for its target, FliM, 20-fold. The interaction between BeF(3)(-)-CheY, a phosphorylated CheY (CheY approximately P) analog, and the FliM sequence that it binds has been described previously in molecular detail. Although the conformation that unphosphorylated CheY adopts in complex with FliM was unknown, some evidence suggested that it is similar to that of CheY approximately P. To resolve the issue, we have solved the crystallographic structure of unphosphorylated, magnesium(II)-bound CheY in complex with a synthetic peptide corresponding to the target region of FliM (the 16 N-terminal residues of FliM [FliM(16)]). While the peptide conformation and binding site are similar to those of the BeF(3)(-)-CheY-FliM(16) complex, the inactive CheY conformation is largely retained in the unphosphorylated Mg(2+)-CheY-FliM(16) complex. Communication between the target binding site and the phosphorylation site, observed previously in biochemical experiments, is enabled by a network of conserved side chain interactions that partially mimic those observed in BeF(3)(-)-activated CheY. This structure makes clear the active role that the beta4-alpha4 loop plays in the Tyr(87)-Tyr(106) coupling mechanism that enables allosteric communication between the phosphorylation site and the target binding surface. Additionally, this structure provides a high-resolution view of an intermediate conformation of a response regulator protein, which had been generally assumed to be two state.     

The structure-activity relationship of the series of non-peptide small antagonists for p56lck SH2 domain

The antagonists for the SH2 domain are regarded as novel therapeutic candidates for cancer, autoimmune disease, and chronic inflammatory disease. Previously, we identified rosmarinic acid (alpha-o-caffeoyl-3,4-dihydroxyphenyl-lactic acid; RosA) from Prunella vulgaris as an antagonist for the p56lck SH2 domain by screening natural products. RosA not containing phosphotyrosine surrogate had a considerable inhibitory activity for T-cell antigen receptor (TCR)-induced interleukin (IL)-2 expression, and subsequent T-cell proliferation in vitro cell assay. To investigate the structure-activity relationship of RosA and to identify a novel p56lck SH2 antagonist with more potent in vitro T-cell inhibitory activity, we synthesized several analogs of RosA by using rational design. All synthesized compounds were tested in vitro binding activity for the SH2 domain and in vitro T-cell inhibitory activity. All four hydroxyl groups of RosA were essential for binding with the p56lck SH2 domain and T-cell inhibitory activity. Unexpectedly, conformationally less constrained analogs 4 and 9 showed a more potent binding affinity for the SH2 domain than that of RosA, and chirality of the analog did not play an important role in protein binding. We successfully identified several RosA analogs with a more potent T-cell inhibitory activity than that of RosA. Overall results revealed important structural requirements of the p56lck SH2 antagonists for in vitro T-cell inhibitory activity and in vitro protein binding activity.     

Cyclobutane analogs of GABA

Bothcis-andtrans-3-aminocyclobutane-1-carboxylic acid have been synthesized as conformationally restricted analogs of GABA. The cis isomer displayed weak to moderate GABA-like activity with respect to (1) inhibition of GABA uptake in rat brain minislices, (2) inhibition of sodium-independent binding of GABA to rat brain membranes, (3) activity as a substrate for GABA aminotransferase, and (4) depression of the firing rate of cat spinal neurons in vivo. The trans isomer was less effective on all four assays. The result has been interpreted in terms of the conformational pinning back of the polar groups by the cyclobutane ring in the trans GABA analog so that unfavorable steric interactions would occur between one of the methylene groups and a region of steric hindrance at the active sites for particular GABA processes.     

RecA protein-promoted cleavage of LexA repressor in the presence of ADP and structural analogues of inorganic phosphate, the fluoride complexes of aluminum and beryllium

Complexes formed from A13+ or Be2+ and fluoride inhibit the single-stranded DNA-dependent ATPase activity of RecA protein. In contrast, poly(dT)-RecA-ADP complexes, which are inactive for cleavage of LexA protein, become fully active in the presence of AlF4- or BeF3- ions. These data suggest that fluoride complexes of aluminum and beryllium (called herein X) convert RecA-ADP complexes, which bind weakly to single-stranded DNA, into RecA-ADP-X complexes, which bind tightly to single-stranded DNA, the ADP-X moiety behaving as a nonhydrolyzable analogue of ATP. We propose that AlF4- and BeF3- ions act as analogues of inorganic phosphate by binding to the site of the gamma-phosphate of ATP on RecA-ADP complexes, hence mimicking the single-stranded DNA-RecA-ADP-Pi transition state. We conclude that the elementary reaction that switches RecA protein from a high affinity single-stranded DNA binding state to a low affinity single-stranded DNA binding state is not ATP hydrolysis per se but Pi release.