Oxindole-based inhibitors of cyclin-dependent kinase 2 (CDK2): design, synthesis, enzymatic activities, and X-ray crystallographic analysis.

Two closely related classes of oxindole-based compounds, 1H-indole-2,3-dione 3-phenylhydrazones and 3-(anilinomethylene)-1,3-dihydro-2H-indol-2-ones, were shown to potently inhibit cyclin-dependent kinase 2 (CDK2). The initial lead compound was prepared as a homologue of the 3-benzylidene-1,3-dihydro-2H-indol-2-one class of kinase inhibitor. Crystallographic analysis of the lead compound bound to CDK2 provided the basis for analogue design. A semiautomated method of ligand docking was used to select compounds for synthesis, and a number of compounds with low nanomolar inhibitory activity versus CDK2 were identified. Enzyme binding determinants for several analogues were evaluated by X-ray crystallography. Compounds in this series inhibited CDK2 with a potency approximately 10-fold greater than that for CDK1. Members of this class of inhibitor cause an arrest of the cell cycle and have shown potential utility in the prevention of chemotherapy-induced alopecia.

Solution studies of recombinant human stromal-cell-derived factor-1.

Stromal-cell-derived factor-1 (SDF-1alpha) is an 8-kDa chemokine that is constitutively expressed in bone-marrow-derived stromal cells and has been identified as a ligand for the CXCR4 receptor. We produced the chemokine recombinantly as methionine-SDF-1alpha in Escherichia coli without the leader peptide sequence. The protein was denatured, refolded, and further purified by reversed-phase HPLC. SDF-1alpha was shown to be >95% pure as judged by SDS-PAGE. The final yield of purified and refolded SDF-1alpha was 1-2 mg per gram of wet cell paste. The refolded protein is a ligand for the CXCR4 receptor and has been used to block HIV-mediated cell fusion and downmodulates the CXCR4 receptor. Our ability to purify hundreds of milligrams of refolded protein allowed us to conduct detailed studies of the biophysical properties of the protein. We have used a combination of biophysical techniques to study the solution properties of SDF-1alpha. The average mass of SDF-1alpha, as determined by static light scattering, gave us the first indications that the chemokine may self-associate. Further investigation with sedimentation velocity ultracentrifugation confirmed the existence of two species. The measured s(20, W) values defined two masses corresponding to monomer and dimer. Finally, sedimentation equilibrium ultracentrifugation and dynamic light scattering yielded a composite value of 150 +/- 30 microM for the dimerization constant. We conclude that SDF-1alpha exists in a monomer-dimer equilibrium.

Prevention of chemotherapy-induced alopecia in rats by CDK inhibitors.

Most traditional cytotoxic anticancer agents ablate the rapidly dividing epithelium of the hair follicle and induce alopecia (hair loss). Inhibition of cyclin-dependent kinase 2 (CDK2), a positive regulator of eukaryotic cell cycle progression, may represent a therapeutic strategy for prevention of chemotherapy-induced alopecia (CIA) by arresting the cell cycle and reducing the sensitivity of the epithelium to many cell cycle-active antitumor agents. Potent small-molecule inhibitors of CDK2 were developed using structure-based methods. Topical application of these compounds in a neonatal rat model of CIA reduced hair loss at the site of application in 33 to 50% of the animals. Thus, inhibition of CDK2 represents a potentially useful approach for the prevention of CIA in cancer patients.

Atomic structure of PDE4: insights into phosphodiesterase mechanism and specificity.

Cyclic nucleotides are second messengers that are essential in vision, muscle contraction, neurotransmission, exocytosis, cell growth, and differentiation. These molecules are degraded by a family of enzymes known as phosphodiesterases, which serve a critical function by regulating the intracellular concentration of cyclic nucleotides. We have determined the three-dimensional structure of the catalytic domain of phosphodiesterase 4B2B to 1.77 angstrom resolution. The active site has been identified and contains a cluster of two metal atoms. The structure suggests the mechanism of action and basis for specificity and will provide a framework for structure-assisted drug design for members of the phosphodiesterase family.

The E1 helicase of human papillomavirus type 11 binds to the origin of replication with low sequence specificity.

Expression of the human papillomavirus type 11 E1 and E2 genes is necessary and sufficient to support viral DNA replication. The full-length E2 protein is a transcriptional modulator that also interacts with the E1 helicase to form an E1/E2 complex at the viral origin of replication. Previous studies indicated that efficient binding of this complex to the replication origin is site-specific and that the E2 homodimer was required for efficient E1 binding. Human papillomavirus type 11 E2 and E1 proteins have been purified and their cooperative binding to the HPV type 11 viral replication origin has been characterized. Low-affinity E1 binding to the HPV type 11 replication origin was demonstrated and found to be largely nonspecific. DNA binding by E1 does not require complex formation with E2 and appears to be independent of ATP binding or hydrolysis. E1 binding quantitatively increased with the addition of increasing amounts of E2 and mutations in the E2 binding site demonstrated that the E2BS site is required for E1 and E2 to specifically bind as a high-affinity complex at the replication origin. Analysis of the A/T-rich E1 binding site via mutation showed that it was nonessential for high-affinity E1/E2 complex formation. Thus, although the replication functions between the animal and the human papillomaviruses are well conserved, there are subtle differences in the DNA binding requirements for E1, which may portend mechanistic differences among the DNA replication systems of various papillomavirus types.

Replication-associated activities of purified human papillomavirus type 11 E1 helicase.

Replication of human papillomavirus type11 (HPV11) requires both the E1 and the E2 proteins. E1 is structurally and functionally similar to SV40 large T-antigen and is a DNA helicase/NTPase that binds to the origin of replication and initiates viral DNA replication. The biochemical characterization of HPV E1 is incompletely documented in the literature in part because of difficulties in expressing and purifying the protein. Herein, we report a method for the overexpression of full-length, untagged E1 (73.5 kDa) in baculovirus-infected Trichoplusia ni insect cells and the purification to homogeneity using a two-step procedure. The purified protein is a nonspecific NTPase that hydrolyzes ATP, dATP, UTP, or GTP equally well. Point mutations were made in the putative NTPase domain to verify that the activities observed were encoded by E1. Purified mutant D523N had negligible ATPase and helicase activities but retained DNA-binding activity. Sedimentation equilibrium ultracentrifugation and glycerol gradient centrifugation demonstrated that the wild-type protein is primarily a hexamer in its purified form. Secondary structure determination by circular dichroism revealed a large percentage of alpha-helical structure consistent with secondary structure predictions. These data define a fundamental set of biochemical and kinetic parameters for HPV E1 which are a critical prerequisite to future mechanistic studies of the enzyme.

Subunit affinities and stoichiometries of the human papillomavirus type 11 E1:E2:DNA complex.

The association between the papillomavirus E1 and E2 proteins is an important regulatory interaction, imparting coordinated control of viral transcription and replication. Using fluorescence polarization, we have characterized the interactions between HPV-11 E1, HPV-11 E2, and DNA in solution at equilibrium. For these studies, two double-stranded fluorescein-labeled oligonucleotides were prepared. The first fluorescent oligonucleotide, designated Fl-E2BS and containing a single E2 binding-site palindrome (ACCGN6CGGT), was used to determine the affinity of E2 for its DNA binding site. HPV-11 E2 bound Fl-E2BS with an apparent Kd of 0.84 nM. Binding was saturable and consistent with a single class of noninteracting sites. The second oligonucleotide, designated Fl-E1E2BS, contained both E1 and E2 sites in sequence derived directly from the HPV-11 origin of replication. Under titration conditions identical to those used for Fl-E2BS, the E2 protein exhibited reduced affinity for Fl-E1E2BS (Kd > 100 nM). E1 binding to Fl-E1E2BS was of very low affinity. Addition of excess HPV-11 E1 to Fl-E1E2BS lowered the dissociation constant for the E2:Fl-E1E2BS interaction to 2 nM. This effect was not dependent upon ATP or magnesium ion. Fluorescence polarization and other data suggest formation of a complex containing six E1 molecules and a single dimer of E2 bound to a single Fl-E1E2BS oligonucleotide; E2 dissociation from the final complex did not occur. In summary, physical interaction between E1 and E2 increases the DNA binding affinity of each. The role of this energy coupling may be to promote origin-specific binding of both E1 and E2 to DNA.

Dual inhibition of human type 4 phosphodiesterase isostates by (R, R)-(+/-)-methyl 3-acetyl-4-[3-(cyclopentyloxy)-4-methoxyphenyl]-3- methyl-1-pyrrolidinecarboxylate.

Purified recombinant human type 4 phosphodiesterase B2B (HSPDE4B2B) exists in both a low- and a high-affinity state that bind (R)-rolipram with Kd's of ca. 500 and 1 nM, respectively [Rocque, W. J., Tian, G., Wiseman, J. S., Holmes, W. D., Thompson, I. Z., Willard, D. H., Patel, I. R., Wisely, G. B., Clay, W. C., Kadwell, S. H., Hoffman, C. R., and Luther, M. A. (1997) Biochemistry 36, 14250-14261]. Since the tissue distribution of the two isostates may be significantly different, development of inhibitors that effectively inhibit both forms may be advantageous pharmacologically. In this study, enzyme inhibition and binding of HSPDE4B2B by (R, R)-(+/-)-methyl 3-acetyl-4-[3-(cyclopentyloxy)-4-methoxyphenyl]-3-methyl-1-pyrrolidin ecarboxylate (1), a novel inhibitor of phosphodiesterase 4 (PDE 4), were investigated. Binding experiments demonstrated high-affinity binding of 1 to HSPDE4B2B with a stoichiometry of 1:1. Inhibition of PDE activity showed only a single transition with an observed Ki similar to the apparent Kd determined by the binding experiments. Deletional mutants of HSPDE4B2B, which have been shown to bind (R)-rolipram with low affinity, were shown to interact with 1 with high affinity, indistinguishable from the results obtained with the full-length enzyme. Bound 1 was completely displaced by (R)-rolipram, and the displacement showed a biphasic transition that resembles the biphasic inhibition of HSPDE4B2B by (R)-rolipram. Theoretical analysis of the two transitions exemplified in the interaction of (R)-rolipram with HSPDE4B2B indicated that the two isostates were nonexchangeable. Phosphorylation at serines 487 and 489 on HSPDE4B2B had no effect on the stoichiometry of binding, the affinity for binding, or the inhibition of the enzyme by 1. These data further illustrate the presence of two isostates in PDE 4 as shown previously for (R)-rolipram binding and inhibition. In contrast to (R)-rolipram, where only one of the two isostates of PDE 4 binds with high affinity, 1 is a potent, dual inhibitor of both of the isostates of PDE 4. Kinetic and thermodynamic models describing the interactions between the nonexchangeable isostates of PDE 4 and its ligands are discussed.

Crystal structure of the catalytic domain of the human cell cycle control phosphatase, Cdc25A.

Cdc25 phosphatases activate the cell division kinases throughout the cell cycle. The 2.3 A structure of the human Cdc25A catalytic domain reveals a small alpha/beta domain with a fold unlike previously described phosphatase structures but identical to rhodanese, a sulfur-transfer protein. Only the active-site loop, containing the Cys-(X)5-Arg motif, shows similarity to the tyrosine phosphatases. In some crystals, the catalytic Cys-430 forms a disulfide bond with the invariant Cys-384, suggesting that Cdc25 may be self-inhibited during oxidative stress. Asp-383, previously proposed to be the general acid, instead serves a structural role, forming a conserved buried salt-bridge. We propose that Glu-431 may act as a general acid. Structure-based alignments suggest that the noncatalytic domain of the MAP kinase phosphatases will share this topology, as will ACR2, a eukaryotic arsenical resistance protein.

Human recombinant phosphodiesterase 4B2B binds (R)-rolipram at a single site with two affinities.

The interactions between (R)-rolipram and purified human recombinant low-Km, cAMP-specific phosphodiesterase (HSPDE4B2B) constructs were investigated using biochemical, kinetic, and biophysical approaches. The full-length protein (amino acids 1-564) and an N-terminal truncated protein (amino acids 81-564) exhibited high-affinity (R)-rolipram binding, whereas an N-terminal and C-terminal truncated protein (amino acids 152-528) lacked high-affinity (R)-rolipram binding. The 152-528 and 81-564 proteins had similar Km's and kcat/Km's and differed less than 4-fold compared with the 1-564 protein. (R)-Rolipram inhibition plots were biphasic for the 1-564 and 81-564 proteins and fit to two states, a high-affinity (Ki = 5-10 nM) state and a low-affinity (Ki = 200-400 nM) state, whereas the 152-528 protein fit to a single state (Ki = 350-400 nM). The stoichiometry for high-affinity binding using a filter binding assay was found to be <1 mol of (R)-rolipram per mole of 1-564 or 81-564 protein. Titration microcalorimetric studies revealed both a high-affinity state with a stoichiometry of 0.3 mol of (R)-rolipram per mole of protein and a low-affinity state with a stoichiometry of 0.6 mol of (R)-rolipram per mole of protein for the 81-564 protein. A single low-affinity state with a stoichiometry of 0.9 mol of (R)-rolipram per mole of protein was seen using the 152-528 protein. The data indicate that purified HSPDE4B2B 1-564 and 81-564 proteins contain a single binding site for (R)-rolipram and suggest that the proteins exist in two different states distinguishable by their affinity for (R)-rolipram. Furthermore, the high-affinity binding state of the protein requires amino acid residues at the N-terminus (81-151) of the protein and catalytic domain (152-528), whereas the low-affinity binding state only requires residues in the catalytic domain (152-528). Phosphorylation at residues 487 and 489 of the 81-564 protein does not appear to alter the substrate kinetics or the stoichiometry and binding affinity of (R)-rolipram.

Scanning alanine mutagenesis and de-peptidization of a Candida albicans myristoyl-CoA:protein N-myristoyltransferase octapeptide substrate reveals three elements critical for molecular recognition.

Candida albicans produces a single myristoyl-CoA:protein N-myristoyltransferase (Nmt) that is essential for its viability. An ADP-ribosylation factor (Arf) is included among the few cellular protein substrates of this enzyme. An octapeptide (GLYASKLS-NH2) derived from a N-terminal Arf sequence was used as the starting point to identify elements critical for recognition by the acyltransferases's peptide-binding site. In vitro kinetic studies, employing purified Nmt and a panel of peptides with single Ala substitutions at each position of GLYASKLS-NH2, established that its Gly1, Ser5, and Lys6 residues play predominant roles in binding. ALYASKLS-NH2 was found to be an inhibitor competitive for peptide (Ki = 15.3 +/- 6.4 microM) and noncompetitive for myristoyl-CoA (Ki = 31.2 +/- 0.7 microM). A survey of 26 derivatives of this inhibitor, representing (i) a complete alanine scan, (ii) progressive C-terminal truncations, and (iii) manipulation of the physical-chemical properties of its residues 1, 5, and 6, confirmed the important stereochemical requirements for the N-terminal amine, the beta-hydroxyl of Ser5, and the epsilon-amino group of Lys6. Remarkably, replacement of the the N-terminal tetrapeptide of ALYASKLS-NH2 with an 11-aminoundecanoyl group produced a competitive inhibitor, 11-aminoundecanoyl-SKLS-NH2, that was 38-fold more potent (Ki = 0.40 +/- 0.03 microM) than the starting octapeptide. Removing the primary amine (undecanoyl-SKLS-NH2), or replacing it with a methyl group (dodecanoyl-SKLS-NH2), resulted in 26- and 34-fold increases in IC50, confirming the important contribution of the amine to recognition. Removal of LeuSer from the C terminus (11-aminoundecanoyl-SK-NH2) yielded a competitive dipeptide inhibitor with a Ki (11.7 +/- 0.4 microM) equivalent to that of the starting octapeptide, ALYASKLS-NH2. Substitution of Ser with homoserine, cis-4-hydroxyproline, or tyrosine reduces potency by 3-70-fold, emphasizing the requirement for proper presentation of the hydroxyl group in the dipeptide inhibitor. Substituting D- for L-Lys decreases its inhibitory activity >100-fold, while deletion of the epsilon-amino group (Nle) or masking its charge (epsilon-N-acetyl-lysine) produces 4-7-fold attenuations. L-His, but not its D-isomer, can fully substitute for L-Lys, producing a competitive dipeptide inhibitor with similar potency (Ki = 11.9 +/- 1.0 microM). 11-Aminoundecanoyl-SK-NH2 and 11-aminoundecanoyl-SH-NH2 establish that a simple alkyl backbone can maintain an appropriate distance between three elements critical for recognition by the fungal enzyme's peptide-binding site: a simple omega-terminal amino group, a beta-hydroxyl, and an epsilon-amino group or an imidazole. These compounds contain one peptide bond and two chiral centers, suggesting that it may be feasible to incorporate these elements of recognition, or functionally equivalent mimics, into a fully de-peptidized Nmt inhibitor.

Detailed characterization of a purified type 4 phosphodiesterase, HSPDE4B2B: differentiation of high- and low-affinity (R)-rolipram binding.

We have overexpressed in a baculovirus expression system, and purified to > 95% homogeneity, milligram quantities of a human recombinant rolipram-sensitive cAMP phosphodiesterase, HSPDE4B2B (amino acid residues 81-564). The protein expression levels were approximately 8 mg of HSPDE4B2B (81-564) per liter of Sf9 cells. The Km of the purified enzyme for cAMP was 4 microM and the Ki for the Type 4 phosphodiesterase-specific inhibitor (R)-rolipram was 0.6 microM. The specific activity of the purified protein was 40 mumol/min/mg protein. A nonequilibrium filter binding assay revealed a high-affinity (R)-rolipram binding site on the purified enzyme with a Kd of 1.5 nM and a stoichiometry of 0.05-0.3 mol of (R)-rolipram per mol of HSPDE4B2B (81-564). Equilibrium dialysis experiments revealed a single binding constant of 140 nM with a stoichiometry of 0.75 mol of (R)-rolipram per mol of HSPDE4B2B (81-564). Size exclusion chromatography and analytical ultracentrifugation experiments suggest that the protein exists in multiple association states larger than a monomer. Proteolysis experiments revealed a 43-kDa fragment that contained catalytic and rolipram-inhibitable activities, but the fragment showed no high-affinity (R)-rolipram binding. Based on the proteolytic cleavage studies a 43-kDa protein was constructed, expressed, and purified. This protein, HSPDE4B2B (152-528), had Km and Vmax similar to those of the HSPDE4B2B (81-564) protein, but did not exhibit high-affinity (R)-rolipram binding. The protein did show low-affinity (R)-rolipram binding using the equilibrium binding assay. These results show that a low-affinity binding site for (R)-rolipram is solely contained within the catalytic domain of HSPDE4B2B, whereas high-affinity (R)-rolipram binding requires residues within the catalytic domain and residues flanking N- and/or C-terminal to the catalytic region.

Phosphorylation of a cAMP-specific phosphodiesterase (HSPDE4B2B) by mitogen-activated protein kinase.

A cAMP-specific phosphodiesterase, HSPDE4B2B, was found to be phosphorylated when expressed in Sf9 cells or yeast. Deletion of amino acids 81-151 and 529-564 had no effect on the phosphorylation of HSPDE4B2B. Mass spectrometric analysis of purified HSPDE4B2B(1-564). HSPDE4B2B(81-564) and HSPDE4B2B(152-528) showed that phosphorylation occurred predominantly on Ser487 and Ser489. The stoicheiometry of phosphorylation was 1.2:1 (Ser487:Ser487, 489). There was no evidence by MS for a non-phosphorylated form of HSPDE4B2B(81-564) or HSPDE4B2B(152-528) when expressed in Sf9 cells. There was no detectable phosphorylation of purified HSPDE4B2B(152-528) expressed in Escherichia coli. Radiolabelling experiments with 32P revealed that phosphorylation of HSPDE4B2B(152-528) expressed in Sf9 cells was abolished when Ser487 and Ser489 were mutated to alanines. Analysis of the amino acid sequence revealed that Ser487 and Ser489 of HSPDE4B2B conform to the consensus motifs for phosphorylation by mitogen-activated protein kinase (MAP kinase) and casein kinase II respectively. Kinetic experiments in vitro showed that MAP kinase-phosphorylated E.coli expressed and purified HSPDE4B2B(151-528) with a K(m) of 63 microM and a Vmax of 3.0 mumol/min per mg. In comparison, MAP kinase phosphorylated myelin basic protein with a Km of 26.0 microM and a Vmax of 5.5 mumol/min per mg under the same conditions. Using MS and mutational analysis we found that MAP kinase-phosphorylated E. coli expressed HSPDE4B2B(152-528) exclusively at Ser487. These results suggest that phosphodiesterases could contribute to the cross-talk between the cAMP and MAP kinase signalling pathways.

A comparative analysis of the kinetic mechanism and peptide substrate specificity of human and Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase.

Human myristoyl-CoA:protein N-myristoyltransferase (hNmt) catalyzes the transfer of myristate from CoA to the amino-terminal Gly residue of a number of cellular proteins involved in signal transduction pathways, to structural and nonstructural proteins encoded by retroviruses, hepadnaviruses, picornaviruses, and reoviruses, as well as to several transforming tyrosine kinases. hNmt has been purified 230-fold from an erythroleukemia cell line. The monomeric enzyme has no associated methionyl aminopeptidase activity. To determine the enzyme's kinetic mechanism, we examined the effect of covariation of subsaturating concentrations of myristoyl-CoA and peptide substrate on initial velocity. Double-reciprocal plots excluded a double displacement (ping-pong) mechanism. Product inhibition studies indicated that CoA was a noncompetitive inhibitor against myristoyl-CoA and a mixed-type inhibitor against peptide substrates. Together these results are consistent with a sequential ordered mechanism where, in a typical catalytic cycle, myristoyl-CoA binds to apoenzyme before peptide followed by release of the CoA and then myristoylpeptide products. This kinetic mechanism is identical to that described for Saccharomyces cerevisiae N-myristoyl-transferase (Nmt1p) and emphasizes the impact that regulation of myristoyl-CoA pool size and accessibility may have in modulating protein N-myristoylation in these two species. Comparative studies of the peptide substrate specificities of hNmt and Nmt1p using a panel of 12 octapeptides revealed distinct differences in their tolerance for amino acid substitutions at positions 3, 4, 7, and 8 of parental peptides derived from the amino-terminal sequences of known N-myristoyl-proteins. This finding contrasts with our recent observation that the acyl-CoA substrate specificities of hNmt and Nmt1p are highly conserved and suggests that these differences in peptide recognition provide an opportunity to develop species-specific enzyme inhibitors.

Use of photoactivatable peptide substrates of Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (Nmt1p) to characterize a myristoyl-CoA-Nmt1p-peptide ternary complex and to provide evidence for an ordered reaction mechanism.

Nmt1p (EC 2.3.1.97) catalyzes the transfer of myristate (C14:0) from coenzyme A to the N-terminal glycine residue of a variety of eukaryotic cellular and viral proteins. Our recent studies of the 455-amino acid Saccharomyces cerevisiae acyltransferase (Nmt1p) suggested that its mechanism of catalysis is ordered Bi Bi with myristoyl-CoA binding occurring prior to binding of peptide and release of CoA occurring prior to release of the myristoyl-peptide. The interaction between enzyme and peptide has now been examined in greater detail by using photoactivatable octapeptide substrates containing 125I-labeled azidosalicyclic acid attached via an amide bond to the gamma-amino group of a diaminobutyrate residue located at position 2 or the epsilon-amino group of a lysine residue located at position 8. The photopeptides can be specifically crosslinked to chymotryptic fragments of Nmt1p in the presence but not in the absence of a nonhydrolyzable myristoyl-CoA analog, S-(2-oxo)pentadecyl-CoA. Labeling of the chymotryptic fragments is markedly reduced when GLYASKLS, a high-affinity substrate derived from residues 2-9 of S. cerevisiae ADP-ribosylation factor 2, or ALYASKLS, a competitive inhibitor (for peptide), is added with the iodinated photopeptide. These findings suggest that peptide affinity for the acyl-CoA-Nmt1p binary complex is much greater than it is for apoNmt1p, consistent with the ordered Bi Bi mechanism ascribed to Nmt1p. Finally, automated sequential Edman degradation of these chymotryptic fragments suggests that the peptide binding domain of Nmt1p may be composed of elements from two protease-resistant domains, Arg42-Try219 and Thr220-Leu455.

Effects of pH on bacterial porin function.

Porin is a trimeric channel-forming protein in the outer membrane of Gram-negative bacteria. Functions of the porins OmpF, OmpC, and PhoE from Escherichia coli K12 were analyzed at various pHs. Preliminary results from bilayer lipid membrane and liposome swelling assays indicated that in vitro porin has at least two open-channel configurations with a small and a large size. The small channels were stabilized at low pH while the larger channels were detected under basic conditions. The size switch occurred over a very narrow range near neutral pH, and the two major open-channel configurations responded differently to variations in voltage. The presence of two or more pH-dependent substates of porin could explain the variability in pore diameter measured by others and suggests a more dynamic role for porin in the cell.

Kinetic and structural evidence for a sequential ordered Bi Bi mechanism of catalysis by Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase.

The mechanism of catalysis of Escherichia coli-derived Saccharomyces cerevisiae myristoyl-CoA: protein N-myristoyltransferase (NMT) has been characterized. Previous studies indicated that a high affinity reaction intermediate forms between NMT and myristoyl-CoA in the absence of a peptide substrate. This complex has been further characterized using S-(2-oxo)pentadecyl-CoA, a nonhydrolyzable myristoyl-CoA analog. Binding studies involving this analog, as well as myristoylpeptide and CoA, have indicated that the CoA moiety of the acyl substrate is retained in the acyl-NMT complex prior to peptide addition. These structural data, along with kinetic studies of myristoylpeptide and CoA product inhibition, indicate that the mechanism of catalysis of NMT is ordered Bi Bi, with myristoyl-CoA binding to NMT occurring prior to peptide binding and CoA release taking place before release of acyl peptide. Further analyses of the interactions between NMT, acyl peptide, and CoA demonstrate that NMT is able to deacylate a myristoylpeptide in the presence of CoA.

Structure and function of an OmpC deletion mutant porin from Escherichia coli K-12.

Escherichia coli K-12 strain RAM122 contains a mutation in the ompC gene that results in an eight amino acid deletion, delta 103-110, in the porin protein. Since this strain is capable of growing on maltodextrins in the absence of a functional lamB gene, the mutant protein is thought to have a larger channel size. The stability and structure/function properties of the mutant OmpC porin were investigated and compared to wild-type porin. Isolated unheated RAM122 porin was characterized as a trimer on sodium dodecyl sulfate-polyacrylamide gels. The RAM122 trimer was less stable to temperature when compared to the wild-type porin. In addition, the overall enthalpy for thermal denaturation was lower for the mutant than the wild-type porin as determined by using differential scanning microcalorimetry. Both the proteins' secondary structures, monitored by circular dichroism, were high in beta-sheet content, but the spectra were slightly different in their crossover points as well as their minima. When the proteins were reconstituted and channel activity was assayed by using a liposome swelling technique, the size-exclusion limit of the mutant porin was twice that of the wild-type porin. Conductance measurements across bilayer lipid membranes showed that the mutant porin was voltage gated at much lower membrane potentials, 50 and 75 mV, than the wild-type sample. The closing events of the mutant porin were predominantly of monomer size. The channels detected by using the mutant protein were larger in size than those measured for the wild-type porin monomer. These data suggest that the OmpC mutant porin has a channel size capable of allowing maltodextrins to enter and that this channel is highly voltage regulated.

Isolation and preliminary characterization of wild-type OmpC porin dimers from Escherichia coli K-12.

Escherichia coli K-12 strain PLB3255 contains a mutation in the ompF gene that results in a 15 amino acid deletion in the porin protein. The mutation (dex) appears to increase the OmpF channel size, allowing the PLB3255 cells to grow on maltodextrins in the absence of a functional maltoporin. Porin isolated from strain PLB3255, which contains a wild-type ompC gene, was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and shown to contain 50-60% trimer aggregates and 35-40% of a 50-kDa "dimer". When the porin isolate was heated to 100 degrees C and separated on SDS gels containing 8 M urea, both the trimer and the "dimer" were recovered in a single band at 36 kDa that corresponded in mobility to wild-type OmpC porin. An analysis of the temperature stability of the isolate showed that the OmpC "dimer" was less stable and denatured at 66 degrees C compared to 81 degrees C for the trimer. To separate these aggregates, the unheated porin was suspended in 30% SDS, applied to a Sephadex G-200 gel filtration column, and eluted with 0.5% sodium deoxycholate. Two peaks were recovered containing separated trimers and "dimers". Circular dichroism spectra of isolated dimers and trimers indicated similar amounts of beta-structure. The isolated dimers and trimers were reconstituted into artificial membranes. Electrical conductance across planar bilayer lipid membranes and liposome swelling assays showed that the two isolates had similar channel-forming activity. Four other ompF deletion mutants of the same phenotype were also shown to produce 50-kDa OmpC porin "dimers".(ABSTRACT TRUNCATED AT 250 WORDS)

Polycation binding to isolated lipopolysaccharide from antibiotic-hypersusceptible mutant strains of Escherichia coli.

Lipopolysaccharide (LPS) samples isolated from a parent and two antibiotic-hypersusceptible mutant strains of Escherichia coli were analyzed for polycation affinity and level of binding. Purified salts of the LPSs from the parent strain, UB1005, and from one of the mutant strains, DC1, bound similar amounts of sodium and magnesium, but the samples from the second mutant strain, DC2, had significantly greater amounts of counterions bound per phosphate than did the other two isolates. The 31P nuclear magnetic resonance spectra indicated that, compared with LPS from the parental strain, the sample from strain DC1 was similar but the DC2 sample contained fewer diphosphodiester and more diphosphomonoester groups. Motion within the lipid A head group regions of the magnesium salts of the three isolates was dramatically different, as revealed by an electron spin resonance probe. The binding of the cations to the LPS aggregates was measured by the displacement of this cationic spin probe from the LPS samples. The polycations polymyxin, gentamicin, and spermine displaced more probe from samples of the two mutant strains than from that of the parental strain. The sample from the most antibiotic-susceptible strain, DC2, had the highest affinity for all the polyvalent cations tested. The results indicate that antibiotic hypersusceptibility can result from at least two distinct alterations in LPS structure. The decrease in diphosphodiesters and increase in diphosphomonoesters in the LPS of the DC2 sample resulted in more acidic phosphate moieties and a more antibiotic-susceptible cell. In contrast, the alterations in the LPS of DC1 that resulted in antibiotic hypersusceptibility of the cell were not in the phosphate substituents. In both mutants, however, hypersusceptibility resulted in an alteration in LPS structure that increased the affinity of the molecules for polycations.