Alan Peterkofsky

The Escherichia coli glucose transporter enzyme IICB(Glc) recruits the global repressor Mlc.

In addition to effecting the catalysis of sugar uptake, the bacterial phosphoenolpyruvate:sugar phosphotransferase system regulates a variety of physiological processes. Exposure of cells to glucose can result in repression or induction of gene expression. While the mechanism for carbon catabolite repression by glucose was well documented, that for glucose induction was not clearly understood in Escherichia coli. Recently, glucose induction of several E.coli genes has been shown to be mediated by the global repressor Mlc. Here, we elucidate a general mechanism for glucose induction of gene expression in E.coli, revealing a novel type of regulatory circuit for gene expression mediated by the phosphorylation state‐dependent interaction of a membrane‐bound protein with a repressor. The dephospho‐form of enzyme IICBGlc, but not its phospho‐form, interacts directly with Mlc and induces transcription of Mlc‐regulated genes by displacing Mlc from its target sequences. Therefore, the glucose induction of Mlc‐regulated genes is caused by dephosphorylation of the membrane‐bound transporter enzyme IICBGlc, which directly recruits Mlc to derepress its regulon.

Mechanistic and physiological consequences of HPr(ser) phosphorylation on the activities of the phosphoenolpyruvate:sugar phosphotransferase system in gram-positive bacteria: studies with site-specific mutants of HPr.

The bacterial phosphotransferase system (PTS) catalyzes the transport and phosphorylation of its sugar substrates. The protein‐kinase‐catalyzed phosphorylation of serine 46 in the phosphocarrier protein, HPr, inhibits PTS activity, but neither the mechanism of this inhibition nor its physiological significance is known. Site‐specific HPr mutants were constructed in which serine 46 was replaced by alanine (S46A), threonine (S46T), tyrosine (S46Y) or aspartate (S46D). The purified S46D protein exhibited markedly lower Vmax and higher Km values than the wild‐type, S46T or S46A protein for the phosphoryl transfer reactions involving HPr(His approximately P). Interactions of HPr with the enzymes catalyzing phosphoryl transfer to and from HPr regulated the kinase‐catalyzed reaction. These results establish the inhibitory effect of a negative charge at position 46 on PTS‐mediated phosphoryl transfer and suggest that HPr is phosphorylated on both histidyl and seryl residues by enzymes that recognize its tertiary rather than its primary structure. In vivo studies showed that a negative charge on residue 46 of HPr strongly inhibits PTS‐mediated sugar uptake, but that competition of two PTS permeases for HPr(His approximately P) is quantitatively more important to the regulation of PTS function than serine 46 phosphorylation.

The first step in sugar transport: crystal structure of the amino terminal domain of enzyme I of the E. coli PEP: sugar phosphotransferase system and a model of the phosphotransfer complex with HPr

BACKGROUND The bacterial phosphoenolpyruvate (PEP): sugar phosphotransferase system (PTS) transports exogenous hexose sugars through the membrane and tightly couples transport with phosphoryl transfer from PEP to the sugar via several phosphoprotein intermediates. The phosphate group is first transferred to enzyme I, second to the histidine-containing phosphocarrier protein HPr, and then to one of a number of sugar-specific enzymes II. The structures of several HPrs and enzymes IIA are known. Here we report the structure of the N-terminal half of enzyme I from Escherichia coli (EIN). RESULTS The crystal structure of EIN (MW approximately 30 kDa) has been determined and refined at 2.5 A resolution. It has two distinct structural subdomains; one contains four alpha helices arranged as two hairpins in a claw-like conformation. The other consists of a beta sandwich containing a three-stranded antiparallel beta sheet and a four-stranded parallel beta sheet, together with three short alpha helices. Plausible models of complexes between EIN and HPr can be made without assuming major structural changes in either protein. CONCLUSIONS The alpha/beta subdomain of EIN is topologically similar to the phosphohistidine domain of the enzyme pyruvate phosphate dikinase, which is phosphorylated by PEP on a histidyl residue but does not interact with HPr. It is therefore likely that features of this subdomain are important in the autophosphorylation of enzyme I. The helical subdomain of EIN is not found in pyruvate phosphate dikinase; this subdomain is therefore more likely to be involved in phosphoryl transfer to HPr.

Escherichia coli enzyme IIANtr regulates the K+ transporter TrkA

The maintenance of ionic homeostasis in response to changes in the environment is essential for all living cells. Although there are still many important questions concerning the role of the major monovalent cation K+, cytoplasmic K+ in bacteria is required for diverse processes. Here, we show that enzyme IIANtr (EIIANtr) of the nitrogen-metabolic phosphotransferase system interacts with and regulates the Escherichia coli K+ transporter TrkA. Previously we reported that an E. coli K-12 mutant in the ptsN gene encoding EIIANtr was extremely sensitive to growth inhibition by leucine or leucine-containing peptides (LCPs). This sensitivity was due to the requirement of the dephosphorylated form of EIIANtr for the derepression of ilvBN expression. Whereas the ptsN mutant is extremely sensitive to LCPs, a ptsN trkA double mutant is as resistant as WT. Furthermore, the sensitivity of the ptsN mutant to LCPs decreases as the K+ level in culture media is lowered. We demonstrate that dephosphorylated EIIANtr, but not its phosphorylated form, forms a tight complex with TrkA that inhibits the accumulation of high intracellular concentrations of K+. High cellular K+ levels in a ptsN mutant promote the sensitivity of E. coli K-12 to leucine or LCPs by inhibiting both the expression of ilvBN and the activity of its gene products. Here, we delineate the similarity of regulatory mechanisms for the paralogous carbon and nitrogen phosphotransferase systems. Dephosphorylated EIIAGlc regulates a variety of transport systems for carbon sources, whereas dephosphorylated EIIANtr regulates the transport system for K+, which has global effects related to nitrogen metabolism.

Solution structure of the phosphoryl transfer complex between the signal transducing proteins HPr and IIA(glucose) of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system.

The solution structure of the second protein–protein complex of the Escherichia coli phosphoenolpyruvate: sugar phosphotransferase system, that between histidine‐containing phosphocarrier protein (HPr) and glucose‐specific enzyme IIAGlucose (IIAGlc), has been determined by NMR spectroscopy, including the use of dipolar couplings to provide long‐range orientational information and newly developed rigid body minimization and constrained/restrained simulated annealing methods. A protruding convex surface on HPr interacts with a complementary concave depression on IIAGlc. Both binding surfaces comprise a central hydrophobic core region surrounded by a ring of polar and charged residues, positive for HPr and negative for IIAGlc. Formation of the unphosphorylated complex, as well as the phosphorylated transition state, involves little or no change in the protein backbones, but there are conformational rearrangements of the interfacial side chains. Both HPr and IIAGlc recognize a variety of structurally diverse proteins. Comparisons with the structures of the enzyme I–HPr and IIAGlc–glycerol kinase complexes reveal how similar binding surfaces can be formed with underlying backbone scaffolds that are structurally dissimilar and highlight the role of redundancy and side chain conformational plasticity.

Thermoregulation in rats: opposing effects of thyrotropin releasing hormone and its metabolite histidyl-proline diketopiperazine.

Abstract Intraventricular administration of histidyl-proline diketopiperazine to rats produces a dose-dependent hypothermia at 4° or 24°, but not at 31°. At 4°, administration of thyrotropin releasing hormone elicits a dose-dependent hypothermia up to 0.1 μmole/kg which is not evoked at higher doses. At 24°, thyrotropin releasing hormone administration results in no change in core temperature, whereas it induces hyperthermia at 31°. At 4°, thyrotropin releasing hormone antagonizes and thyrotropin releasing hormone antiserum potentiates the hypothermic effect of histidyl-proline diketopiperazine, suggesting opposing actions of thyrotropin releasing hormone and histidyl-proline diketopiperazine on thermoregulation.

High affinity binding and allosteric regulation of Escherichia coli glycogen phosphorylase by the histidine phosphocarrier protein, HPr.

The histidine phosphocarrier protein (HPr) is an essential element in sugar transport by the bacterial phosphoenolpyruvate:sugar phosphotransferase system. Ligand fishing, using surface plasmon resonance, was used to show the binding of HPr to a nonphosphotransferase protein in extracts ofEscherichia coli; the protein was subsequently identified as glycogen phosphorylase (GP). The high affinity (association constant ∼108 m −1), species-specific interaction was also demonstrated in electrophoretic mobility shift experiments by polyacrylamide gel electrophoresis. Equilibrium ultracentrifugation analysis indicates that HPr allosterically regulates the oligomeric state of glycogen phosphorylase. HPr binding increases GP activity to 250% of the level in control assays. Kinetic analysis of coupled enzyme assays shows that the binding of HPr to GP causes a decrease in the K m for glycogen and an increase in the V max for phosphate, indicating a mixed type activation. The stimulatory effect of E. coliHPr on E. coli GP activity is species-specific, and the unphosphorylated form of HPr activates GP more than does the phosphorylated form. Replacement of specific amino acids in HPr results in reduced GP activation; HPr residues Arg-17, Lys-24, Lys-27, Lys-40, Ser-46, Gln-51, and Lys-72 were established to be important. This novel mechanism for the regulation of GP provides the first evidence directly linking E. coli HPr to the regulation of carbohydrate metabolism.

Effect of in vitro methylation on the chromatographic and coding properties of methyl-deficient leucine transfer RNA

Abstract Whereas normal Escherichia coli leucyl-tRNA § , which contains several subspecies, responds equally efficiently in codon recognition studies with poly UG or poly UC, leucyl-tRNA preparations containing methyl-deficient tRNALeu react more efficiently with poly UC than poly UG ( Capra & Peterkofsky, 1966 ). The increased reactivity with poly UC in these preparations is primarily associated with a chromatographic species of tRNALeu singular to methyl-deficient tRNA ( Peterkofsky, 1964 ). The interpretation of these observations has, until now, been ambiguous. They could indicate that methyl-deficient tRNA preparations are enriched with those subspecies of tRNALeu which normally interact with poly UC. Alternatively, it might be suggested that species of tRNALeu which normally recognize poly UG exhibit a changed codon response to poly UC when they are methyl-deficient. The studies herein support the latter hypothesis. Whereas in vitro methylation of the subspecies of tRNALeu corresponding to those found in normally methylated preparations has no effect on their properties, such a treatment of subspecies of tRNALeu restricted to methyl-deficient tRNA leads to a change in their Chromatographic and coding properties.

Solution structure of the phosphoryl transfer complex between the cytoplasmic A domain of the mannitol transporter IIMannitol and HPr of the Escherichia coli phosphotransferase system.

The solution structure of the complex between the cytoplasmic A domain (IIAMtl) of the mannitol transporter IIMannitol and the histidine-containing phosphocarrier protein (HPr) of theEscherichia coli phosphotransferase system has been solved by NMR, including the use of conjoined rigid body/torsion angle dynamics, and residual dipolar couplings, coupled with cross-validation, to permit accurate orientation of the two proteins. A convex surface on HPr, formed by helices 1 and 2, interacts with a complementary concave depression on the surface of IIAMtlformed by helix 3, portions of helices 2 and 4, and β-strands 2 and 3. The majority of intermolecular contacts are hydrophobic, with a small number of electrostatic interactions at the periphery of the interface. The active site histidines, His-15 of HPr and His-65 of IIAMtl, are in close spatial proximity, and a pentacoordinate phosphoryl transition state can be readily accommodated with no change in protein-protein orientation and only minimal perturbations of the backbone immediately adjacent to the histidines. Comparison with two previously solved structures of complexes of HPr with partner proteins of the phosphotransferase system, the N-terminal domain of enzyme I (EIN) and enzyme IIAGlucose(IIAGlc), reveals a number of common features despite the fact that EIN, IIAGlc, and IIAMtl bear no structural resemblance to one another. Thus, entirely different underlying structural elements can form binding surfaces for HPr that are similar in terms of both shape and residue composition. These structural comparisons illustrate the roles of surface and residue complementarity, redundancy, incremental build-up of specificity and conformational side chain plasticity in the formation of transient specific protein-protein complexes in signal transduction pathways.

Identification of Novel Human Immunodeficiency Virus Type 1-Inhibitory Peptides Based on the Antimicrobial Peptide Database

ABSTRACT To identify novel anti-HIV-1 peptides based on the antimicrobial peptide database (APD; http://aps.unmc.edu/AP/main.php ), we have screened 30 candidates and found 11 peptides with 50% effective concentrations (EC50) of <10 μM and therapeutic indices (TI) of up to 17. Furthermore, among the eight peptides (with identical amino acid compositions but different sequences) generated by shuffling the sequence of an aurein 1.2 analog, two had a TI twice that of the original sequence. Because antiviral peptides in the database have an arginine/lysine (R/K) ratio of >1, increases in the Arg contents of amphibian maximin H5 and dermaseptin S9 peptides and the database-derived GLK-19 peptide improved the TIs. These examples demonstrate that the APD is a rich resource and a useful tool for developing novel HIV-1-inhibitory peptides.