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Proteins interact with one another across a broad spectrum of affinities. Our understanding of the low end of this spectrum, as characterized by millimolar dissociation constants, relies on a handful of cases in which weak encounters have experimentally been identified. These weak interactions away from the specific target binding site can lead toward a higher-affinity complex. Recently, we detected weak encounters between two paralogous phosphotransferase pathways of Escherichia coli, which regulate various metabolic processes and stress responses. In addition to encounters that are known to occur between cognate proteins, i.e., those that can exchange phosphate groups with each other, surprisingly, encounters involving noncognates were also observed. It is not clear whether these “futile” encounters have a cooperative or competitive role. Using agent-based simulations, we find that the encounter complexes can be cooperative or competitive so as to increase or lower the effective binding affinity of the specific complex under different circumstances. This finding invites further questions into how organisms might exploit such low affinities to connect their signaling components.

Paralogous enzymes arise from gene duplication events that confer a novel function, although it is unclear how cross-reaction between the original and duplicate protein interaction network is minimized. We investigated HPr:EIsugar and NPr:EINtr, the initial complexes of paralogous phosphorylation cascades involved in sugar import and nitrogen regulation in bacteria, respectively. Although the HPr:EIsugar interaction has been well characterized, involving multiple complexes and transient interactions, the exact nature of the NPr:EINtr complex was unknown. We set out to identify the key features of the interaction by performing binding assays and elucidating the structure of NPr in complex with the phosphorylation domain of EINtr (EINNtr), using a hybrid approach involving X-ray, homology, and sparse nuclear magnetic resonance. We found that the overall fold and active-site structure of the two complexes are conserved in order to maintain productive phosphorylation, however, the interface surface potential differs between the two complexes, which prevents cross-reaction.

While many eukaryotic and some prokaryotic proteins show a phosphorylation-dependent mobility shift (PDMS) on SDS-PAGE, the molecular mechanism for this phenomenon had not been elucidated. We have recently shown that the distribution of negatively charged amino acids around the phosphorylation site is important for the PDMS of some proteins. Here, we show that replacement of the phosphorylation site with a negatively charged amino acid results in a similar degree of the mobility shift of a protein as phosphorylation, indicating that the PDMS is due to the introduction of a negative charge by phosphorylation. Compared with a protein showing no shift, one showing a retarded mobility on SDS-PAGE had a decreased capacity for SDS binding. The elucidation of the consensus sequence (ΘX1-3ΘX1-3Θ, where Θ corresponds to an acidic function) for a PDMS suggests a general strategy for mutagenizing a phosphorylatable protein resulting in a PDMS.

Summary In addition to the phosphoenolpyruvate:sugar phosphotransferase system (sugar PTS), most proteobacteria possess a paralogous system (nitrogen phosphotransferase system, PTSNtr). The first proteins in both pathways are enzymes (enzyme Isugar and enzyme INtr) that can be autophosphorylated by phosphoenolpyruvate. The most striking difference between enzyme Isugar and enzyme INtr is the presence of a GAF domain at the N-terminus of enzyme INtr. Since the PTSNtr was identified in 1995, it has been implicated in a variety of cellular processes in many proteobacteria and many of these regulations have been shown to be dependent on the phosphorylation state of PTSNtr components. However, there has been little evidence that any component of this so-called PTSNtr is directly involved in nitrogen metabolism. Moreover, a signal regulating the phosphorylation state of the PTSNtr had not been uncovered. Here, we demonstrate that glutamine and α-ketoglutarate, the canonical signals of nitrogen availability, reciprocally regulate the phosphorylation state of the PTSNtr by direct effects on enzyme INtr autophosphorylation and the GAF signal transduction domain is necessary for the regulation of enzyme INtr activity by the two signal molecules. Taken together, our results suggest that the PTSNtr senses nitrogen availability.

Bacterial phosphoenolpyruvate-dependent phosphotransferase systems (PTS) play multiple roles in addition to sugar transport. Recent studies revealed that enzyme IIA(Ntr) of the nitrogen PTS regulates the intracellular concentration of K(+) by direct interaction with TrkA and KdpD. In this study, we show that dephosphorylated NPr of the nitrogen PTS interacts with Escherichia coli LpxD which catalyzes biosynthesis of lipid A of the lipopolysaccharide (LPS) layer. Mutations in lipid A biosynthetic genes such as lpxD are known to confer hypersensitivity to hydrophobic antibiotics such as rifampin; a ptsO (encoding NPr) deletion mutant showed increased resistance to rifampin and increased LPS biosynthesis. Taken together, our data suggest that unphosphorylated NPr decreases lipid A biosynthesis by inhibiting LpxD activity.

An Escherichia coli mutant devoid of enzyme IIA(Ntr) (EIIA(Ntr) ) of the nitrogen PTS is extremely sensitive to leucine-containing peptides due to decreased expression of acetohydroxy acid synthase. This decreased expression is due to defective potassium homeostasis. We further elucidate here the mechanism for regulation of gene expression by the intracellular level of K(+) . The leucine hypersensitivity of a ptsN (encoding EIIA(Ntr) ) mutant was suppressed by deleting rpoS, encoding the stationary phase σ factor. Despite intracellular levels of sigma factors comparable to the wild-type strain, most of the genes downregulated in a ptsN mutant are controlled by σ(70) , while all the upregulated genes are controlled by σ(S) , implying that the balance of sigma activities is modified by ptsN deletion. This change of sigma factor activities in the deletion mutant was found to be due to increased levels of K(+) . In vitro transcription assays demonstrated that a σ(70) controlled gene and a σ(S) controlled gene were differentially affected by potassium concentration. Biochemical studies revealed that K(+) is responsible for sigma factor competition by differentially influencing the binding of σ(70) and σ(S) to core RNA polymerase. Taken together, the data suggest that EIIA(Ntr) controls sigma factor selectivity by regulating the intracellular K(+) level.

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 (EC 50 ) 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.

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 IIA Ntr (EIIA Ntr ) 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 EIIA Ntr 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 EIIA Ntr 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 EIIA Ntr , 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 EIIA Glc regulates a variety of transport systems for carbon sources, whereas dephosphorylated EIIA Ntr regulates the transport system for K + , which has global effects related to nitrogen metabolism.

In a variety of bacteria, the phosphotransferase protein IIA(Glc) plays a key regulatory role in catabolite repression in addition to its role in the vectorial phosphorylation of glucose catalyzed by the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). The lactose permease (LacY) of Escherichia coli catalyzes stoichiometric symport of a galactoside with an H(+), using a mechanism in which sugar- and H(+)-binding sites become alternatively accessible to either side of the membrane. Both the expression (via regulation of cAMP levels) and the activity of LacY are subject to regulation by IIA(Glc) (inducer exclusion). Here we report the thermodynamic features of the IIA(Glc)-LacY interaction as measured by isothermal titration calorimetry (ITC). The studies show that IIA(Glc) binds to LacY with a Kd of about 5 μM and a stoichiometry of unity and that binding is driven by solvation entropy and opposed by enthalpy. Upon IIA(Glc) binding, the conformational entropy of LacY is restrained, which leads to a significant decrease in sugar affinity. By suppressing conformational dynamics, IIA(Glc) blocks inducer entry into cells and favors constitutive glucose uptake and utilization. Furthermore, the studies support the notion that sugar binding involves an induced-fit mechanism that is inhibited by IIA(Glc) binding. The precise mechanism of the inhibition of LacY by IIA(Glc) elucidated by ITC differs from the inhibition of melibiose permease (MelB), supporting the idea that permeases can differ in their thermodynamic response to binding IIA(Glc).

Bacterial transport of many sugars, coupled to their phosphorylation, is carried out by the phosphoenolpyruvate (PEP):sugar phosphotransferase system and involves five phosphoryl group transfer reactions. Sugar translocation initiates with the Mg 2+ -dependent phosphorylation of enzyme I (EI) by PEP. Crystals of Escherichia coli EI were obtained by mixing the protein with Mg 2+ and PEP, followed by oxalate, an EI inhibitor. The crystal structure reveals a dimeric protein where each subunit comprises three domains: a domain that binds the partner PEP:sugar phosphotransferase system protein, HPr; a domain that carries the phosphorylated histidine residue, His-189; and a PEP-binding domain. The PEP-binding site is occupied by Mg 2+ and oxalate, and the phosphorylated His-189 is in-line for phosphotransfer to/from the ligand. Thus, the structure represents an enzyme intermediate just after phosphotransfer from PEP and before a conformational transition that brings His-189∼P in proximity to the phosphoryl group acceptor, His-15 of HPr. A model of this conformational transition is proposed whereby swiveling around an α-helical linker disengages the His domain from the PEP-binding domain. Assuming that HPr binds to the HPr-binding domain as observed by NMR spectroscopy of an EI fragment, a rotation around two linker segments orients the His domain relative to the HPr-binding domain so that His-189∼P and His-15 are appropriately stationed for an in-line phosphotransfer reaction.

A widely accepted model for catabolite repression posits that phospho-IIAGlc of the bacterial phosphotransferase system activates adenylyl cyclase (AC) activity. For many years, attempts to observe such regulatory properties of AC in vitro have been unsuccessful. To further study the regulation, AC was produced fused to the transmembrane segments of the serine chemoreceptor Tsr. Cells harboring Tsr-AC and normal AC, expressed from the cya promoter on a low copy number vector, exhibit similar behavior with respect to elevation of cAMP levels resulting from deletion of crp, expressing the catabolite regulatory protein. Membrane-bound Tsr-AC exhibits activity comparable with the native form of AC. Tsr-AC binds IIAGlc specifically, regardless of its phosphorylation state, but not the two general phosphotransferase system proteins, enzyme I and HPr; IIAGlc binding is localized to the C-terminal region of AC. Binding to membranes of either dephospho- or phospho-IIAGlc has no effect on AC activity. However, in the presence of an Escherichia coli extract, P-IIAGlc, but not IIAGlc, stimulates AC activity. Based on these findings of a direct interaction of IIAGlc with AC, but activity regulation only in the presence of E. coli extract, a revised model for AC activity regulation is proposed.

Summary While the proteins of the phosphoenolpyruvate:carbohydrate phosphotransferase system (carbohydrate PTS) have been shown to regulate numerous targets, little such information is available for the nitrogen-metabolic phosphotransferase system (nitrogen-metabolic PTS). To elucidate the physiological role of the nitrogen-metabolic PTS, we carried out phenotype microarray (PM) analysis with Escherichia coli K-12 strain MG1655 deleted for the ptsP gene encoding the first enzyme of the nitrogen-metabolic PTS. Together with the PM data, growth studies revealed that a ptsN (encoding enzyme IIANtr) mutant became extremely sensitive to leucine-containing peptides (LCPs), while both ptsP (encoding enzyme INtr) and ptsO (encoding NPr) mutants were more resistant than wild type. The toxicity of LCPs was found to be due to leucine and the dephospho-form of enzyme IIANtr was found to be necessary to neutralize leucine toxicity. Further studies showed that the dephospho-form of enzyme IIANtr is required for derepression of the ilvBN operon encoding acetohydroxy acid synthase I catalysing the first step common to the biosynthesis of the branched-chain amino acids.

The solution structure of the 48-kDa IIA(Man)-HPr complex of the mannose branch of the Escherichia coli phosphotransferase system has been solved by NMR using conjoined rigid body/torsion angle-simulated annealing on the basis of intermolecular nuclear Overhauser enhancement data and residual dipolar couplings. IIA(Man) is dimeric and has two symmetrically related binding sites per dimer for HPr. A convex surface on HPr, formed primarily by helices 1 and 2, interacts with a deep groove at the interface of the two subunits of IIA(Man). The interaction surface on IIA(Man) is predominantly helical, comprising helix 3 from the subunit that bears the active site His-10 and helices 1, 4, and 5 from the other subunit. The total buried accessible surface area at the protein-protein interface is 1450 A(2). The binding sites on the two proteins are complementary in terms of shape and distribution of hydrophobic, hydrophilic, and charged residues. The active site histidines, His-10 of IIA(Man) and His-15 (italics indicate HPr residues) of HPr, are in close proximity. An associative transition state involving a pentacoordinate phosphoryl group with trigonal bipyramidal geometry bonded to the N-epsilon2 atom of His-10 and the N-delta1 atom of His-15 can be readily formed with negligible displacement in the backbone coordinates of the residues immediately adjacent to the active site histidines. Comparing the structures of complexes of HPr with three other structurally unrelated phosphotransferase system proteins, enzymes I, IIA(glucose), and IIA(mannitol), reveals a number of common features that provide a molecular basis for understanding how HPr specifically recognizes a wide range of diverse proteins.

The bacterial phosphoenolpyruvate:sugar phosphotransferase system regulates a variety of physiological processes as well as effecting sugar transport. The crr gene product (enzyme IIA(Glc) (IIA(Glc))) mediates some of these regulatory phenomena. In this report, we characterize a novel IIA(Glc)-binding protein from Escherichia coli extracts, discovered using ligand-fishing with surface plasmon resonance spectroscopy. This protein, which we named FrsA (fermentation/respiration switch protein), is the 47-kDa product of the yafA gene, previously denoted as "function unknown." FrsA forms a 1:1 complex specifically with the unphosphorylated form of IIA(Glc), with the highest affinity of any protein thus far shown to interact with IIA(Glc). Orthologs of FrsA have been found to exist only in facultative anaerobes belonging to the gamma-proteobacterial group. Disruption of frsA increased cellular respiration on several sugars including glucose, while increased FrsA expression resulted in an increased fermentation rate on these sugars with the concomitant accumulation of mixed-acid fermentation products. These results suggest that IIA(Glc) regulates the flux between respiration and fermentation pathways by sensing the available sugar species via a phosphorylation state-dependent interaction with FrsA.

Enzyme IIA G l c of the Escherichia coli phosphoenolpyruvate:glucose phosphotransferase system plays a direct role in regulating inducible transport systems. Dephosphorylated IIA G l c binds directly to lactose permease in a reaction that requires binding of a galactosidic substrate. A double-Cys mutation (Ile 129 → Cys/Lys131 → Cys) was introduced into helix IV of the permease near the IIA G l c binding site in cytoplasmic loop IV/V and in the vicinity of the galactoside binding site at the interface of helices IV, V, and VIII. The mutant no longer requires galactoside for IIA G l c binding as demonstrated by both a [ 1 2 5 I]IIA G l c binding assay and a newly developed fluorescence anisotropy assay. Further characterization of the mutant shows that it binds substrate with high affinity, but is almost completely defective in all modes of translocation across the cytoplasmic membrane. The data are consistent with the interpretation that the double mutant is locked in an inward-facing conformation.

The solution structure of the final phosphoryl transfer complex in the glucose-specific arm of the Escherichia coli phosphotransferase system, between enzyme IIAGlucose (IIAGlc) and the cytoplasmic B domain (IIBGlc) of the glucose transporter IICBGlc, has been solved by NMR. The interface (approximately 1200-A2 buried surface) is formed by the interaction of a concave depression on IIAGlc with a convex protrusion on IIBGlc. The phosphoryl donor and acceptor residues, His-90 of IIAGlc and Cys-35 of IIBGlc (residues of IIBGlc are denoted in italics) are in close proximity and buried at the center of the interface. Cys-35 is primed for nucleophilic attack on the phosphorus atom by stabilization of the thiolate anion (pKa approximately 6.5) through intramolecular hydrogen bonding interactions with several adjacent backbone amide groups. Hydrophobic intermolecular contacts are supplemented by peripheral electrostatic interactions involving an alternating distribution of positively and negatively charged residues on the interaction surfaces of both proteins. Salt bridges between the Asp-38/Asp-94 pair of IIAGlc and the Arg-38/Arg-40 pair of IIBGlc neutralize the accumulation of negative charge in the vicinity of both the Sgamma atom of Cys-35 and the phosphoryl group in the complex. A pentacoordinate phosphoryl transition state is readily accommodated without any change in backbone conformation, and the structure of the complex accounts for the preferred directionality of phosphoryl transfer between IIAGlc and IIBGlc. The structures of IIAGlc.IIBGlc and the two upstream complexes of the glucose phosphotransferase system (EI.HPr and IIAGlc.HPr) reveal a cascade in which highly overlapping binding sites on HPr and IIAGlc recognize structurally diverse proteins.

The N-terminal domain of enzyme IIA(Glc) of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system confers amphitropism to the protein, allowing IIA(Glc) to shuttle between the cytoplasm and the membrane. To further understand this amphitropic protein, we have elucidated, by NMR spectroscopy, the solution structure of a synthetic peptide corresponding to the N-terminal domain of IIA(Glc). In water, this peptide is predominantly disordered, consistent with previous data obtained in the absence of membranes. In detergent micelles of dihexanoylphosphatidylglycerol (DHPG) or sodium dodecylsulfate (SDS), however, residues Phe 3-Val 10 of the peptide adopt a helical conformation in the ensemble of structures calculated on the basis of NOE-derived distance restraints. The root mean square deviations for superimposing the backbone atoms of the helical region are 0.18 A in DHPG and 0.22 A in SDS. The structure, chemical shifts, and spin-spin coupling constants all indicate that, of the four lysines in the N-terminal domain of IIA(Glc), only Lys 5 and Lys 7 in the amphipathic helical region interact with DHPG. In addition, the peptide-detergent interactions were investigated using intermolecular NOESY experiments. The aliphatic chains of anionic detergents DHPG, SDS, and 2,2-dimethyl-2-silapentane-5-sulfonate sodium salt (DSS) all showed intermolecular NOE cross-peaks to the peptide, providing direct evidence for the putative membrane anchor of IIA(Glc) in binding to the membrane-mimicking micelles.

Pairwise interactions are intuitive to our understanding on protein-ligand binding; however in vivo this is rarely true. Most intracellular proteins operate in a highly cooperative manner to perform tasks ranging from metabolic turnover to intricate signaling regulation. In some cases, one substrate needs to simultaneously interact with more than one binding partner to carry out faithful signal transductions. While one of these binding partners is the determinant of such signal transduction, it can share very similar tertiary structure with the other but might differ in functional role and abundance. Motivated by this observation, we explore the physical consequences of the mere steric presence of a non-specific ligand, the “competitor”, crowding the surface of a “target” ligand. The specific interaction occurs between the protein and the “target” ligand, which explores the same surface as well, albeit for its unique binding site. A simple lattice model incorporating these elements along with the natural rules of exclusion and hopping reveals the regimes for when recruitment (turnover) or residence (transition state stabilization) are favored. Exploration of the search dynamics of the two ligands along the protein surface provides further insight.

The bacterial PEP:sugar phosphotransferase system couples the phosphorylation and translocation of specific sugars across the membrane. The activity of the first protein in this pathway, enzyme I (EI), is regulated by a monomer-dimer equilibrium where a Mg(2+)-dependent autophosphorylation by PEP requires the dimer. Dimerization constants for dephospho- and phospho-EI and inactive mutants EI(H189E) and EI(H189A) (in which Glu or Ala is substituted for the active site His189) have been measured under a variety of conditions by sedimentation equilibrium at pH 7.5 and 4 and 20 degrees C. Concurrently, thermal unfolding of these forms of EI has been monitored by differential scanning calorimetry and by changes in the intrinsic tryptophanyl residue fluorescence. Phosphorylated EI and EI(H189E) have 10-fold increased dimerization constants [ approximately 2 x 10(6) (M monomer)(-1)] compared to those of dephospho-EI and EI(H189A) at 20 degrees C. Dimerization is strongly promoted by 1 mM PEP with 2 mM MgCl(2) [K(A)' > or = 10(8) M(-1) at 4 or 20 degrees C], as demonstrated with EI(H189A) which cannot undergo autophosphorylation. Together, 1 mM PEP and 2 mM Mg(2+) also markedly stabilize and couple the unfolding of C- and N-terminal domains of EI(H189A), increasing the transition temperature (T(m)) for unfolding the C-terminal domain by approximately 18 degrees C and that for the N-terminal domain by approximately 9 degrees C to T(max) congruent with 63 degrees C, giving a value of K(D)' congruent with 3 microM PEP at 45 degrees C. PEP alone also promotes the dimerization of EI(H189A) but only increases T(m) approximately 5 degrees C for C-terminal domain unfolding without affecting N-terminal domain unfolding, giving an estimated value of K(D)' congruent with 0.2 mM for PEP dissociation in the absence of Mg(2+) at 45 degrees C. In contrast, the dimerization constant of phospho-EI at 20 degrees C is the same in the absence and presence of 5 mM PEP and 2 mM MgCl(2). Thus, the separation of substrate binding effects from those of phosphorylation by studies with the inactive EI(H189A) has shown that intracellular concentrations of PEP and Mg(2+) are important determinants of both the conformational stability and dimerization of dephospho-EI.

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 IICB(Glc), 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 IICB(Glc), which directly recruits Mlc to derepress its regulon.

Enzyme IIA(Glucose) (IIA(Glc)) is a signal-transducing protein in the phosphotransferase system of Escherichia coli. Structural studies of free IIA(Glc) and the HPr-IIA(Glc) complex have shown that IIA(Glc) comprises a globular beta-sheet sandwich core (residues 19-168) and a disordered N-terminal tail (residues 1-18). Although the presence of the N-terminal tail is not required for IIA(Glc) to accept a phosphorus from the histidine phosphocarrier protein HPr, its presence is essential for effective phosphotransfer from IIA(Glc) to the membrane-bound IIBC(Glc). The sequence of the N-terminal tail suggests that it has the potential to form an amphipathic helix. Using CD, we demonstrate that a peptide, corresponding to the N-terminal 18 residues of IIA(Glc), adopts a helical conformation in the presence of either the anionic lipid phosphatidylglycerol or a mixture of anionic E. coli lipids phosphatidylglycerol (25%) and phosphatidylethanolamine (75%). The peptide, however, is in a random coil state in the presence of the zwitterionic lipid phosphatidylcholine, indicating that electrostatic interactions play a role in the binding of the lipid to the peptide. In addition, we show that intact IIA(Glc) also interacts with anionic lipids, resulting in an increase in helicity, which can be directly attributed to the N-terminal segment. From these data we propose that IIA(Glc) comprises two functional domains: a folded domain containing the active site and capable of weakly interacting with the peripheral IIB domain of the membrane protein IIBC(Glc); and the N-terminal tail, which interacts with the negatively charged E. coli membrane, thereby stabilizing the complex of IIA(Glc) with IIBC(Glc). This stabilization is essential for the final step of the phosphoryl transfer cascade in the glucose transport pathway.

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 IIA(Glucose) (IIA(Glc)), 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 IIA(Glc). Both binding surfaces comprise a central hydrophobic core region surrounded by a ring of polar and charged residues, positive for HPr and negative for IIA(Glc). 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 IIA(Glc) recognize a variety of structurally diverse proteins. Comparisons with the structures of the enzyme I-HPr and IIA(Glc)-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.

Mediated by the protein IIA Glc , the phospho enol pyruvate:sugar phosphotransferase system plays a role in the regulation of activity of other sugar transport systems in Escherichia coli . By using a direct binding assay, a collection of single-Cys replacement mutants in cytoplasmic loops of lactose permease were evaluated for their capacity to bind IIA Glc . Selected Cys replacements in loops IV/V or VI/VII result in loss of binding activity. Analysis of the mutagenesis results together with multiple sequence alignments of a family of proteins that interacts with IIA Glc provides the basis for developing two regions of consensus sequence in those partner proteins necessary for binding to IIA Glc . The requirement for two interaction regions is interpreted in the regulatory framework of a substrate-dependent conformational change that brings those two regions into an orientation optimal for binding IIA Glc .

The solution structure of the first protein-protein complex of the bacterial phosphoenolpyruvate: sugar phosphotransferase system between the N-terminal domain of enzyme I (EIN) and the histidine-containing phosphocarrier protein HPr has been determined by NMR spectroscopy, including the use of residual dipolar couplings that provide long-range structural information. The complex between EIN and HPr is a classical example of surface complementarity, involving an essentially all helical interface, comprising helices 2, 2', 3 and 4 of the alpha-subdomain of EIN and helices 1 and 2 of HPr, that requires virtually no changes in conformation of the components relative to that in their respective free states. The specificity of the complex is dependent on the correct placement of both van der Waals and electrostatic contacts. The transition state can be formed with minimal changes in overall conformation, and is stabilized in favor of phosphorylated HPr, thereby accounting for the directionality of phosphoryl transfer.

The phosphorylated form of the N-terminal domain of enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli has been investigated by one-bond and long-range 1H-15N correlation spectroscopy. The active site His 189 is phosphorylated at the Nepsilon2 position and has a pKa of 7.3, which is one pH unit higher than that of unphosphorylated His 189. Because the neutral form of unphosphorylated His 189 is in the Ndelta1-H tautomer, and its Nepsilon2 atom is solvent inaccessible and accepts a hydrogen bond from the hydroxyl group of Thr 168, both protonation and phosphorylation of His 189 must be accompanied by a change in the side-chain conformation of His 189, specifically from a chi(2) angle in the g+ conformer in the unphosphorylated state to the g- conformer in the phosphorylated state.

Sugar transport by some permeases in Escherichia coli is allosterically regulated by the phosphorylation state of the intracellular regulatory protein, enzyme IIA glc of the phospho enol pyruvate:sugar phosphotransferase system. A sensitive radiochemical assay for the interaction of enzyme IIA glc with membrane-associated lactose permease was used to characterize the binding reaction. The binding is stimulated by transportable substrates such as lactose, melibiose, and raffinose, but not by sugars that are not transported (maltose and sucrose). Treatment of lactose permease with N -ethylmaleimide, which blocks ligand binding and transport by alkylating Cys-148, also blocks enzyme IIA glc binding. Preincubation with the substrate analog β- d -galactopyranosyl 1-thio-β- d -galactopyranoside protects both lactose transport and enzyme IIA glc binding against inhibition by N -ethylmaleimide. A collection of lactose permease replacement mutants at Cys-148 showed, with the exception of C148V, a good correlation of relative transport activity and enzyme IIA glc binding. The nature of the interaction of enzyme IIA glc with the cytoplasmic face of lactose permease was explored. The N- and C-termini, as well as five hydrophilic loops in the permease, are exposed on the cytoplasmic surface of the membrane and it has been proposed that the central cytoplasmic loop of lactose permease is the major determinant for interaction with enzyme IIA glc . Lactose permease mutants with polyhistidine insertions in cytoplasmic loops IV/V and VI/VII and periplasmic loop VII/VIII retain transport activity and therefore substrate binding, but do not bind enzyme IIA glc , indicating that these regions of lactose permease may be involved in recognition of enzyme IIA glc . Taken together, these results suggest that interaction of lactose permease with substrate promotes a conformational change that brings several cytoplasmic loops into an arrangement optimal for interaction with the regulatory protein, enzyme IIA glc . A topological map of the proposed interaction is presented.

In addition to the phosphoenolpyruvate:sugar phosphotransferase system (sugar PTS), most proteobacteria possess a paralogous system (nitrogen phosphotransferase system, PTS(Ntr)). The first proteins in both pathways are enzymes (enzyme I(sugar) and enzyme I(Ntr)) that can be autophosphorylated by phosphoenolpyruvate. The most striking difference between enzyme I(sugar) and enzyme I(Ntr) is the presence of a GAF domain at the N-terminus of enzyme I(Ntr). Since the PTS(Ntr) was identified in 1995, it has been implicated in a variety of cellular processes in many proteobacteria and many of these regulations have been shown to be dependent on the phosphorylation state of PTS(Ntr) components. However, there has been little evidence that any component of this so-called PTS(Ntr) is directly involved in nitrogen metabolism. Moreover, a signal regulating the phosphorylation state of the PTS(Ntr) had not been uncovered. Here, we demonstrate that glutamine and α-ketoglutarate, the canonical signals of nitrogen availability, reciprocally regulate the phosphorylation state of the PTS(Ntr) by direct effects on enzyme I(Ntr) autophosphorylation and the GAF signal transduction domain is necessary for the regulation of enzyme I(Ntr) activity by the two signal molecules. Taken together, our results suggest that the PTS(Ntr) senses nitrogen availability.

The region of the genome of Mycoplasma capricolum upstream of the portion encompassing the genes for Enzymes I and IIAglc of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) was cloned and sequenced. Examination of the sequence revealed open reading frames corresponding to numerous genes involved with the oxidation of pyruvate. The deduced gene organization is naox (encoding NADH oxidase)-lplA (encoding lipoate-protein ligase)-odpA (encoding pyruvate dehydrogenase EI alpha)-odpB (encoding pyruvate dehydrogenase EI beta)-odp2(encoding pyruvate dehydrogenase EII)-dldH (encoding dihydrolipoamide dehydrogenase)-pta (encoding phosphotransacetylase)-ack (encoding acetate kinase)-orfA (an unknown open reading frame)-kdtB-ptsI-crr. Analysis of the DNA sequence suggests that the naox and lplA genes are part of a single operon, odpA and odpB constitute an additional operon, odp2 and dldH a third operon, and pta and ack an additional transcription unit. Phylogenetic analyses of the protein products of the odpA and odpB genes indicate that they are most similar to the corresponding proteins from Mycoplasma genitalium, Acholeplasma laidlawii, and Gram-positive organisms. The product of the odp2 gene contains a single lipoyl domain, as is the case with the corresponding proteins from M. genitalium and numerous other organisms. An evolutionary tree places the M. capricolum odp2 gene product in close relationship to the corresponding proteins from A. laidlawii and M.genitalium. The dldH gene encodes an unusual form of dihydrolipoamide dehydrogenase that contains an aminoterminal extension corresponding to a lipoyl domain, a property shared by the corresponding proteins from Alcaligenes eutrophus and Clostridium magnum. Aside from that feature, the protein is related phylogenetically to the corresponding proteins from A. laidlawii and M. genitalium. The phosphotransacetylase from M. capricolum is related most closely to the corresponding protein from M. genitalium and is distinguished easily from the enzymes from Escherichia coli and Haemophilus influenzae by the absence of the characteristic amino-terminal extension. The acetate kinase from M. capricolum is related evolutionarily to the homologous enzyme from M. genitalium. Map position comparisons of genes encoding proteins involved with pyruvate metabolism show that, whereas all the genes are clustered in M. capricolum, they are scattered in M. genitalium.

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 ∼30 kDa) has been determined and refined at 2.5 a resolution. It has two distinct structural subdomains; one contains four α helices arranged as two hairpins in a claw-like conformation. The other consists of a β sandwich containing a three-stranded antiparallel β sheet and a four-stranded parallel β sheet, together with three short α helices. Plausible models of complexes between EIN and HPr can be made without assuming major structural changes in either protein. Conclusion The α / β subdomain of EIN is topologically similar to the phospho-histidine 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.

The gene encoding enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system from an Escherichia coli enzyme I mutant was cloned and sequenced. The mutation was shown to be a guanine to adenine transition resulting in an altered protein in which glycine-338 was replaced by aspartic acid. The enzyme I structural gene was mutated to change glycine-338 to a variety of other amino acid residues. Fermentation tests indicated that glycine-338 could be mutated to alanine with no gross loss in phosphotransferase activity, while mutation to valine, glutamic acid, aspartic acid, arginine, histidine, or asparagine led to significant loss of activity. An expression vector for enzyme I was mutated to change glycine-338 to a variety of other amino acid residues and highly purified mutant proteins were prepared. Analysis of phosphorylation of the proteins by PEP indicated that mutation of glycine-338 to alanine had little effect on phosphorylation, mutation to valine substantially decreased phosphorylation, change to histidine or arginine drastically diminished phosphorylation, and mutation to aspartic or glutamic acids abolished phosphorylation activity. Mutation at glycine-338 influences the autophosphorylation rather than the phosphoryl transfer activity of enzyme I.

GTP, as well as other nucleoside triphosphates, stimulates the activity of Escherichia coli adenylyl cyclase in permeable cells; the stimulatory effect is lost when the cells are disrupted by passage through a French pressure cell. These data suggested that the allosteric regulation by GTP of adenylyl cyclase activity requires an interaction of the enzyme with other protein factors. Strains deleted for genes encoding proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) failed to show an activity stimulation by GTP. With a view to localizing the site of interaction of GTP with the adenylyl cyclase complex, a variety of studies using purified PTS proteins were performed using the photoaffinity labeling reagent, 8-azidoGTP. These studies showed that 8-azidoGTP bound specifically to HPr. A species specificity study showed that the photoaffinity reagent labeled E. coli HPr but not HPr proteins from Mycoplasma capricolum or Bacillus subtilis. A variety of site-directed mutations of E. coli HPr were evaluated for interaction with GTP by photoaffinity labeling as well as by nuclear magnetic resonance; the results of these studies indicate that the lysine residues at positions 24 and 27, serine-46, the threonine at position 36, and the aspartate at position 69 are important for the binding of GTP to HPr. Molecular modeling has been used to formulate a model for the binding of GTP to HPr involving electrostatic interaction of the phosphate groups of the nucleotide with the side chains of lysine residues 27 and 45 and serine-43, interaction of the sugar with serine-46, and interaction of the base with lysine-24. From these data, it is hypothesized that the binding of GTP to HPr is required for the GTP-dependent stimulation of the activity of the adenylyl cyclase complex.

The gene (ptsH) for the phosphocarrier protein, HPr, of the phosphoenolpyruvate:sugar phosphotransferase system from Mycoplasma capricolum was previously cloned and sequenced. We present here the results of experiments in which the ptsH gene was cloned into a vector for high level expression in Escherichia coli of the phosphocarrier protein. Conditions were developed for overproduction and purification of HPr by a two-column procedure. The purified protein, analyzed by Edman degradation and mass spectrometry, was found to have been processed by removal of the N-terminal methionine residue. Examination of the purified protein by gel electrophoresis under isoelectric focusing conditions revealed that it has an unusually high isoelectric point.

The region of the genome of Mycoplasma capricolum encompassing the gene (ptsH) encoding HPr, a general energy-coupling protein of the phosphoenolpyruvate:sugar phosphotransferase system, was cloned and sequenced. Examination of the sequence revealed a unique arrangement of the ptsH gene. In all other bacterial species characterized thus far, the ptsH gene is part of a polycistronic operon that includes the gene (ptsI) encoding Enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system; the M. capricolum ptsH gene is part of a monocistronic operon that is situated between two open reading frames unrelated to phosphoenolpyruvate:sugar phosphotransferase system function. The gene immediately upstream of ptsH codes for a helicase, and the open reading frame immediately downstream of ptsH, although not homologous to any previously identified protein, contains a signature sequence characteristic of [C-5] cytosine-specific DNA methylases. The product of the ptsH gene has characteristics similar to the HPr protein produced by Gram-positive organisms: it has a greater sequence similarity to HPrs of Gram-positive bacteria than to those of Gram-negative organisms, it is phosphorylated by a protein kinase derived from Gram-positive organisms, and it complements sugar phosphorylation activity in Gram-positive extracts. The high calculated isoelectric point (pI = 9.18) and the absence of glutamate residues in the C-terminal region distinguish the M. capricolum HPr from all previously described HPrs.

Escherichia coli cells permeabilized by treatment with low concentrations of toluene contain an adenylyl cyclase activity that can be stimulated 3.6-7.6-fold by GTP. The stimulatory effect of GTP is maximal at concentrations of the nucleotide in the physiological range (above 0.7 mM). Studies of the dependence of velocity on substrate (ATP) concentration indicate that the velocity vs. substrate plots are sigmoid in the absence of GTP but hyperbolic in the presence of GTP, suggesting an allosteric regulatory site that can be occupied by either ATP or GTP. Replacement of ATP by AMPPNP as substrate results in velocity vs. substrate plots that are hyperbolic in the absence or presence of GTP, although GTP increases the Vmax by a factor of 2.2; these findings indicate that AMPPNP specifically occupies the substrate site and GTP exclusively occupies the regulatory site. A test of the capacity of other guanine nucleotides to stimulate adenylyl cyclase activity showed that 2'-deoxy-GTP was almost as effective as GTP, but that GDP, GMP, ppGpp, and 3',5'-cGMP were not stimulatory effectors; GTP-gamma-S and GMPPNP stimulated adenylyl cyclase activity but to a lesser degree than did GTP. In addition to the previous indication that ATP can occupy the regulatory site on adenylyl cyclase, it was found that CTP and UTP were potent stimulators. Thus, all the naturally occurring RNA precursor nucleoside triphosphates are capable of stimulating adenylyl cyclase activity. In contrast, PPPi inhibits adenylyl cyclase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzyme INtr is the first protein in the nitrogen phosphotransferase pathway. Using an array of biochemical and biophysical tools, we characterized the protein, compared its properties to that of EI of the carbohydrate PTS and, in addition, examined the effect of substitution of all nonexchangeable protons by deuterium (perdeuteration) on the properties of EINtr. Notably, we find that the catalytic function (autophosphorylation and phosphotransfer to NPr) remains unperturbed while its stability is modulated by deuteration. In particular, the deuterated form exhibits a reduction of approximately 4 °C in thermal stability, enhanced oligomerization propensity, as well as increased sensitivity to proteolysis in vitro. We investigated tertiary, secondary, and local structural changes, both in the absence and presence of PEP, using near- and far-UV circular dichroism and Trp fluorescence spectroscopy. Our data demonstrate that the aromatic residues are particularly sensitive probes for detecting effects of deuteration with an enhanced quantum yield upon PEP binding and apparent decreases in tertiary contacts for Tyr and Trp side chains. Trp mutagenesis studies showed that the region around Trp522 responds to binding of both PEP and NPr. The significance of these results in the context of structural analysis of EINtr are evaluated.

Escherichia coli delta cya-283 is a 75-bp in-frame deletion overlapping the 5' end of delta cya-854; delta cya-201 is a 41-bp frameshift deletion overlapping the 3' end of delta cya-854. Sequence repeats were found at the boundaries of delta cya-283 and delta cya-201, suggesting a mechanism for deletion formation. Recombinant DNA procedures were used to construct a strain in which the total cya structural gene in the chromosome was replaced by the kanamycin resistance gene.

A nitrogen-related signal transduction pathway, consisting of the three phosphotransfer proteins EI(Ntr), NPr, and IIA(Ntr), was discovered recently to regulate the uptake of K(+) in Escherichia coli. In particular, dephosphorylated IIA(Ntr) inhibits the activity of the K(+) transporter TrkA. Since the phosphorylation state of IIA(Ntr) is partially determined by its reversible phosphorylation by NPr, we have determined the three-dimensional structure of NPr by solution NMR spectroscopy. In total, we obtained 973 NOE-derived distance restraints, 112 chemical shift-derived backbone angle restraints, and 35 hydrogen-bond restraints derived from temperature coefficients (wave). We propose that temperature wave is useful for identifying exposed beta-strands and assists in establishing protein folds based on chemical shifts. The deduced structure of NPr contains three alpha-helices and four beta-strands with the three helices all packed on the same face of the beta-sheet. The active site residue His16 of NPr for phosphoryl transfer was found to be neutral and in the N epsilon 2-H tautomeric state. There appears to be increased motion in the active site region of NPr compared to HPr, a homologous protein involved in the uptake and regulation of carbohydrate utilization.

Carbohydrate uptake in certain bacteria is under the regulation of the phosphotransfer protein IIAGlc. IIAGlc regulates the expression and activity of various inducible transporters, including the Na+-coupled melibiose permease (MelB) and H+-lactose permease (LacY). We characterized the thermodynamic features of IIAGlc binding to both the permeases by applying chemical cross-link and isothermal titration calorimetry. Here, we show that IIAGlc directly binds to MelB or LacY in the absence or presence of melibiose, a sugar substrate for both permeases. The protein-protein interaction in both cases exhibits dissociation constant between 3-10 μM at a stoichiometry of one. Strikingly, the thermodynamic strategy for the binding is different. The binding to MelB, which is crystallized in an outside-open conformation, is driven by enthalpy with compensation of entropy; the binding to LacY, which is captured in an inside-open conformation, is driven by entropy with compensation of enthalpy; however, the binding in both cases restrains the conformational entropy of the permease. Consistently, we show that IIAGlc-bound MelB or LacY exhibits a largely decreased affinity for sugar substrate. The data allow us to conclude that IIAGlc regulates activity of MelB and LacY via restraining conformational dynamics and an induced-fit process. In all cases studied, IIAGlc blocks inducer entry into the cells thus preferentially favoring constitutive glucose uptake.

Aurein 1.2 is an antimicrobial and anticancer peptide isolated from an Australian frog. To improve our understanding of the mechanism of action, two series of peptides were designed. The first series includes the N-terminal membrane anchor of bacterial glucose-specific enzyme IIA, aurein 1.2, and a newly identified aurein 1.2 analog from human LL-37 (LLAA). The order of antibacterial activity is LLAA>aurein 1.2>>the membrane anchor (inactive). The structure of LLAA in detergent micelles was determined by (1)H NMR spectroscopy, including structural refinement by natural abundance (13)C(alpha), (13)C(beta), and (15)N chemical shifts. The hydrophobic surface area of the 3D structure is related to the retention time of the peptide on a reverse-phase HPLC column. The higher activity of LLAA compared to aurein 1.2 was attributed to additional cationic residues that enhance the membrane perturbation potential. The second peptide series was created by changing the C-terminal phenylalanine (F13) of aurein 1.2 to either phenylglycine or tryptophan. A closer or further location of the aromatic rings to the peptide backbone in the mutants relative to F13 is proposed to cause a drop in activity. Phenylglycine with unique chemical shifts may be a useful NMR probe for structure-activity relationship studies of antimicrobial peptides. To facilitate potential future use for NMR studies, random-coil chemical shifts for phenylglycine (X) were measured using the synthetic peptide GGXGG. Aromatic rings of phenylalanines in all the peptides penetrated 2-5 A below the lipid head group and are essential for membrane targeting as illustrated by intermolecular peptide-lipid NOE patterns.

While the proteins of the phosphoenolpyruvate:carbohydrate phosphotransferase system (carbohydrate PTS) have been shown to regulate numerous targets, little such information is available for the nitrogen-metabolic phosphotransferase system (nitrogen-metabolic PTS). To elucidate the physiological role of the nitrogen-metabolic PTS, we carried out phenotype microarray (PM) analysis with Escherichia coli K-12 strain MG1655 deleted for the ptsP gene encoding the first enzyme of the nitrogen-metabolic PTS. Together with the PM data, growth studies revealed that a ptsN (encoding enzyme IIA(Ntr)) mutant became extremely sensitive to leucine-containing peptides (LCPs), while both ptsP (encoding enzyme I(Ntr)) and ptsO (encoding NPr) mutants were more resistant than wild type. The toxicity of LCPs was found to be due to leucine and the dephospho-form of enzyme IIA(Ntr) was found to be necessary to neutralize leucine toxicity. Further studies showed that the dephospho-form of enzyme IIA(Ntr) is required for derepression of the ilvBN operon encoding acetohydroxy acid synthase I catalysing the first step common to the biosynthesis of the branched-chain amino acids.

The solution structure of the cytoplasmic B domain of the mannitol (Mtl) transporter (II(Mtl)) from the mannitol branch of the Escherichia coli phosphotransferase system has been solved by multidimensional NMR spectroscopy with extensive use of residual dipolar couplings. The ordered IIB(Mtl) domain (residues 375-471 of II(Mtl)) consists of a four-stranded parallel beta-sheet flanked by two helices (alpha(1) and alpha(3)) on one face and helix alpha(2) on the opposite face with a characteristic Rossmann fold comprising two right-handed beta(1)alpha(1)beta(2) and beta(3)alpha(2)beta(4) motifs. The active site loop is structurally very similar to that of the eukaryotic protein tyrosine phosphatases, with the active site cysteine (Cys-384) primed in the thiolate state (pK(a)5.6) for nucleophilic attack at the phosphorylated histidine (His-554) of the IIA(Mtl) domain through stabilization by hydrogen bonding interactions with neighboring backbone amide groups at positions i + 2/3/4 from Cys-384 and with the hydroxyl group of Ser-391 at position i + 7. Modeling of the phosphorylated state of IIB(Mtl) suggests that the phosphoryl group can be readily stabilized by hydrogen bonding interactions with backbone amides in the i + 2/4/5/6/7 positions as well as with the hydroxyl group of Ser390 at position i + 6. Despite the absence of any significant sequence identity, the structure of IIB(Mtl) is remarkably similar to the structures of bovine protein tyrosine phosphatase (which contains two long insertions relative to IIB(Mtl)) and the cytoplasmic B component of enzyme II(Chb), which fulfills an analogous role to IIB(Mtl) in the N,N'-diacetylchitobiose branch of the phosphotransferase system. All three proteins utilize a cysteine residue in the nucleophilic attack of a phosphoryl group covalently bound to another protein.

The effects of phospholipid or detergent chain length on the structure and translational diffusion coefficient of the membrane-targeting peptide corresponding to the N-terminal amphipathic sequence of Escherichia coli enzyme IIA(Glc) were investigated by nuclear magnetic resonance (NMR) spectroscopy. Three anionic phospholipids (dihexanoyl phosphatidylglycerol, dioctanoyl phosphatidylglycerol, and didecanoyl phosphatidylglycerol) and four lipid-mimicking anionic detergents (sodium hexanesulfonate, 2,2-dimethyl-silapentane-5-sulfonate, sodium nonanesulfonate, and sodium dodecylsulfate) were evaluated. In all cases, the cationic peptide adopts an amphipathic helical structure. While the chain length of the two-chain phospholipids has a negligible effect on the peptide conformation, the effect of chain length of those single-chain detergents on the helix length is more pronounced. The diffusion coefficients of the peptide/micelle complexes were found to correlate with the chain lengths of both the lipid and the detergent groups. Taken together, short-chain anionic phospholipids are proposed to be useful membrane-mimetic models for the structural elucidation of membrane-binding peptides such as cationic antimicrobial peptides. DSS does not form micelles by itself according to the diffusion coefficient data, but it does associate with this cationic peptide. Consequently, both DSS and its analog may be chosen as NMR chemical shift reference compounds depending on the nature of the biomolecules under investigation.

The solution structure of the complex between the cytoplasmic A domain (IIA(Mtl)) of the mannitol transporter II(Mannitol) and the histidine-containing phosphocarrier protein (HPr) of the Escherichia 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 IIA(Mtl) formed by helix 3, portions of helices 2 and 4, and beta-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 IIA(Mtl), 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 IIA(Glucose) (IIA(Glc)), reveals a number of common features despite the fact that EIN, IIA(Glc), and IIA(Mtl) 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.

Enzyme IIA(Glc), encoded by the crr gene of the phosphoenolpyruvate:sugar phosphotransferase system, plays an important role in regulating intermediary metabolism in Escherichia coli ("catabolite repression"). One function involves inhibition of inducible transport systems ("inducer exclusion"), and with lactose permease, a galactoside is required for unphosphorylated IIA(Glc) binding to cytoplasmic loops IV/V and VI/VII [Sondej, M., Sun, J. et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 3525-3530]. With inside-out membrane vesicles containing the permease, [(125)I]IIA(Glc) binding promoted by melibiose exhibits an affinity (K(D)(IIA)) of approximately 1 microM and a stoichiometry of one mole of IIA(Glc) per six moles of lactose permease. Both the quantity of [(125)I]IIA(Glc) bound and the sugar concentration required for half-maximal IIA(Glc) binding (K(0.5)(IIA)(sug)) was measured for eight permease substrates. Differences in maximal IIA(Glc) binding are observed, and the K(0.5)(IIA)(sug) does not correlate with the affinity of LacY for sugar. Furthermore, K(0.5)(IIA)(sug) does not correlate with sugar affinities for various permease mutants. IIA(Glc) does not bind to a mutant (Cys154 --Gly), which is locked in an outwardly facing conformation, binds with increased stoichiometry to mutant Lys131 --Cys, and binds only weakly to two other mutants which appear to be predominantly in either an outwardly or an inwardly facing conformation. When the latter two mutations are combined, sugar-dependent IIA(Glc) binding returns to near wild-type levels. The findings suggest that binding of various substrates to lactose permease results in a collection of unique conformations, each of which presents a specific surface toward the inner face of the membrane that can interact to varying degrees with IIA(Glc).

The bacterial phosphoenolpyruvate:sugar phosphotransferase system accomplishes both the transport and phosphorylation of sugars as well as the regulation of some cellular processes. An important component of this system is the histidine-containing phosphocarrier protein, HPr, which accepts a phosphoryl group from enzyme I, transfers a phosphoryl group to IIA proteins, and is an allosteric regulator of glycogen phosphorylase. Because the nature of the surface on HPr that interacts with this multiplicity of proteins from Escherichia coli was previously undefined, we investigated these interactions by nuclear magnetic resonance spectroscopy. The chemical shift changes of the backbone and side-chain amide (1)H and (15)N nuclei of uniformly (15)N-labeled HPr in the absence and presence of natural abundance glycogen phosphorylase, glucose-specific enzyme IIA, or the N-terminal domain of enzyme I have been determined. Mapping these chemical shift perturbations onto the three-dimensional structure of HPr permitted us to identify the binding surface(s) of HPr for interaction with these proteins. Here we show that the mapped interfaces on HPr are remarkably similar, indicating that HPr employs a similar surface in binding to its partners.

The unphosphorylated form of enzyme IIAglc of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system inhibits transport catalyzed by lactose permease. We (Seok et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 13515-13519) previously characterized the area on the cytoplasmic face of lactose permease that interacts with enzyme IIAglc, using radioactive enzyme IIAglc. Subsequent studies (Sondej et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 3525-3530) suggested consensus binding sequences on proteins that interact with enzyme IIAglc. The present study characterizes a region on the surface of enzyme IIAglc that interfaces with lactose permease. Acetylation of lysine residues by sulfosuccinimidyl acetate treatment of enzyme IIAglc, but not lactose permease, reduced the degree of interaction between the two proteins. To localize the lysine residue(s) on enzyme IIAglc that is(are) involved in the regulatory interaction, selected lysine residues were mutagenized. Conversion of nine separate lysines to glutamic acid resulted in proteins that were still capable of phosphoryl acceptance from HPr. Except for Lys69, all the modified proteins were as effective as the wild-type enzyme IIAglc in a test for binding to lactose permease. The Lys69 mutant was also defective in phosphoryl transfer to glucose permease. To derive further information concerning the contact surface, additional selected residues in the vicinity of Lys69 were mutagenized and tested for binding to lactose permease. On the basis of these studies, a model for the region of the surface of enzyme IIAglc that interacts with lactose permease is proposed.

Enzyme I of the bacterial phosphoenolpyruvate:sugar phosphotransferase system can be phosphorylated by PEP on an active-site histidine residue, localized to a cleft between an alpha-helical domain and an alpha/beta domain on the amino terminal half of the protein. The phosphoryl group on the active-site histidine can be passed to an active-site histidine residue of HPr. It has been proposed that the major interaction between enzyme I and HPr occurs via the alpha-helical domain of enzyme I. The isolated recombinant alpha-helical domain (residues 25-145) with approximately 80% alpha-helices as well as enzyme I deficient in that domain [EI(DeltaHD)] with approximately 50% alpha-helix content from M. capricolum were used to further elucidate the nature of the enzyme I-HPr complex. Isothermal titration calorimetry demonstrated that HPr binds to the alpha-helical domain and intact enzyme I with = 5 x 10(4) and 1.4 x 10(5) M(-)(1) at pH 7.5 and 25 degrees C, respectively, but not to EI(DeltaHD), which contains the active-site histidine of enzyme I and can be autophosphorylated by PEP. In vitro reconstitution experiments with proteins from both M. capricolum and E. coli showed that EI(DeltaHD) can donate its bound phosphoryl group to HPr in the presence of the isolated alpha-helical domain. Furthermore, M. capricolum recombinant C-terminal domain of enzyme I (EIC) was shown to reconstitute phosphotransfer activity with recombinant N-terminal domain (EIN) approximately 5% as efficiently as the HD-EI(DeltaHD) pair. Recombinant EIC strongly self-associates ( approximately 10(10) M(-)(1)) in comparison to dimerization constants of 10(5)-10(7) M(-)(1) measured for EI and EI(DeltaHD).