Phage display selection of peptides against enzyme I of the phosphoenolpyruvate–sugar phosphotransferase system (PTS)

Mukhija, Seema; Erni, Bernhard

doi:10.1046/j.1365-2958.1997.5501910.x

Cited by 22 publications

(17 citation statements)

References 41 publications

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“…The transition state apparently is stabilized by the phosphoryl donor rather than the phosphoryl acceptor protein. This was demonstrated with short basic peptides containing either a histidine or a cysteine that are rapidly phosphorylated by enzyme I but cannot donate the phosphoryl group further to an acceptor protein (37). The inverse mechanism has been observed with CheY, the response regulator of the chemotactic signaling cascade.…”

Section: Discussionmentioning

confidence: 81%

Mutational Analysis of Invariant Arginines in the IIABMan Subunit of the Escherichia coliPhosphotransferase System

Gutknecht

Lanz

Erni

1998

Journal of Biological Chemistry

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The mannose transporter of bacterial phosphotransferase system mediates uptake of mannose, glucose, and related hexoses by a mechanism that couples translocation with phosphorylation of the substrate. Protein phosphorylation plays an important role in energy transduction, signal transduction and enzyme regulation. Ptype ATPases transport cations across the cell membrane and are transiently phosphorylated on an aspartic acid during turnover (1). Protein kinases and protein phosphatases regulate the activity of enzymes and membrane bound receptors by phosphorylation and dephosphorylation (2, 3). In the phosphoenolpyruvate-dependent phosphotransferase system of bacteria (PTS) 1 active transport and signaling are two functions of a protein phosphorylation cascade comprising four phosphoprotein units. The four components, enzyme I, HPr, IIA, and IIB sequentially transfer phosphoryl groups from phosphoenolpyruvate to carbohydrates that are accumulated across the cell membrane by a mechanism coupling translocation to phosphorylation. Whereas enzyme I and HPr are two energy coupling components, IIA and IIB together with the transmembrane IIC (and sometimes an additional IID) units form the sugar-specific transport complexes. IIA, IIB, and IIC occur either as protein subunits or as domains of a multidomain protein. Escherichia coli for example has over 30 genes for transporter complexes that differ in substrate specificity, amino acid sequence, and subunit/domain composition. The phosphorylation sites are histidines in enzyme I, HPr and the different IIA components, cysteines in the IIBs belonging to the glucose, mannitol, and lactose family, and histidines in the IIBs belonging to the mannose family of PTS transporters (for reviews see Refs. 4 and 5).Phosphate-binding sites and phosphate-catalytic sites frequently contain arginines, which by their guanidino group can stabilize phosphate through hydrogen bonding and electrostatic interactions (6). Amino acid sequence comparisons with respect to invariant arginines in PTS proteins from different bacteria reveal the following picture. The N-terminal domain of enzyme I has , which are completely invariant in 17 sequences. are close to the phosphorylation site His-189 (7). Arg-131 is conserved in 14 and Arg-136 is conserved in 15 sequences. The C-terminal domain has 7 completely invariant arginines (Protein Domain Data Base: http://protein.toulouse.inra.fr/prodom/ prodom.html). The functional role of these residues in enzyme I has not been explored. HPr is a 9-kDa open faced fourstranded antiparallel ␤-sheet with three ␣-helices on one face. Arg-17, which is close to the active site His-15, is invariant in all 22 known sequences. When Arg-17 is replaced by Ser or Glu, phospho-donor and -acceptor activity of HPr are reduced to between 6% and less than 0.1% of the control (8). The phosphoryl group bound to His-15 of HPr is most likely complexed by the guanidino group of Arg-17, as inferred from its restricted conformational freedom observed upon phosphorylation of HPr, and fr...

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Section: Discussionmentioning

confidence: 81%

Mutational Analysis of Invariant Arginines in the IIABMan Subunit of the Escherichia coliPhosphotransferase System

Gutknecht

Lanz

Erni

1998

Journal of Biological Chemistry

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“…In the control experiments, the EI-1 peptide (50 μM) and tetracycline (45 μM) inhibit cell growth drastically. The EI-1 peptide was identified previously as an inhibitor of enzyme I by phage display (Mukhija and Erni 1997). To estimate the IC 50 value of peptide AP1, various concentrations of AP1 (12.5,25,50,100,and 200μM) were grown with the cells.…”

Section: Dna Sequencing Peptide Synthesis and Characterizationmentioning

confidence: 99%

“…Peptides with relatively high-affinity for a variety of interacting molecules can be affinity-isolated from the phage-displayed random peptide libraries (Scott and Smith 1990;Yang et al 2005). Several studies have demonstrated the potential of this technique in discovering new peptides with antimicrobial capability using either whole cells or specific enzymes as the targets (Mukhija and Erni 1997;Paradis-Bleau et al 2008;El Zoeiby et al 2003;BishopHurley et al 2005). In the present studies, the recombinant HPr protein was prepared and used as the target in screening for the peptides that bind tightly with HPr.…”

Section: Introductionmentioning

confidence: 97%

A dodecapeptide (YQVTQSKVMSHR) exhibits antibacterial effect and induces cell aggregation in Escherichia coli

Lin

Chen

Chang

et al. 2012

Appl Microbiol Biotechnol

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Antimicrobial peptides play an important role in the innate immune response and host defense mechanism. In the present study, we employed phage display technique to screen for inhibitors which may block the phosphoenolpyruvatedependent phosphotransferase system (PTS) pathway and hence retard cell growth. The recombinant histidine-containing phosphocarrier HPr protein was prepared as the target to screen for the tight binders from the phage-displayed random peptide library Ph.D.-12. The biopanning processes were performed and the binding capabilities of the selected phage were further estimated by enzyme-linked immunosorbent assay (ELISA). The single-stranded DNAs of the 20 selected phages were isolated, sequenced, and five corresponding peptides were synthesized. Only one of the five peptides, AP1 (YQVTQSK VMSHR) was found to inhibit the growth of Escherichia coli cells efficiently (IC₅₀~50 μM). Molecular modeling reveals that AP1 may block the EI-HPr interaction and phosphotransfer. Interestingly, AP1 was also found to induce cell aggregation in a concentration-dependent manner. Since glycogen accumulation has been attributed to biofilm formation, the effects of AP1 on the intracellular glycogen levels were measured. The results strongly indicate that the cell aggregation may be caused by the binding of peptide AP1 with HPr to block the interaction of dephosphorylated HPr with glycogen phosphorylase (GP). Because glycogen phosphorylase activity can be activated by HPr-GP interaction, the binding of AP1 to HPr would cause a decreasing rate of glycogen breakdown in M9 medium and accumulation of glycogen, which may lead to eventual cell aggregation. To the best of our knowledge, this is the first study to demonstrate that an inhibitor bound to a dephosphorylated HPr can decouple its regulatory function and induce cell aggregation.

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“…In the case of HPr, inactivation is biphasic, initially fast, comparable with what happens with IIA Glc , then slow and linear (not shown). This second phase is probably the result of the continuous regeneration of HPr by slow hydrolysis of its phosphorylated form (56).…”

Section: Pep Analogs Asmentioning

confidence: 99%

Mechanism-based Inhibition of Enzyme I of the Escherichia coli Phosphotransferase System

Garcia-Alles¹,

Flükiger²,

Hewel³

et al. 2002

Journal of Biological Chemistry

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The bacterial phosphoenolpyruvate:glycose phosphotransferase system (PTS) 1 catalyzes the uptake and phosphorylation of carbohydrates (1). It is further involved in signal transduction (2), e.g. catabolite repression (3), chemotaxis (4), and allosteric regulation of metabolic enzymes and transporters in response to the availability of carbohydrates (5, 6). The PTS is widely distributed in eubacteria and absent from eukaryotes. It generally consists of two central phosphoryl carriers, EI and HPr, and depending on the species, between 1 and 20 peripheral phosphotransferases, the integral membrane components that recognize and transport hexoses, hexitols, and disaccharides. EI and HPr are the proteins at the top of this divergent phosphorylation cascade (7). EI is a 64-kDa two-domain protein (8, 9). In the aminoterminal domain (EIN, residues 1-250) is located His-189, the amino acid transiently phosphorylated by PEP. The threedimensional structure of EIN has been elucidated (10, 11) and its mode of interaction with HPr characterized by NMR spectroscopy (12, 13). It is composed of a HPr binding ␣-helical subdomain and an ␣/␤ subdomain, structurally similar to the phosphohistidine swivel domain of pyruvate phosphate dikinase (PPDK). Phospho-EIN transfers the phosphoryl group to HPr, but it requires the presence of the carboxyl-terminal domain (EIC) to become phosphorylated by PEP (8,14). EIC contains the PEP binding site (15) and plays a crucial role in dimerization (16,17). It also confers species and subunit specificity during the phosphoryl transfer to the next phosphocarrier protein (14). EIC is proteolytically unstable and flexible. Its structure is not yet known. However, the amino acid sequence similarity between EIC and the PEP binding domain of PPDK of Clostridium symbiosum points toward similar fold and structure. There is also sequence similarity with PEP synthase (18).The mode of action of EI and the way its activity is controlled are not yet fully understood. The protein dimerizes in a temperature-dependent manner (19). The dissociation constant has been reported to shift from 20 M at 6°C to 0.9 M at 30°C. The presence of divalent cations (Mg 2ϩ or Mn 2ϩ ) and PEP also affects the equilibrium and the association and dissociation rate constants (20,21). The association rate constant is 2-3 orders of magnitude slower than in other dimeric proteins, * This work was supported by Grant 31-45838.95 from the Swiss National Science Foundation and a fellowship from the Secretaría de Estado de Educación y Universidades, Spain (to L. F. G.-A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.‡ To whom correspondence should be addressed. Tel.: 41-0-31-631-3792; Fax: 41-0-31-631-4887; E-mail:garcia@ibc.unibe.ch.1 The abbreviations used are: PTS, phosphoenolpyruvate:glucose phosphotransferase system; Z-and E-Cl-PEP, (Z)-3-and (E)-3-chlorophos...

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Phage display selection of peptides against enzyme I of the phosphoenolpyruvate–sugar phosphotransferase system (PTS)

Cited by 22 publications

References 41 publications

Mutational Analysis of Invariant Arginines in the IIABMan Subunit of the Escherichia coliPhosphotransferase System

Mutational Analysis of Invariant Arginines in the IIABMan Subunit of the Escherichia coliPhosphotransferase System

A dodecapeptide (YQVTQSKVMSHR) exhibits antibacterial effect and induces cell aggregation in Escherichia coli

Mechanism-based Inhibition of Enzyme I of the Escherichia coli Phosphotransferase System

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