Sequence Requirements of the GPNG β-Turn of the Ecballium elaterium Trypsin Inhibitor II Explored by Combinatorial Library Screening

Wentzel, Alexander; Christmann, Andreas; Krätzner, Ralph; Kolmar, Harald

doi:10.1074/jbc.274.30.21037

Cited by 64 publications

(56 citation statements)

References 31 publications

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“…This time window may be sufficient for the outer membrane passage of REI v in an unfolded state. The same may hold true for EETI-II, in which disulfide bond formation is also a rather slow process (48). The situation for ␤-lactamase export is quite different.…”

Section: Discussionmentioning

confidence: 89%

Intimin-Mediated Export of Passenger Proteins Requires Maintenance of a Translocation-Competent Conformation

2005

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Intimins from pathogenic bacteria promote intimate bacterial adhesion to epithelial cells. Several structurally similar domains form on the bacterial cell surface an extended rigid rod that exposes the carboxy-terminal domain, which interacts with the translocated intimin receptor. We constructed a series of intimin-derived fusion proteins consisting of carboxy-terminally truncated intimin and the immunoglobulin light-chain variable domain REI v , ubiquitin, calmodulin, ␤-lactamase inhibitor protein, or ␤-lactamase. By systematically investigating the intimin-mediated cell surface exposure of these passenger domains in the presence or absence of compounds that interfere with outer membrane stability or passenger domain folding, we acquired experimental evidence that intimin-mediated protein export across the outer membrane requires, prior to export, the maintenance of a translocation-competent conformation that may be distinct from the final protein structure. We propose that, during export, competition exists between productive translocation and folding of the passenger domain in the periplasm into a stable conformation that is not compatible with translocation through the bacterial outer membrane. These results may expand understanding of the mechanism by which intimins are inserted into the outer membrane and expose extracellular domains on the cell surface.The dual membrane envelopes of gram-negative bacteria provide two barriers of unlike nature that pose formidable problems concerning the transport of molecules into and out of these organisms. Nutrients and essential cofactors must be actively transported into the cells, and end products of metabolism, toxic molecules, and proteins need to be extruded. While gram-positive bacteria, eukaryotes, and archaea exhibit just three known secretory systems for protein transport across the cytoplasmic and endoplasmic reticulum membranes (7, 37, 45), gram-negative bacteria have evolved multiple systems for protein transport across the whole-cell envelope; the proteins may remain attached to the surface or be released into the extracellular milieu (8,25,40,50). They serve as, for example, substrate-degrading enzymes, adhesion anchors, or pathogenicity factors that interfere with host metabolism or immune defense.Most machineries for translocating proteins across gramnegative bacterial membranes are composed of numerous proteins that form heterooligomeric structures, which mediate the simultaneous export of a passenger protein across both membranes (25). Two exceptions are known: the type V secretion pathway (19) and the autodisplay of intimins and invasins (34). In these cases, all of the necessary elements for translocation across the outer membrane are located within their own polypeptide sequences. Members of the family of type V secreted virulence factors comprise three functional domains in a single autoexport protein: an N-terminal targeting sequence, a C-terminal translocation domain, and the passenger domain in between. The C-terminal domain is supposed to for...

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Section: Discussionmentioning

confidence: 89%

Intimin-Mediated Export of Passenger Proteins Requires Maintenance of a Translocation-Competent Conformation

2005

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“…To determine whether the amino-terminal fragment of intimin was sufficient for outer membrane translocation of a heterologous passenger domain using E. coli K-12 as the expression host, a derivative of EETI-II was used as a reporter. This protein is a member of the cystine knot family of protease inhibitors and provides a stable framework for the display of conformationally constrained peptides of various length and sequence (9,49). The variant used here (EETI-CK Send ) contains a 13-residue epitope sequence from Sendai virus L protein in place of the original inhibitor loop and can easily be detected using a monoclonal anti-Sendai virus antibody (11).…”

Section: Resultsmentioning

confidence: 99%

“…The Ecballium elaterium trypsin inhibitor was chosen as a model passenger domain, since it was shown to be successfully translocated through the outer membrane when fused to LppOmpA or the C-terminal domain of the N. gonorrhoeae IgA protease precursor protein IgA␤ (9,49). EETI-II is a 28-residue peptide which is stabilized by three intramolecular disulfide bonds.…”

Section: Discussionmentioning

confidence: 99%

Display of Passenger Proteins on the Surface ofEscherichia coliK-12 by the EnterohemorrhagicE. coliIntimin EaeA

et al. 2001

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Intimins are members of a family of bacterial adhesins from pathogenic Escherichia coli which specifically interact with diverse eukaryotic cell surface receptors. The EaeA intimin from enterohemorrhagic E. coli O157:H7 contains an N-terminal transporter domain, which resides in the bacterial outer membrane and promotes the translocation of four C-terminally attached passenger domains across the bacterial cell envelope. We investigated whether truncated EaeA intimin lacking two carboxy-terminal domains could be used as a translocator for heterologous passenger proteins. We found that a variant of the trypsin inhibitor Ecballium elaterium trypsin inhibitor II (EETI-II), interleukin 4, and the Bence-Jones protein REI v were displayed on the surface of E. coli K-12 via fusion to truncated intimin. Fusion protein net accumulation in the outer membrane could be regulated over a broad range by varying the cellular amount of suppressor tRNA that is necessary for translational readthrough at an amber codon residing within the truncated eaeA gene. Intimin-mediated adhesion of the bacterial cells to eukaryotic target cells could be mimicked by surface display of a short fibrinogen receptor binding peptide containing an arginine-glycine-aspartic acid sequence motif, which promoted binding of E. coli K-12 to human platelets. Cells displaying a particular epitope sequence fused to truncated intimin could be enriched 200,000-fold by immunofluorescence staining and fluorescence-activated cell sorting in three sorting rounds. These results demonstrate that truncated intimin can be used as an anchor protein that mediates the translocation of various passenger proteins through the cytoplasmic and outer membranes of E. coli and their exposure on the cell surface. Intimin display may prove a useful tool for future protein translocation studies with interesting biological and biotechnological ramifications.In recent years there has been considerable progress in the development of expression systems for the display of heterologous peptides and proteins on the surfaces of bacteria and yeasts (6,16,31). Cells displaying peptides and proteins such as receptors, antibodies, and enzymes are of considerable value for various biotechnological applications, such as bioseparations, vaccine development, and combinatorial library screening. Numerous anchor proteins that mediate the translocation of passenger proteins through the cytoplasmic and outer membranes of Escherichia coli and their exposure on the cell surface have been used. Short peptides (less than approximately 50 amino acids [aa]) were successfully displayed on the cell surface by insertion into surface-exposed loops of fimbrial proteins (24) or outer membrane proteins like LamB (7) or PhoE (2). Larger passenger domains could be presented on the E. coli cell surface by insertion into a surface-exposed domain of the E. coli flagellin FliC (50), by carboxy-terminal fusion to Lpp-OmpA (a hybrid protein consisting of parts of the E. coli lipoprotein Lpp and OmpA protein [9,12]), by usi...

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“…Also, to maintain the important disulfide bridges, the sequence of the inhibitory peptide PMT-LEYR was modified in position 2 to PCTLEYR. Furthermore, to avoid problems with an adjacent positive charge, and since a substitution for methionine is possible without decreasing inhibition (2,8), the Arg-7 was changed to methionine, a conserved position in most squash inhibitors. This inhibitor with the N-terminal sequence now reading PCTLEYM (see Fig.…”

Section: Resultsmentioning

confidence: 99%

“…able amino acid exchanges and length variations are incorporated within its binding loop (8). Natural squash inhibitors that inhibit porcine pancreatic elastase are also known from the literature.…”

mentioning

confidence: 99%

Design and Characterization of a Hybrid Miniprotein That Specifically Inhibits Porcine Pancreatic Elastase

Hilpert

Wessner

Schneider‐Mergener

et al. 2003

Journal of Biological Chemistry

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Studying protease/peptide inhibitor interactions is a useful tool for understanding molecular recognition in general and is particularly relevant for the rational design of inhibitors with therapeutic potential. An inhibitory peptide (PMTLEYR) derived from the third domain of turkey ovomucoid inhibitor and optimized for specific porcine pancreatic elastase inhibition was introduced into an inhibitor scaffold to increase the proteolytic stability of the peptide. The trypsinspecific squash inhibitor EETI II from Ecballium elaterium was chosen as the scaffold. The resulting hybrid inhibitor HEI-TOE I (hybrid inhibitor from E. elaterium and the optimized binding loop of the third domain of turkey ovomucoid inhibitor) shows a specificity and affinity to porcine pancreatic elastase similar to the free inhibitory peptide but with significantly higher proteolytic stability. Isothermal titration calorimetry revealed that elastase binding of HEI-TOE I occurs with a small unfavorable positive enthalpy contribution, a large favorable positive entropy change, and a large negative heat capacity change. In addition, the inhibitory peptide and the hybrid inhibitor HEI-TOE I protected endothelial cells against degradation following treatment with porcine pancreatic elastase.

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Sequence Requirements of the GPNG β-Turn of the Ecballium elaterium Trypsin Inhibitor II Explored by Combinatorial Library Screening

Cited by 64 publications

References 31 publications

Intimin-Mediated Export of Passenger Proteins Requires Maintenance of a Translocation-Competent Conformation

Intimin-Mediated Export of Passenger Proteins Requires Maintenance of a Translocation-Competent Conformation

Display of Passenger Proteins on the Surface ofEscherichia coliK-12 by the EnterohemorrhagicE. coliIntimin EaeA

Design and Characterization of a Hybrid Miniprotein That Specifically Inhibits Porcine Pancreatic Elastase

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