Binding of peptides in solution by the Escherichia coli chaperone PapD as revealed using an inhibition ELISA and NMR spectroscopy

Abstract

PapD is the prototype member of a family of periplasmic chaperones which are required for assembly of virulence associated pili in pathogenic, gram-negative bacteria. In the present investigation, an ELISA has been developed for evaluation of compounds as inhibitors of PapD. Synthetic peptides, including an octamer, derived from the C-terminus of the pilus adhesin PapG were able to inhibit PapD in the ELISA. Evaluation of a panel of octapeptides in the ELISA, in combination with NMR studies, showed that the peptides were bound as extended β-strands by PapD in aqueous solution. The PapD–peptide complex was stabilized by backbone to backbone hydrogen bonds and interactions involving three hydrophobic peptide side chains. This structural information, together with previous crystal structure data, provides a starting point in efforts to design and synthesize compounds which bind to chaperones and interfere with pilus assembly in pathogenic bacteria.

Introduction

Many gram-negative bacteria assemble extracellular adhesive organelles termed pili on their surfaces that allow them to colonize host tissue and give rise to infections.[1] This group of bacteria includes pathogens that cause a variety of diseases, such as urinary tract infection and the more severe pyelonephritis, meningitis, whooping cough, pneumonia and diarrhoea.[2] Pili are rodlike, supramolecular protein fibers that present adhesins at their tip for specific attachment to receptors found in the host.3, 4 The P pilus of uropathogenic Escherichia coli is the best characterized of all bacterial pili and carries the adhesin PapG at the end of a thin tip fibrillum joined to a thicker pilus rod.[1] The mode of binding of the PapG adhesin to the receptor-active disaccharide, α-d-galactopyranosyl-(1-4)-β-d-galactopyranose [Galα(1-4)Galβ], present in the globoseries of glycolipids on kidney epithelium cells,5, 6 has been revealed in detail using synthetic receptor analogues.7, 8, 9 The P pilus tip fibrillar structure is composed of repeating PapE subunits whereas the pilus rod consists of large numbers of the major pilin subunit PapA. The adaptor proteins PapF, PapK, and PapH join the different parts of the pilus to each other and anchor the pilus in the outer cell membrane of the bacteria. All bacterial pilus assembly requires the presence of periplasmic chaperones. P pili are assembled by the chaperone PapD that binds to and caps interactive surfaces on each pilus subunit thus preventing premature aggregation during their secretion into the periplasmic space.10, 11, 12 The crystal structure of PapD has been solved at 2.5 Å resolution and reveals that PapD consists of two β-barrel domains oriented in the shape of a boomerang.[13] Furthermore, PapD has been found to have significant sequence homology (25–56% identity) with chaperones that assemble other types of pili suggesting that all chaperones have closely related structures.2, 14

The C-terminus of the P pilus proteins has been indicated to constitute an essential part of the epitope on each subunit that is recognized by the PapD chaperone.10, 12, 15, 16 Thus, truncation of the C-terminal 13 residues in the adhesin PapG abolished complex formation with PapD.[10] Moreover, synthetic 19-mer peptides from the C-terminus of P-pilus proteins bound to PapD and some of these peptides also inhibited chaperone mediated folding of the adhesin PapG in an in vitro assay.[15] The crystal structure of PapD bound to the C-terminal 19-mer peptide from PapG (PapG296-314), determined at 3.0 Å resolution, suggested a structural basis for chaperone-subunit interactions.[15] It was found that the peptide was anchored by hydrogen and ionic bonding of the C-terminal carboxylate group to residues Arg[8] and Lys¹¹², which are found in the cleft of PapD and are invariant in the chaperone superfamily. Seven main-chain hydrogen bonds were also formed between the peptide backbone and the G1 β-strand on the surface of PapD resulting in a parallel β-strand interaction. Furthermore, the hydrophobic side chains of Phe³¹³, Leu³¹¹, Met³⁰⁹, and Met³⁰⁷ in the peptide made significant contact with PapD. Interestingly, these hydrophobic residues are part of a conserved pattern of alternating hydrophobic and hydrophilic residues present in the C-terminus of pilus proteins.[15] However, within the crystal, the PapD-peptide β-sheet was extended even further, since a second PapD-peptide complex was placed adjacent to the first, so that the two bound peptide chains interacted as antiparallel β-strands. It can not be ruled out that the structure of the complex was significantly influenced by this dimerization.

In addition to providing information on how chaperones recognize pilus proteins, studies of the interactions between synthetic peptides and chaperones provide structural information that may be used for design and synthesis of ligands that bind to chaperones. By inhibiting pilus assembly, such compounds would interfere with bacterial attachment to the host; therefore, they constitute potential novel antibiotics. It was recently pointed out that the development of compounds that interfere with bacterial protein secretion (for instance, in pilus assembly) constitutes an attractive approach to overcome widespread bacterial resistance to existing antibiotics.[17] However, such efforts have been hampered by the lack of bioassays, which properly evaluate compounds as inhibitors of the critical complex formation between chaperone and pilus protein.[15] Therefore, we have developed an enzyme-linked immunosorbent assay (ELISA) in which compounds may be evaluated for their ability to inhibit complex formation between PapD and a fusion protein (MaltBP–PapG175-314)[16] which consists of the chaperone binding domain of the PapG linked to the maltose binding protein. Evaluation of a panel of synthetic peptides derived from the C-terminus of P pilus proteins in the assay resulted in the identification of a minimal inhibitory octapeptide. Investigation of alanine and serine scans of this octapeptide in the ELISA, in combination with NMR studies of two peptide–PapD complexes, revealed structural details responsible for the formation of complexes between PapD and peptides in aqueous solution.