Short Proline-rich Peptides Research Articles

Effects of Lipidation on a Proline-Rich Antibacterial Peptide.

The emergence of multidrug-resistant bacteria is a worldwide health problem. Antimicrobial peptides have been recognized as potential alternatives to conventional antibiotics, but still require optimization. The proline-rich antimicrobial peptide Bac7(1-16) is active against only a limited number of Gram-negative bacteria. It kills bacteria by inhibiting protein synthesis after its internalization, which is mainly supported by the bacterial transporter SbmA. In this study, we tested two different lipidated forms of Bac7(1-16) with the aim of extending its activity against those bacterial species that lack SbmA. We linked a C12-alkyl chain or an ultrashort cationic lipopeptide Lp-I to the C-terminus of Bac7(1-16). Both the lipidated Bac-C12 and Bac-Lp-I forms acquired activity at low micromolar MIC values against several Gram-positive and Gram-negative bacteria. Moreover, unlike Bac7(1-16), Bac-C12, and Bac-Lp-I did not select resistant mutants in E. coli after 14 times of exposure to sub-MIC concentrations of the respective peptide. We demonstrated that the extended spectrum of activity and absence of de novo resistance are likely related to the acquired capability of the peptides to permeabilize cell membranes. These results indicate that C-terminal lipidation of a short proline-rich peptide profoundly alters its function and mode of action and provides useful insights into the design of novel broad-spectrum antibacterial agents.

Structural enzymology binding studies of the peptide-substrate-binding domain of human collagen prolyl 4-hydroxylase (type-II): High affinity peptides have a PxGP sequence motif.

The peptide-substrate-binding (PSB) domain of collagen prolyl 4-hydroxylase (C-P4H, an α2 β2 tetramer) binds proline-rich procollagen peptides. This helical domain (the middle domain of the α subunit) has an important role concerning the substrate binding properties of C-P4H, although it is not known how the PSB domain influences the hydroxylation properties of the catalytic domain (the C-terminal domain of the α subunit). The crystal structures of the PSB domain of the human C-P4H isoform II (PSB-II) complexed with and without various short proline-rich peptides are described. The comparison with the previously determined PSB-I peptide complex structures shows that the C-P4H-I substrate peptide (PPG)3 , has at most very weak affinity for PSB-II, although it binds with high affinity to PSB-I. The replacement of the middle PPG triplet of (PPG)3 to the nonhydroxylatable PAG, PRG, or PEG triplet, increases greatly the affinity of PSB-II for these peptides, leading to a deeper mode of binding, as compared to the previously determined PSB-I peptide complexes. In these PSB-II complexes, the two peptidyl prolines of its central P(A/R/E)GP region bind in the Pro5 and Pro8 binding pockets of the PSB peptide-binding groove, and direct hydrogen bonds are formed between the peptide and the side chains of the highly conserved residues Tyr158, Arg223, and Asn227, replacing water mediated interactions in the corresponding PSB-I complex. These results suggest that PxGP (where x is not a proline) is the common motif of proline-rich peptide sequences that bind with high affinity to PSB-II.

Abstract B190: Allosteric modulation of Src family kinases via SH3 domain displacement

Abstract The Src family of non-receptor protein-tyrosine kinases (SFKs) plays a crucial role in cancer progression by affecting multiple signaling pathways linked to tumor cell adhesion, invasion and survival. Recent work from our group and others has shown that individual Src-family members may have unique and in some cases opposing roles in the regulation of cell growth and differentiation. Discovery and development of SFK isoform-selective inhibitors and agonists is therefore essential for the most effective therapeutic targeting of this ubiquitous kinase family. Current SFK inhibitors target the ATP-binding site of the kinase domain or an allosteric pocket nearby. However, the highly conserved nature of the active site across the Src kinase family limits identification of isoform-selective compounds. One possible solution to this problem involves small molecule modulation of SFK activity via the regulatory SH3 and SH2 domains. SFK activity is tightly regulated by intramolecular interactions of the SH3 domain with the SH2:kinase linker and the SH2 domain with the phosphorylated C-terminal tail. Small molecules that either enhance or disrupt these interactions are anticipated to result in kinase inhibition or activation, respectively. Recently, we reported that binding of a short proline-rich peptide to the SH3 domains of several Src-family members is sufficient to induce kinase activation in vitro. This peptide, known as VSL12 (VSLARRPLPLP), was postulated to disrupt the regulatory SH3:linker interaction in the downregulated kinase. In the present study, we used VSL12 as probe to better understand the structural basis of kinase activation by SH3 displacement. We first compared the affinity and kinetics of the VSL12 peptide interaction with SFK SH3 domains vs. the near full-length kinase proteins using surface plasmon resonance. We found that VSL12 bound to the isolated SH3 domains of Src, Hck, Lyn and Fyn with similar micromolar potencies. Surprisingly, the KD values for VSL12 binding to the corresponding near-full-length kinases were in the low nanomolar range, suggesting that the peptide makes additional contacts with residues outside of the SH3 domain that result in the stable, active complex. This idea was tested further using differential scanning fluorimetry to explore the thermal stability of the protein:peptide complexes. Addition of VSL12 enhanced the thermal stability of a recombinant Src SH3-SH2-linker protein, consistent with increased molecular order. However, VSL12 decreased the thermal stability of near full-length c-Src, which may reflect conformational changes in the kinase domain that accompany kinase activation. Addition of the kinase domain inhibitor dasatinib restored thermal stability of the Src:VSL12 complex. Complexes of the Src SH3-SH2-linker protein with the VSL12 peptide readily formed crystals that diffract X-rays to 2.6 Å resolution, providing further support for the stabilization of the complex by VSL12 binding. Determining the structures of the Src SH3-SH2-linker protein and the near-full-length kinase in complex with VSL12 will provide fresh insight as to the mechanism of c-Src activation by SH3 domain displacement and aid in the design of potent and selective non-peptide allosteric modulators. Citation Format: Heather L. Rust, Jamie A. Moroco, John J. Alvarado, John J. Engen, Thomas E. Smithgall. Allosteric modulation of Src family kinases via SH3 domain displacement. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B190.

The impact of either 4-R-hydroxyproline or 4-R-fluoroproline on the conformation and SH3m-cort binding of HPK1 proline-rich peptide

SH3 domains are probably the most abundant molecular-recognition modules of the proteome. A common feature of these domains is their interaction with ligand proteins containing Pro-rich sequences. Crystal and NMR structures of SH3 domains complexes with Pro-rich peptides show that the peptide ligands are bound over a range of up to seven residues in a PPII helix conformation. Short proline-rich peptides usually adopt little or no ordered secondary structure before binding interactions, and consequently their association with the SH3 domain is characterized by unfavorable binding entropy due to a loss of rotational freedom on forming the PPII helix. With the aim to stabilize the PPII helix conformation into the proline-rich decapeptide PPPLPPKPKF (P2), we replaced some proline residues either with the 4(R)-4-fluoro-L-proline (FPro) or the 4(R)-4-hydroxy-L-proline (Hyp). The interactions of P2 analogues with the SH3 domain of cortactin (SH3(m-cort)) were analyzed by circular dichroism spectroscopy, while CD thermal transition experiments have been used to determine their propensity to adopt a PPII helix conformation. Results show that the introduction of three residues of Hyp efficiently stabilizes the PPII helix conformation, while it does not improve the affinity towards the SH3 domain, suggesting that additional forces, e.g., electrostatic interactions, are involved in the SH3(m-cort) substrate recognition.

Tunable Growth Factor Delivery from Injectable Hydrogels for Tissue Engineering

Current sustained delivery strategies of protein therapeutics are limited by the fragility of the protein, resulting in minimal quantities of bioactive protein delivered. In order to achieve prolonged release of bioactive protein, an affinity-based approach was designed which exploits the specific binding of the Src homology 3 (SH3) domain with short proline-rich peptides. Specifically, methyl cellulose was modified with SH3-binding peptides (MC-peptide) with either a weak affinity or strong affinity for SH3. The release profile of SH3-rhFGF2 fusion protein from hyaluronan MC-SH3 peptide (HAMC-peptide) hydrogels was investigated and compared to unmodified controls. SH3-rhFGF2 release from HAMC-peptide was extended to 10 days using peptides with different binding affinities compared to the 48 h release from unmodified HAMC. This system is capable of delivering additional proteins with tunable rates of release, while maintaining bioactivity, and thus is broadly applicable.

Highly effective serodiagnosis for Chagas' disease.

Many proteins of Trypanosoma cruzi, the causative agent of Chagas' disease, contain characteristic arrays of highly repetitive immunogenic amino acid motifs. Diagnostic tests using these motifs in monomeric or dimeric form have proven to provide markedly improved specificity compared to conventional tests based on crude parasite extracts. However, in many cases the available tests still suffer from limited sensitivity. In this study we produced stable synthetic genes with maximal codon variability for the four diagnostic antigens, B13, CRA, TcD, and TcE, each containing between three and nine identical amino acid repeats. These genes were combined by linker sequences encoding short proline-rich peptides, giving rise to a 24-kDa fusion protein which was used as a novel diagnostic antigen in an enzyme-linked immunosorbent assay setup. Validation of the assay with a large number of well-characterized patient sera from Bolivia and Brazil revealed excellent diagnostic performance. The high sensitivity of the new test may allow future studies to use blood collected by finger prick and dried on filter paper, thus dramatically reducing the costs and effort for the detection of T. cruzi infection.

Structural Insights into the Specific Binding of Huntingtin Proline-Rich Region with the SH3 and WW Domains

The interactions of huntingtin (Htt) with the SH3 domain- or WW domain-containing proteins have been implicated in the pathogenesis of Huntington's disease (HD). We report the specific interactions of Htt proline-rich region (PRR) with the SH3GL3-SH3 domain and HYPA-WW1-2 domain pair by NMR. The results show that Htt PRR binds with the SH3 domain through nearly its entire chain, and that the binding region on the domain includes the canonical PxxP-binding site and the specificity pocket. The C terminus of PRR orients to the specificity pocket, whereas the N terminus orients to the PxxP-binding site. Htt PRR can also specifically bind to WW1-2; the N-terminal portion preferentially binds to WW1, while the C-terminal portion binds to WW2. This study provides structural insights into the specific interactions between Htt PRR and its binding partners as well as the alteration of these interactions that involve PRR, which may have implications for the understanding of HD.

Polyproline II Helix Conformation in a Proline-Rich Environment: A Theoretical Study

Interest centers here on whether a polyproline II helix can propagate through adjacent non-proline residues, and on shedding light on recent experimental observations suggesting the presence of significant PP II structure in a short alanine-based peptide with no proline in the sequence. For this purpose, we explored the formation of polyproline II helices in proline-rich peptides with the sequences Ac-(Pro) 3-X-(Pro) 3-Gly-Tyr-NH 2, with X = Pro (PPP), Ala (PAP), Gln (PQP), Gly (PGP), and Val (PVP), and Ac-(Pro) 3-Ala-Ala-(Pro) 3-Gly-Tyr-NH 2 (PAAP), by using a theoretical approach that includes a solvent effect as well as cis ↔ trans isomerization of the peptide groups and puckering conformations of the pyrrolidine ring of the proline residues. Since 13C chemical shifts have proven to be useful for identifying secondary-structure preferences in proteins and peptides, and because values of the dihedral angles ( ϕ, ψ) are the main determinants of their magnitudes, we have, therefore, computed the Boltzmann-averaged 13C chemical shifts for the guest residues in the PXP peptide ( X = Pro, Ala, Gln, Gly, and Val) with a combination of approaches, involving molecular mechanics, statistical mechanics, and quantum mechanics. In addition, an improved procedure was used to carry out the conformational searches and to compute the solvent polarization effects faster and more accurately than in previous work. The current theoretical work and additional experimental evidence show that, in short proline-rich peptides, alanine decreases the polyproline II helix content. In particular, the theoretical evidence accumulated in this work calls into question the proposal that alanine has a strong preference to adopt conformations in the polyproline II region of the Ramachandran map.

Flexible docking of a ligand peptide to a receptor protein by multicanonical molecular dynamics simulation

A new method for flexible docking by multicanonical molecular dynamics simulation is presented. The method was applied to the binding of a short proline-rich peptide to a Src homology 3 (SH3) domain. The peptide and the side-chains at the ligand binding cleft of SH3 were completely flexible and the large number of possible conformations and dispositions of the peptide were sampled. The reweighted canonical resemble at 300 K resulted in only a few predominant binding modes, one of which was similar to the complex crystal structure. The inverted peptide orientation was also observed in the other binding modes.