Peptoid-Peptide Hybrid Analogs of the Enterococcus faecalis Fsr Auto-Inducing Peptide (AIP) Reveal Crucial Structure-Activity Relationships.

As multidrug-resistant bacteria become a more pressing risk to human health, alternate approaches to treating bacterial infections are being increasingly investigated. Enterococcus faecalis is an opportunistic pathogen responsible for a large percentage of secondary enterococci infections. Its pathogenicity has been shown to be largely dependent on a cell-density communication mechanism, termed quorum sensing. In this study, we conducted a systematic investigation of the lactone-containing macrocyclic signaling peptide used by E. faecalis for Fsr-mediated communication, termed gelatinase biosynthesis activating pheromone (GBAP). Specifically, through a combination of the on-resin sub-monomer and solution phase peptoid building block synthesis approaches, we successfully synthesized a library of peptoid-peptide hybrid analogs of GBAP and determined the biological effects associated with the introduction of the peptoid (N-alkyl glycine derivative) modifications. Within the macrocycle region of the peptide, as have been seen with other modifications, the F7 site was unusually tolerant toward peptoid modification, compared with other macrocyclic sites. Interestingly, within the exocyclic tail, peptoid modification at the N2 site completely abolished activity, a first for a single tail modification.

Metamorphic Proteins: Emergence of Dual Protein Folds from One Primary Sequence.

Every amino acid exhibits a different propensity for distinct structural conformations. Hence, decoding how the primary amino acid sequence undergoes the transition to a defined secondary structure and its final three-dimensional fold is presently considered predictable with reasonable certainty. However, protein sequences that defy the first principles of secondary structure prediction (they attain two different folds) have recently been discovered. Such proteins, aptly named metamorphic proteins, decrease the conformational constraint by increasing flexibility in the secondary structure and thereby result in efficient functionality. In this review, we discuss the major factors driving the conformational switch related both to protein sequence and to structure using illustrative examples. We discuss the concept of an evolutionary transition in sequence and structure, the functional impact of the tertiary fold, and the pressure of intrinsic and external factors that give rise to metamorphic proteins. We mainly focus on the major components of protein architecture, namely, the α-helix and ß-sheet segments, which are involved in conformational switching within the same or highly similar sequences. These chameleonic sequences are widespread in both cytosolic and membrane proteins, and these folds are equally important for protein structure and function. We discuss the implications of metamorphic proteins and chameleonic peptide sequences in de novo peptide design.

Solvation driven conformational transitions in the second transmembrane domain of mycobacteriophage holin.

Holins are pore-forming membrane proteins synthesized by lytic phages. The second transmembrane domain (TM2) of Mycobacteriophage D29 holin presents an Ala- and Gly-rich sequence, with a currently unknown structure and function. In this study, we present the spectroscopic characterization of synthetic TM2 in various solvents, detergents, and lipids. We find that TM2 adopts α-helical conformation under conditions that promote intra-strand hydrogen bonding, such as organic solvents and detergent micelles. When we transfer the peptide to a well-hydrated environment, a polyproline II-like structure is obtained. Surprisingly, we find that the polyproline II-like conformation is retained in lipid vesicles. Based on our results, we present a putative role for TM2 in the process of pore formation by holin. © 2016 The Authors. Peptide Science Published by Wiley Periodicals, Inc. Biopolymers (Pept Sci) 108: 1-10, 2017.

Attenuating the Streptococcus pneumoniae Competence Regulon Using Urea-Bridged Cyclic Dominant-Negative Competence-Stimulating Peptide Analogs.

Streptococcus pneumoniae (pneumococcus) is a prevalent human pathogen that utilizes the competence regulon quorum sensing circuitry to acquire antibiotic resistance and initiate its attack on the human host. Therefore, targeting the competence regulon can be applied as an anti-infective approach with minimal pressure for resistance development. Herein, we report the construction of a library of urea-bridged cyclic dominant-negative competence-stimulating peptide (dnCSP) derivatives and their evaluation as competitive inhibitors of the competence regulon. Our results reveal the first pneumococcus dual-action CSPs that inhibit the group 1 pneumococcus competence regulon while activating the group 2 pneumococcus competence regulon. Structural analysis indicates that the urea-bridge cyclization stabilizes the bioactive α-helix conformation, while in vivo studies using a mouse model of infection exhibit that the lead dual-action dnCSP, CSP1-E1A-cyc(Dab6Dab10), attenuates group 1-mediated mortality without significantly reducing the bacterial burden. Overall, our results pave the way for developing novel therapeutics against this notorious pathogen.

Pharmacological Evaluation of Synthetic Dominant-Negative Peptides Derived from the Competence-Stimulating Peptide of Streptococcus pneumoniae.

The competence regulon of Streptococcus pneumoniae (pneumococcus) is a quorum-sensing circuitry that regulates the ability of this pathogen to acquire antibiotic resistance or perform serotype switching, leading to vaccine-escape serotypes, via horizontal gene transfer, as well as initiate virulence. Induction of the competence regulon is centered on binding of the competence-stimulating peptide (CSP) to its cognate receptor, ComD. We have recently synthesized multiple dominant-negative peptide analogs capable of inhibiting competence induction and virulence in S. pneumoniae. However, the pharmacodynamics and safety profiles of these peptide drug leads have not been characterized. Therefore, in this study, we compared the biostability of cyanine-7.5-labeled wild-type CSPs versus dominant-negative peptide analogs (dnCSPs) spatiotemporally by using an IVIS Spectrum in vivo imaging system. Moreover, in vitro cytotoxicity and in vivo toxicity were evaluated. We conclude that our best peptide analog, CSP1-E1A-cyc(Dap6E10), is an attractive therapeutic agent against pneumococcal infection with superior safety and pharmacokinetics profiles.

Optimizing CSP1 analogs for modulating quorum sensing in Streptococcus pneumoniae with bulky, hydrophobic nonproteogenic amino acid substitutions.

The prompt appearance of multiantibiotic-resistant bacteria necessitates finding alternative treatments that can attenuate bacterial infections while minimizing the rate of antibiotic resistance development. Streptococcus pneumoniae, a notorious human pathogen, is responsible for severe antibiotic-resistant infections. Its pathogenicity is influenced by a cell-density communication system, termed quorum sensing (QS). As a result, controlling QS through the development of peptide-based QS modulators may serve to attenuate pneumococcal infections. Herein, we set out to evaluate the impact of the introduction of bulkier, nonproteogenic side-chain residues on the hydrophobic binding face of CSP1 to optimize receptor-binding interactions in both of the S. pneumoniae specificity groups. Our results indicate that these substitutions optimize the peptide-protein binding interactions, yielding several pneumococcal QS modulators with high potency. Moreover, pharmacological evaluation of lead analogs revealed that the incorporation of nonproteogenic amino acids increased the peptides' half-life towards enzymatic degradation while remaining nontoxic. Overall, our data convey key considerations for SAR using nonproteogenic amino acids, which provide analogs with better pharmacological properties.

Strategies to Attenuate the Competence Regulon in Streptococcus pneumoniae.

Streptococcus pneumoniae is an opportunistic respiratory human pathogen that poses a continuing threat to human health. Natural competence for genetic transformation in S. pneumoniae plays an important role in aiding pathogenicity and it is the best-characterized feature to acquire antimicrobial resistance genes by a frequent process of recombination. In S. pneumoniae, competence, along with virulence factor production, is controlled by a cell-density communication mechanism termed the competence regulon. In this review, we present the recent advances in the development of alternative methods to attenuate the pathogenicity of S. pneumoniae by targeting the various stages of the non-essential competence regulon communication system. We mainly focus on new developments related to competitively intercepting the competence regulon signaling through the introduction of promising dominant-negative Competence Stimulating Peptide (dnCSP) scaffolds. We also discuss recent reports on antibiotics that can block CSP export by disturbing the proton motive force (PMF) across the membrane and various ways to control the pneumococcal pathogenicity by activating the counter signaling circuit and targeting the pneumococcal proteome.

Direct Structural Annotation of Membrane Protein Aggregation Loci using Peptide-Based Reverse Mapping.

Membrane protein aggregation is associated with neurodegenerative diseases. Despite remarkable advances to map protein aggregation, molecular elements that drive the structural transition from functional to amyloidogenic ß-sheet polymers remain elusive. Here, we report a simple and reliable reverse-mapping method to identify the molecular elements. We validate our approach by obtaining molecular details of aggregation loci of human ß-barrel nanopore ion channels that are vital for cell survival. By coupling bottom-up synthesis with time-resolved aggregation kinetics and high-resolution imaging, we identify molecular elements that switch folded channels to polymeric ß-rich aggregates. We prove that intrinsic protein aggregation and amyloidogenicity does not depend on total hydrophobicity but on single residue differences in the primary sequence. Our method offers effective strategies for sequence-based design of aggregation inhibitors in biomedicine for neurodegenerative diseases.

Engineering a Transmembrane Nanopore Ion Channel from a Membrane Breaker Peptide.

Re-engineering nature's molecules is an ideal strategy to obtain explicit functionality such as synthetic molecular machines, yet novel strategies for producing engineered molecular channels are few. Here we report a peptide engineering strategy through sequence reversal, which we applied on the first transmembrane peptide of the mycobacteriophage membranoporin protein holin. We have successfully redesigned the membrane rupture property of this peptide to form specific nanopore ion channels. We report the structural characterization and electrophysiology measurements of a library of 28-residue engineered membrane peptides, with remarkable ion channel behavior. We further identify that key residues at the peptide terminus, the central proline, charge distribution, and hydropathy index of the peptide together contribute to the channel properties that we measure. Our sequence reversal strategy for peptide engineering to successfully obtain nanopore channels can pave the way for better biobased design of controlled nanopores, using only natural amino acids.

Molecular Mechanism of Holin Transmembrane Domain I in Pore Formation and Bacterial Cell Death.

Bacterial cell lysis during bacteriophage infection is timed by perfect orchestration between components of the holin-endolysin cassette. In bacteria, progressively accumulating holin in the inner membrane, retained in its inactive form by antiholin, is triggered into active hole formation, resulting in the canonical host cell lysis. However, the molecular mechanism of regulation and physical basis of pore formation in the mycobacterial cell membrane by D29 mycobacteriophage holin, particularly in the nonexistence of a known antiholin, is poorly understood. In this study, we report, for the first time, the use of fluorescence resonance transfer measurements to demonstrate that the first transmembrane domain (TM1) of D29 holin undergoes a helix ↔ ß-hairpin conformational interconversion. We validate that this structural malleability is mediated by a centrally positioned proline and is responsible for controlled TM1 self-association in membrana, in the presence of a proton gradient across the lipid membrane. We demonstrate that TM1 is sufficient for bacterial growth inhibition. The biological effect of D29 holin structural alteration is presented as a holin self-regulatory mechanism, and its implications are discussed in the context of holin function.

Pro-Gly mediated conformational switch of mycobacteriophage D29 holin transmembrane domain I is lipid concentration driven.

Biophysical and spectroscopic analysis of synthetic transmembrane domain I (1) of mycobacteriophage D29 holin shows a lipid concentration dependent conformational switch from an α-helix to a ß-sheet structure. The reversibility of this switch, upon change in the lipid-to-peptide ratio, requires a central Pro-Gly segment, and is abolished upon mutation to Ala-Ala or (D)Pro-Gly.