Hyung-Sik Won

Antimicrobial Peptides for Therapeutic Applications: A Review

Antimicrobial peptides (AMPs) have been considered as potential therapeutic sources of future antibiotics because of their broad-spectrum activities and different mechanisms of action compared to conventional antibiotics. Although AMPs possess considerable benefits as new generation antibiotics, their clinical and commercial development still have some limitations, such as potential toxicity, susceptibility to proteases, and high cost of peptide production. In order to overcome those obstacles, extensive efforts have been carried out. For instance, unusual amino acids or peptido-mimetics are introduced to avoid the proteolytic degradation and the design of short peptides retaining antimicrobial activities is proposed as a solution for the cost issue. In this review, we focus on small peptides, especially those with less than twelve amino acids, and provide an overview of the relationships between their three-dimensional structures and antimicrobial activities. The efforts to develop highly active AMPs with shorter sequences are also described.

Structural overview on the allosteric activation of cyclic AMP receptor protein

Cyclic AMP receptor protein (CRP) is a prokaryotic global transcription regulator that controls the expression of nearly 200 genes. The protein, allosterically activated by cAMP binding, binds to DNA and interacts with RNA polymerase. Current understanding on the allosteric process of the Escherichia coli CRP activation can be summarized into a rigid-body movement that involves subunit realignment and domain rearrangement. The main consequence of that overall transition is protrusion and adjustment of F-helices that recognize specific DNA sites. Although physicochemical and structural studies during the past decades have contributed to a comprehensive understanding of the CRP allostery, a paucity of structural information about the cAMP-free form (apo-CRP) has precluded a definite elucidation of the allosterism. In this respect, recent achievements of structures on other CRP-family proteins provide useful information to fill in the details of the allosteric transition of CRP. Thus, in this paper, accomplishments of CRP-family structures are summarized and inspected comparatively with new findings. This review not only provides a structural overview on the allosteric conformational change of CRP but also suggests a thoughtful discussion about unsolved issues or conflicting arguments. Solving those issues and the apo-CRP structure would enable us to finally define the CRP allostery.

De novo generation of short antimicrobial peptides with simple amino acid composition

As potential therapeutic agents, antimicrobial peptides with shorter length and simpler amino acid composition can be better candidates for clinical and commercial development. Here, we attempted de novo design of short (5- to 11-residue) antimicrobial peptides with three kinds of amino acids. Amphipathic helical properties were conferred by using leucines and lysines and two tryptophan residues were positioned at the critical amphipathic interface between the hydrophilic ending side and the hydrophobic starting side. According to this specified rule, 12 model peptides were generated and their helical propensity was confirmed by circular dichroism spectroscopy. Antimicrobial and hemolytic activities were compared with those of the known 12-residue peptide agent, omiganan, which is currently under therapeutic and commercial development. Antimicrobial activities against Gram-negative and Gram-positive bacteria, including a multi-drug resistant strain, were observed for certain 7- to 11-residue models. Among them, the most potent activity was found for a 9-residue peptide (L₅K₂W₂), although it also had severe hemolytic activity. Alternatively, an 11-residue peptide (L₄K₅W₂) with little hemolytic activity was potentially the most useful agent, as it showed higher antibacterial activity than omiganan. These results not only suggest useful candidates for novel antibiotic development, but also provide an efficient strategy to design such peptides.

Stoichiometry and Structural Effect of the Cyclic Nucleotide Binding to Cyclic AMP Receptor Protein

Cyclic AMP receptor protein (CRP) is a homodimeric protein, which is activated by cAMP binding to function as a transcriptional regulator of many genes in prokaryotes. Until now, the actual number of cAMP molecules that can be bound by CRP in solution has been ambiguous. In this work, we performed a nuclear magnetic resonance study on CRP to investigate the stoichiometry of cyclic nucleotide binding to CRP. A series of1H-15N heteronuclear single quantum coherence (HSQC) spectra of the protein in the absence and in the presence of cAMP or cGMP were analyzed. The addition of cAMP to CRP induced a biphasic spectral change up to 4 equivalents, whereas the cGMP addition made a monophasic change up to 2 equivalents. Altogether, the results not only established for the first time that CRP possesses two cyclic AMP-binding sites in each monomer, even in a solution without DNA, but also suggest that the syn-cAMP binding sites of the CRP dimer can be formed by an allosteric conformational change of the protein upon the binding of two anti-cAMPs at the N-terminal domain. In addition, a residue-specific inspection of the spectral changes provides some new structural information about the cAMP-induced allosteric activation of CRP.

Action mechanism and structural requirements of the antimicrobial peptides, gaegurins.

Gaegurins (GGNs) are a family of cationic, alpha-helical, antimicrobial peptides that were isolated from a Korean frog, Glandirana emeljanovi (formerly classified as Rana rugosa) and represent one of the structurally well-characterized groups. Among six gaegurins, gaegurin 4 (renamed herein esculentin-2EM), gaegurin 5 (brevinin-1EMa), and gaegurin 6 (brevinin-1EMb) have been investigated comprehensively in terms of structure-activity relationships. In this paper, we first suggest renaming of gaegurins according to a recently raised rule of systematic nomenclature. Then, the current understanding of gaegurins is reviewed by summarizing their structure-activity relationships. In particular competing arguments on gaegurins are synthetically inspected. Finally their action mechanism and structural requirements will be discussed.

De novo generation of antimicrobial LK peptides with a single tryptophan at the critical amphipathic interface

De novo design of amphipathic model peptides has been successful for generating many antimicrobial peptides with various lengths and amino acid compositions. Here, we suggest a very simple strategy to design antimicrobial peptides with a short length and a simple amino acid composition. Amphipathic helical properties were conferred by using only leucines and lysines and a single tryptophan was positioned at the critical amphipathic interface between the hydrophilic ending side and the hydrophobic starting side, in the helical wheel projection. According to this rule, the model peptides with 7 to 13 residues exhibited antimicrobial activity. Among them, the most potent activity against both Gram‐positive and Gram‐negative bacteria, covering all of the nine bacterial strains tested in this study, was found for the 11‐mer sequences having a 1:1 (L5K5W6) or a 3:2 (L6K4W6) ratio of leucines to lysines. In particular, the former peptide L5K5W6 could be evaluated as the most useful agent, as it showed no significant hemolytic activity with a broad‐spectrum of antimicrobial activity. Copyright

Functional identification of toxin-antitoxin molecules from Helicobacter pylori 26695 and structural elucidation of the molecular interactions.

Bacterial toxin-antitoxin (TA) systems are associated with many important cellular processes including antibiotic resistance and microorganism virulence. Here, we identify and structurally characterize TA molecules from the gastric pathogen, Helicobacter pylori. The HP0894 protein had been previously suggested, through our structural genomics approach, to be a putative toxin molecule. In this study, the intrinsic RNase activity and the bacterial cell growth-arresting activity of HP0894 were established. The RNA-binding surface was identified at three residue clusters: (Lys8 and Ser9), (Lys50–Lys54 and Glu58), and (Arg80 and His84–Phe88). In particular, the -UA- and -CA- sequences in RNA were preferentially cleaved by HP0894, and residues Lys52, Trp53, and Ser85–Lys87 were observed to be the main contributors to sequence recognition. The action of HP0894 could be inhibited by the HP0895 protein, and the HP0894-HP0895 complex formed an oligomer with a binding stoichiometry of 1:1. The N and C termini of HP0894 constituted the binding sites to HP0895. In contrast, the unstructured C-terminal region of HP0895 was responsible for binding to HP0894 and underwent a conformational change in the process. Finally, DNA binding activity was observed for both HP0895 and the HP0894-0895 complex but not for HP0894 alone. Taken together, it is concluded that the HP0894-HP0895 protein couple is a TA system in H. pylori, where HP0894 is a toxin with an RNase function, whereas HP0895 is an antitoxin functioning by binding to both the toxin and DNA.

Oxidative Dimerization of PHD2 is Responsible for its Inactivation and Contributes to Metabolic Reprogramming via HIF-1α Activation

Prolyl hydroxylase domain protein 2 (PHD2) belongs to an evolutionarily conserved superfamily of 2-oxoglutarate and Fe(II)-dependent dioxygenases that mediates homeostatic responses to oxygen deprivation by mediating hypoxia-inducible factor-1α (HIF-1α) hydroxylation and degradation. Although oxidative stress contributes to the inactivation of PHD2, the precise molecular mechanism of PHD2 inactivation independent of the levels of co-factors is not understood. Here, we identified disulfide bond-mediated PHD2 homo-dimer formation in response to oxidative stress caused by oxidizing agents and oncogenic H-rasV12 signalling. Cysteine residues in the double-stranded β-helix fold that constitutes the catalytic site of PHD isoforms appeared responsible for the oxidative dimerization. Furthermore, we demonstrated that disulfide bond-mediated PHD2 dimerization is associated with the stabilization and activation of HIF-1α under oxidative stress. Oncogenic H-rasV12 signalling facilitates the accumulation of HIF-1α in the nucleus and promotes aerobic glycolysis and lactate production. Moreover, oncogenic H-rasV12 does not trigger aerobic glycolysis in antioxidant-treated or PHD2 knocked-down cells, suggesting the participation of the ROS-mediated PHD2 inactivation in the oncogenic H-rasV12-mediated metabolic reprogramming. We provide here a better understanding of the mechanism by which disulfide bond-mediated PHD2 dimerization and inactivation result in the activation of HIF-1α and aerobic glycolysis in response to oxidative stress.

Effects of salt and nickel ion on the conformational stability of Bacillus pasteurii UreE

UreE, a urease accessory protein, is proposed to be a metallochaperone assisting the nickel incorporation into the urease active site. We investigated the effects of salt and nickel on the conformational stability of the UreE from Bacillus pasteurii (BpUreE), by circular dichroism (CD) and nuclear magnetic resonance spectroscopy accompanying a thermodynamic inspection. Far‐UV CD spectra of BpUreE showed that both salt and nickel stabilized the ordered structure of the protein. The thermal denaturing of BpUreE showed a bimodal feature with an aggregation process before thermal unfolding. This thermally induced aggregation could be suppressed by the addition of salt up to 50 mM, and the further addition of salt increased the thermal resistance of the protein. The nickel addition also elevated the thermal resistance of BpUreE, although it could not prevent the aggregating process. Additionally, the stoichiometry of a specific nickel binding to BpUreE was revealed as one nickel per dimer. Altogether, the present results establish a rather detailed characterization of the thermostability and nickel‐binding property of BpUreE.

Activity Optimization of an Undecapeptide Analogue Derived from a Frog-Skin Antimicrobial Peptide

While natural antimicrobial peptides are potential therapeutic agents, their physicochemical properties and bioactivity generally need to be enhanced for clinical and commercial development. We have previously developed a cationic, amphipathic α-helical, 11-residue peptide (named herein GA-W2: FLGWLFKWASK-NH2) with potent antimicrobial and hemolytic activity, which was derived from a 24-residue, natural antimicrobial peptide isolated from frog skin. Here, we attempted to optimize peptide bioactivity by a rational approach to sequence modification. Seven analogues were generated from GA-W2, and their activities were compared with that of a 12-residue peptide, omiganan, which is being developed for clinical and commercial applications. Most of the modifications reported here improved antimicrobial activity. Among them, the GA-K4AL (FAKWAFKWLKK-NH2) peptide displayed the most potent antimicrobial activity with negligible hemolytic activity, superior to that of omiganan. The therapeutic index of GA-K4AL was improved more than 53- and more than 31-fold against Gram-negative and Gram-positive bacteria, respectively, compared to that of the starting peptide, GA-W2. Given its relatively shorter length and simpler amino acid composition, our sequence-optimized GA-K4AL peptide may thus be a potentially useful antimicrobial peptide agent.