Ji-Hun Kim

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 and biochemical characterization of HP0315 from Helicobacter pylori as a VapD protein with an endoribonuclease activity

VapD-like virulence-associated proteins have been found in many organisms, but little is known about this protein family including the 3D structure of these proteins. Recently, a relationship between the Cas2 family of ribonucleases associated with the CRISPR system of microbial immunity and VapD was suggested. Here, we show for the first time the structure of a member of the VapD family and present a relationship of VapD with Cas2 family and toxin–antitoxin (TA) systems. The crystal structure of HP0315 from Helicobacter pylori was solved at a resolution of 2.8u2009Å. The structure of HP0315, which has a modified ferredoxin-like fold, is very similar to that of the Cas2 family. Like Cas2 proteins, HP0315 shows endoribonuclease activity. HP0315-cleaved mRNA, mainly before A and G nucleotides preferentially, which means that HP0315 has purine-specific endoribonuclease activity. Mutagenesis studies of HP0315 revealed that D7, L13, S43 and D76 residues are important for RNase activity, in contrast, to the Cas2 family. HP0315 is arranged as an operon with HP0316, which was found to be an antitoxin-related protein. However, HP0315 is not a component of the TA system. Thus, HP0315 may be an evolutionary intermediate which does not belong to either the Cas2 family or TA system.

Structural and functional studies of the Mycobacterium tuberculosis VapBC30 toxin-antitoxin system: implications for the design of novel antimicrobial peptides

Toxin-antitoxin (TA) systems play important roles in bacterial physiology, such as multidrug tolerance, biofilm formation, and arrest of cellular growth under stress conditions. To develop novel antimicrobial agents against tuberculosis, we focused on VapBC systems, which encompass more than half of TA systems in Mycobacterium tuberculosis. Here, we report that theMycobacterium tuberculosis VapC30 toxin regulates cellular growth through both magnesium and manganese ion-dependent ribonuclease activity and is inhibited by the cognate VapB30 antitoxin. We also determined the 2.7-Å resolution crystal structure of the M. tuberculosis VapBC30 complex, which revealed a novel process of inactivation of the VapC30 toxin via swapped blocking by the VapB30 antitoxin. Our study on M. tuberculosis VapBC30 leads us to design two kinds of VapB30 and VapC30-based novel peptides which successfully disrupt the toxin-antitoxin complex and thus activate the ribonuclease activity of the VapC30 toxin. Our discovery herein possibly paves the way to treat tuberculosis for next generation.

Structure-based functional identification of Helicobacter pylori HP0268 as a nuclease with both DNA nicking and RNase activities

HP0268 is a conserved, uncharacterized protein from Helicobacter pylori. Here, we determined the solution structure of HP0268 using three-dimensional nuclear magnetic resonance (NMR) spectroscopy, revealing that this protein is structurally most similar to a small MutS-related (SMR) domain that exhibits nicking endonuclease activity. We also demonstrated for the first time that HP0268 is a nicking endonuclease and a purine-specific ribonuclease through gel electrophoresis and fluorescence spectroscopy. The nuclease activities for DNA and RNA were maximally increased by Mn2+ and Mg2+ ions, respectively, and decreased by Cu2+ ions. Using NMR chemical shift perturbations, the metal and nucleotide binding sites of HP0268 were determined to be spatially divided but close to each other. The lysine residues (Lys7, Lys11 and Lys43) are clustered and form the nucleotide binding site. Moreover, site-directed mutagenesis was used to define the catalytic active site of HP0268, revealing that this site contains two acidic residues, Asp50 and Glu54, in the metal binding site. The nucleotide binding and active sites are not conserved in the structural homologues of HP0268. This study will contribute to improving our understanding of the structure and functionality of a wide spectrum of nucleases.

Solution structure of hypothetical protein HP1423 (Y1423_HELPY) reveals the presence of αL motif related to RNA binding

Helicobacter pylori has uniqueness to survive in the extreme acidic environment in stomach. It is an important human bacterial pathogen, causing diverse gastric diseases such as peptic ulcers, chronic gastritis, and mucosa-associated lymphoid tissue lymphoma.1 In addition, the fact that duodenal ulcers are also associated with H. pylori infection has been proposed.2 H. pylori was isolated from human stomachs in 1979 for the first time,3 and whole-genome analysis of H. pylori was completed in the United States in 1997.4 Until now, complete genome sequences for the H. pylori strain 26695, J99 and HPAG1 have been determined. Even though the genomic information is known well, the functions of many genes are still unknown. The functional studies are still required to expand our biological understanding for H. pylori. In this regard, determining three-dimensional structures of unknown proteins can lead to the elucidation of biological function of proteins and the discovery of new drug target candidate for antibiotics. In this study, we determined the solution structure of HP1423 (Y1423_HELPY), which has 84 amino acid residues, conserved hypothetical protein from H. pylori strain 26695. According to Pfam database, HP1423 belongs to S4 (PF01479) superfamily. The S4 domain is a small domain consisting of 60–65 amino acid residues that probably mediates binding to RNA.5 The structure of HP1423 revealed the presence of the aL-RNA binding motif in the protein, which is a general feature of several RNA binding protein families.

NMR solution structure of HP0827 (O25501_HELPY) from Helicobacter pylori: model of the possible RNA-binding site.

The HP0827 protein is an 82-residue protein identified as a putative ss-DNA-binding protein 12RNP2 Precursor from Helicobacter pylori. Here, we have determined 3D structure of HP0827 using Nuclear Magnetic Resonance. It has a ferredoxin-like fold, beta1-alpha1-beta2-beta3-alpha2-beta4 (alpha; alpha-helix and beta; beta-sheet) and ribonucleoprotein (RNP) motifs which are thought to be important in RNA binding. By using structural homologues search and analyzing electrostatic potential of surface, we could compared HP0827 with other RNA-binding proteins (sex-lethal, T-cell restricted intracellular antigen-1, U1A) to predict RNA-binding sites of HP0827. We could predict that beta sheets of HP0827, especially beta1 and beta3, are primary region for RNA binding. Consequently, similar to other RNA-binding proteins, RNP motifs (Y5, F45, F47), positively charged and hydrophobic regions (K32, R37, K40, K41, K43, R70, R73) are proposed as a putative RNA-binding sites. In addition, differences in amino acids composition of RNP motifs, N, C-terminal residues, loop-region fold and the orientation of alpha1-helix with other RNA recognition motif proteins could give specific biological functions to HP0827. Finally, the study on natural RNA target is also important to completely understand the biological function of HP0827.

Application of Solution NMR to Structural Studies on α-Helical Integral Membrane Proteins

A large portion of proteins in living organisms are membrane proteins which play critical roles in the biology of the cell, from maintenance of the biological membrane integrity to communication of cells with their surroundings. To understand their mechanism of action, structural information is essential. Nevertheless, structure determination of transmembrane proteins is still a challenging area, even though recently the number of deposited structures of membrane proteins in the PDB has rapidly increased thanks to the efforts using X-ray crystallography, electron microscopy, and solid and solution nuclear magnetic resonance (NMR) technology. Among these technologies, solution NMR is a powerful tool for studying protein-protein, protein-ligand interactions and protein dynamics at a wide range of time scales as well as structure determination of membrane proteins. This review provides general and useful guideline for membrane protein sample preparation and the choice of membrane-mimetic media, which are the key step for successful structural analysis. Furthermore, this review provides an opportunity to look at recent applications of solution NMR to structural studies on α-helical membrane proteins through some success stories.

Structural characterization of HP1264 reveals a novel fold for the flavin mononucleotide binding protein.

Complex I (NADH-quinone oxidoreductase) is an enzyme that catalyzes the initial electron transfer from nicotinamide adenine dinucleotide (NADH) to flavin mononucleotide (FMN) bound at the tip of the hydrophilic domain of complex I. The electron flow into complex I is coupled to the generation of a proton gradient across the membrane that is essential for the synthesis of ATP. However, Helicobacter pylori has an unusual complex I that lacks typical NQO1 and NQO2 subunits, both of which are generally included in the NADH dehydrogenase domain of complex I. Here, we determined the solution structure of HP1264, one of the unusual subunits of complex I from H. pylori, which is located in place of NQO2, by three-dimensional nuclear magnetic resonance (NMR) spectroscopy and revealed that HP1264 can bind to FMN through UV-visible, fluorescence, and NMR titration experiments. This result suggests that FMN-bound HP1264 could be involved in the initial electron transfer step of complex I. In addition, HP1264 is structurally most similar to Escherichia coli TusA, which belongs to the SirA-like superfamily having an IF3-like fold in the SCOP database, implying that HP1264 adopts a novel fold for FMN binding. On the basis of the NMR titration data, we propose the candidate residues Ile32, Met34, Leu58, Trp68, and Val71 of HP1264 for the interaction with FMN. Notably, these residues are not conserved in the FMN binding site of any other flavoproteins with known structure. This study of the relationship between the structure and FMN binding property of HP1264 will contribute to improving our understanding of flavoprotein structure and the electron transfer mechanism of complex I.

Backbone 1 H, 15 N, and 13 C Resonances Assignment and Secondary Structure Prediction of SAV0506 from Staphylococcus aureus

SAV0506 is an 87 residue hypothetical protein from Staphylococcus aureus strain Mu50 and also predicted to have similar function to ribosome associated heat shock protein, Hsp 15. Hsp15 is thought to be involved in the repair mechanism of erroneously produced 50S ribosome subunit. In this report, we present the sequence specific backbone resonance assignment of SAV0506. About 82.5% of all resonances could be assigned unambiguously. By analyzing deviations of the and chemical shift values, we could predict the secondary structure of SAV0506. This study is an essential step towards the structural characterization of SAV0506.

Backbone 1 H, 15 N, and 13 C resonance assignments and secondary structure prediction of NifU-like protein, HP1492 from Helicobacter Pylori

HP1492 is a NifU-like protein of Helicobacter pylori (H. pylori) and plays a role as a scaffold which transfer Fe-S cluster to Fe-S proteins like Ferredoxin. To understand how to bind to iron ion or iron-sulfur cluster, HP1492 was expressed and purified in Escherichia coli (E. coli). From the NMR measurement, we could carry out the sequence specific backbone resonance assignment of HP1492. Approximately 91% of all resonances could be assigned unambiguously. By analyzing results of CSI and TALOS from NMR data, we could predict the secondary structure of HP1492, which consists of three -helices and three -sheets. This study is an essential step towards the structural characterization of HP1492.