Dae-Won Sim

Anti-Inflammatory Action of an Antimicrobial Model Peptide That Suppresses the TRIF-Dependent Signaling Pathway via Inhibition of Toll-Like Receptor 4 Endocytosis in Lipopolysaccharide-Stimulated Macrophages

Antimicrobial peptides (AMPs), also called host defense peptides, particularly those with amphipathic helical structures, are emerging as target molecules for therapeutic development due to their immunomodulatory properties. Although the antimicrobial activity of AMPs is known to be exerted primarily by permeation of the bacterial membrane, the mechanism underlying its anti-inflammatory activity remains to be elucidated. We report potent anti-inflammatory activity of WALK11.3, an antimicrobial model peptide with an amphipathic helical conformation, in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. This peptide inhibited the expression of inflammatory mediators, including nitric oxide, COX-2, IL-1β, IL-6, INF-β, and TNF-α. Although WALK11.3 did not exert a major effect on all downstream signaling in the MyD88-dependent pathway, toll-like receptor 4 (TLR4)- mediated pro-inflammatory signals were markedly attenuated in the TRIF-dependent pathway due to inhibition of the phosphorylation of STAT1 by attenuation of IRF3 phosphorylation. WALK11.3 specifically inhibited the endocytosis of TLR4, which is essential for triggering TRIF-mediated signaling in macrophage cells. Hence, we suggest that specific interference with TLR4 endocytosis could be one of the major modes of the anti-inflammatory action of AMPs. Our designed WALK11 peptides, which possess both antimicrobial and anti-inflammatory activities, may be promising molecules for the development of therapies for infectious inflammation.

Structural Characterization of de Novo Designed L5K5W Model Peptide Isomers with Potent Antimicrobial and Varied Hemolytic Activities

In an effort to develop short antimicrobial peptides with simple amino acid compositions, we generated a series of undecapeptide isomers having the L5K5W formula. Amino acid sequences were designed to be perfectly amphipathic when folded into a helical conformation by converging leucines onto one side and lysines onto the other side of the helical axis. The single tryptophans, whose positions were varied in the primary structures, were located commonly at the critical amphipathic interface in the helical wheel projection. Helical conformations and the tryptophanyl environments of the 11 L5K5W peptides were confirmed and characterized by circular dichroism, fluorescence and nuclear magnetic resonance spectroscopy. All of the isomers exhibited a potent, broad-spectrum of antibacterial activity with just a slight variance in individual potency, whereas their hemolytic activities against human erythrocytes were significantly diversified. Interestingly, helical dispositions and fluorescence blue shifts of the peptides in aqueous trifluoroethanol solutions, rather than in detergent micelles, showed a marked linear correlation with their hemolytic potency. These results demonstrate that our de novo design strategy for amphipathic helical model peptides is effective for developing novel antimicrobial peptides and their hemolytic activities can be estimated in correlation with structural parameters.

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.

Verification of the interdomain contact site in the inactive monomer, and the domain-swapped fold in the active dimer of Hsp33 in solution.

Upon dimerization by oxidation, Hsp33 functions as a molecular chaperone in prokaryotes. Previously published structures of both the inactive and active species are of doubtful relevance to the solution conformations since the inactive (reduced) crystal structure was dimeric, while the active (oxidized) species was crystallized with a truncation of its regulation domain. The interdomain contact site of the inactive monomer, identified in this work, is consistent with that previously observed in the reduced dimer crystal. In contrast, fluorescence quenching of the active dimer contradicted the results expected from the domain‐swapped fold observed in the truncated dimer crystal. The results of this study provide important new information concerning controversial issues in the activation process of Hsp33.

Semi-Empirical Structure Determination of Escherichia coli Hsp33 and Identification of Dynamic Regulatory Elements for the Activation Process.

The activation process of the redox-regulated chaperone heat shock protein 33 (Hsp33) is constituted by the oxidation-induced unfolding of the C-terminal zinc-binding domain and concomitant oligomerization of the N-terminal core domain. Herein, the semi-empirical solution structure of Escherichia coli Hsp33 in the reduced, inactive form was generated through conformational space annealing calculations, utilizing minimalistic NMR data and multiple homology restraints. The various conformations of oxidized Hsp33 and some mutant forms were also investigated in solution. Interestingly, a specific region concentrated around the interdomain linker stretch and its interacting counterparts, the N-terminal β-strand 1 and α-helix 1, hardly showed up as signals in the NMR measurements. The NMR spectra of an Hsp33 derivative with a six-residue deletion in the disordered N-terminus implied a plausible conformational exchange associated with the identified region, and the corresponding exchange rate appeared slower than that of the wild type. Subsequent mutations that destroyed the structure of the β1 or α1 elements resulted in the formation of a reduced but active monomer, without the unfolding of the zinc-binding domain. Collectively, structural insights into the inactive and active conformations, including wild-type and mutant proteins, suggest that the dynamic interactions of the N-terminal segments with their contacting counterpart, the interdomain linker stretch, in the reduced, inactive state are the structural determinants regulating the activation process of the post-translationally regulated chaperone, Hsp33.

Backbone NMR assignments of a putative secretory protein from Helicobacter pylori, using a high-field (900 MHz) NMR

The HP0902, a homodimeric 22.1 kDa protein, has been suggested as a putative secretory protein from Helicobacter pylori, although the protein possesses no signal peptide for secretion. Since it may be associated with the virulence of the bacterium, NMR study has been initiated in terms of structural genomics. In our previous effort to assign the backbone NMR resonances, using 800MHz NMR machine at pH 7.8, the resonances from eight of the 99 residues could not be assined due to missing of the signals. In this work, to enhance the extent of assignments, a 900 MHz machine was employed and the sample pH was reduced down to 6.5. Finally, almost all signals, except for those from G9 and S24, could be clearly assigned. The determined secondary structure using the assined chemical shifts indicated that the HP0902 consists of 11 -strands with no helices. In our database search result, HP0902 was predicted to interact with VacA (Vacuolating cytotoxin A), which is a representative virulence factor secreted from Helicobacter pylori. Thus, molecular interaction between HP0902 and VacA would be worthy of investigation, on the basis of the present results of NMR assignments.

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.

Bacterial production and structure-functional validation of a recombinant antigen-binding fragment (Fab) of an anti-cancer therapeutic antibody targeting epidermal growth factor receptor.

Fragment engineering of monoclonal antibodies (mAbs) has emerged as an excellent paradigm to develop highly efficient therapeutic and/or diagnostic agents. Engineered mAb fragments can be economically produced in bacterial systems using recombinant DNA technologies. In this work, we established recombinant production in Escherichia coli for monovalent antigen-binding fragment (Fab) adopted from a clinically used anticancer mAB drug cetuximab targeting epidermal growth factor receptor (EGFR). Recombinant DNA constructs were designed to express both polypeptide chains comprising Fab in a single vector and to secrete them to bacterial periplasmic space for efficient folding. Particularly, a C-terminal engineering to confer an interchain disulfide bond appeared to be able to enhance its heterodimeric integrity and EGFR-binding activity. Conformational relevance of the purified final product was validated by mass spectrometry and crystal structure at 1.9xa0Å resolution. Finally, our recombinant cetuximab-Fab was found to have strong binding affinity to EGFR overexpressed in human squamous carcinoma model (A431) cells. Its binding ability was comparable to that of cetuximab. Its EGFR-binding affinity was estimated at approximately 0.7xa0nM of Kd in vitro, which was quite stronger than the binding affinity of natural ligand EGF. Hence, the results validate that our construction could serve as an efficient platform to produce a recombinant cetuximab-Fab with a retained antigen-binding functionality.

Crystallization and X-ray data collection of HP0902 from Helicobacter pylori 26695.

HP0902 from Helicobacter pylori 26695 belongs to the cupin superfamily of proteins, which encompasses proteins with a great diversity in function. In this work, two types of recombinant HP0902 protein were crystallized: one with an N-terminal His(6) tag ((H6)HP0902) and the other with a C-terminal His(6) tag (HP0902(H6)). The (H6)HP0902 crystal diffracted to 1.40 Å resolution and belonged to space group P2(1), with unit-cell parameters a = 33.5, b = 78.6, c = 41.4 Å. The HP0902(H6) crystal belonged to space group P4(3)2(1)2 or P4(1)2(1)2 and diffracted to 2.5 Å resolution, with unit-cell parameters a = b = 50.4, c = 142.0 Å.

C‐terminal dimerization of apo‐cyclic AMP receptor protein validated in solution

Although cyclic AMP receptor protein (CRP) has long served as a typical example of effector‐mediated protein allostery, mechanistic details into its regulation have been controversial due to discrepancy between the known crystal structure and NMR structure of apo‐CRP. Here, we report that the recombinant protein corresponding to its C‐terminal DNA‐binding domain (CDD) forms a dimer. This result, together with structural information obtained in the present NMR study, is consistent with the previous crystal structure and validates its relevance also in solution. Therefore, our findings suggest that dissociation of the CDD may be critically involved in cAMP‐induced allosteric activation of CRP.