Byong-Seok Choi

The structural basis of lipopolysaccharide recognition by the TLR4-MD-2 complex

The lipopolysaccharide (LPS) of Gram negative bacteria is a well-known inducer of the innate immune response. Toll-like receptor (TLR) 4 and myeloid differentiation factor 2 (MD-2) form a heterodimer that recognizes a common ‘pattern’ in structurally diverse LPS molecules. To understand the ligand specificity and receptor activation mechanism of the TLR4–MD-2–LPS complex we determined its crystal structure. LPS binding induced the formation of an m-shaped receptor multimer composed of two copies of the TLR4–MD-2–LPS complex arranged symmetrically. LPS interacts with a large hydrophobic pocket in MD-2 and directly bridges the two components of the multimer. Five of the six lipid chains of LPS are buried deep inside the pocket and the remaining chain is exposed to the surface of MD-2, forming a hydrophobic interaction with the conserved phenylalanines of TLR4. The F126 loop of MD-2 undergoes localized structural change and supports this core hydrophobic interface by making hydrophilic interactions with TLR4. Comparison with the structures of tetra-acylated antagonists bound to MD-2 indicates that two other lipid chains in LPS displace the phosphorylated glucosamine backbone by ∼5 Å towards the solvent area. This structural shift allows phosphate groups of LPS to contribute to receptor multimerization by forming ionic interactions with a cluster of positively charged residues in TLR4 and MD-2. The TLR4–MD-2–LPS structure illustrates the remarkable versatility of the ligand recognition mechanisms employed by the TLR family, which is essential for defence against diverse microbial infection.

CONTRASTING STRUCTURAL IMPACTS INDUCED BY cis-syn CYCLOBUTANE DIMER AND (6–4) ADDUCT IN DNA DUPLEX DECAMERS: IMPLICATION IN MUTAGENESIS AND REPAIR ACTIVITY

Abstract—

The protein shuffle. Sequential interactions among components of the human nucleotide excision repair pathway.

Xeroderma pigmentosum (XP) is an inherited disease in which cells from patients exhibit defects in nucleotide excision repair (NER). XP proteins A–G are crucial in the processes of DNA damage recognition and incision, and patients with XP can carry mutations in any of the genes that specify these proteins. In mammalian cells, NER is a dynamic process in which a variety of proteins interact with one another, via modular domains, to carry out their functions. XP proteins are key players in several steps of the NER process, including DNA strand discrimination (XPA, in complex with replication protein A), repair complex formation (XPC, in complex with hHR23B; XPF, in complex with ERCC1) and repair factor recruitment (transcription factor IIH, in complex with XPG). Through these protein–protein interactions, various types of bulky DNA adducts can be recognized and repaired. Communication between the NER system and other cellular pathways is also achieved by selected binding of the various structural domains. Here, we summarize recent studies on the domain structures of human NER components and the regulatory networks that utilize these proteins. Data provided by these studies have helped to illuminate the complex molecular interactions among NER factors in the context of DNA repair.

Unusually stable helical kink in the antimicrobial peptide — A derivative of gaegurin

The structure of an active analog of the antibacterial peptide gaegurin was investigated by CD and NMR spectroscopy. The NOE connectivities showed that 21 out of 24 residues formed an α‐helix despite the presence of a central proline. CD and NMR analysis indicates that the helix is in fast equilibrium with random coil. From chemical shift analysis of the amide protons, the distances of hydrogen bonding in the helix were calculated, and manifested obvious periodicity which implied a kink in the middle of the helix. 1D amide proton exchange experiments provided further evidence of an exceptionally stable kink. It is inferred that this kink is important not only to the function of the peptide but also to the early stage of the folding as a nucleation site.

Discovery of Hepatitis C Virus NS3 Helicase Inhibitors by a Multiplexed, High‐Throughput Helicase Activity Assay Based on Graphene Oxide

A GO‐to solution: A simple graphene oxide (GO)‐based assay to screen for selective inhibitors of a hepatitis C virus (HCV) helicase along with inhibitors of a severe acute respiratory syndrome coronavirus (SARS CoV) helicase was tested (see scheme). A single screen found five inhibitors highly selective for the HCV helicase orthologous to the SARS CoV helicase. Some of these hits were validated using the same GO‐based assay.WILEY-VCH

NMR Spectroscopic Elucidation of the B−Z Transition of a DNA Double Helix Induced by the Zα Domain of Human ADAR1

The human RNA editing enzyme ADAR1 (double-stranded RNA deaminase I) deaminates adenine in pre-mRNA to yield inosine, which codes as guanine. ADAR1 has two left-handed Z-DNA binding domains, Z alpha and Z beta, at its NH(2)-terminus and preferentially binds Z-DNA, rather than B-DNA, with high binding affinity. The cocrystal structure of Z alpha(ADAR1) complexed to Z-DNA showed that one monomeric Z alpha(ADAR1) domain binds to one strand of double-stranded DNA and a second Z alpha(ADAR1) monomer binds to the opposite strand with 2-fold symmetry with respect to DNA helical axis. It remains unclear how Z alpha(ADAR1) protein specifically recognizes Z-DNA sequence in a sea of B-DNA to produce the stable Z alpha(ADAR1)-Z-DNA complex during the B-Z transition induced by Z alpha(ADAR1). In order to characterize the molecular recognition of Z-DNA by Z alpha(ADAR1), we performed circular dichroism (CD) and NMR experiments with complexes of Zalpha(ADAR1) bound to d(CGCGCG)(2) (referred to as CG6) produced at a variety of protein-to-DNA molar ratios. From this study, we identified the intermediate states of the CG6-Z alpha(ADAR1) complex and calculated their relative populations as a function of the Z alpha(ADAR1) concentration. These findings support an active B-Z transition mechanism in which the Z alpha(ADAR1) protein first binds to B-DNA and then converts it to left-handed Z-DNA, a conformation that is then stabilized by the additional binding of a second Z alpha(ADAR1) molecule.

Residual structure within the disordered C‐terminal segment of p21Waf1/Cip1/Sdi1 and its implications for molecular recognition

Probably the most unusual class of proteins in nature is the intrinsically unstructured proteins (IUPs), because they are not structured yet play essential roles in protein‐protein signaling. Many IUPs can bind different proteins, and in many cases, adopt different bound conformations. The p21 protein is a small IUP (164 residues) that is ubiquitous in cellular signaling, for example, cell cycle control, apoptosis, transcription, differentiation, and so forth; it binds to approximately 25 targets. How does this small, unstructured protein recognize each of these targets with high affinity? Here, we characterize residual structural elements of the C‐terminal segment of p21 encompassing residues 145–164 using a combination of NMR measurements and molecular dynamics simulations. The N‐terminal half of the peptide has a significant helical propensity which is recognized by calmodulin while the C‐terminal half of the peptide prefers extended conformations that facilitate binding to the proliferating cell nuclear antigen (PCNA). Our results suggest that the final bound conformations of p21 (145–164) pre‐exist in the free peptide even without its binding partners. While the conformational flexibility of the p21 peptide is essential for adapting to diverse binding environments, the intrinsic structural preferences of the free peptide enable promiscuous yet high affinity binding to a diverse array of molecular targets.

Intrinsically unstructured N-terminal domain of bZIP transcription factor HY5.

The Arabidopsis HY5 protein is a basic leucine zipper (bZIP) transcription factor that promotes photomorphogenesis. HY5 binds directly to the promoters of light responsible element containing the G‐box and thus regulates their transcriptional activity. The level and activity of HY5 are negatively regulated, in a light‐dependent manner, by interaction with the COP1 protein, which targets HY5 for proteasome‐mediated degradation in the nucleus. Despite its essential roles in plant development, no structural information exists for HY5. In this article, we report the first structural and biophysical characterization of HY5. Using limited proteolysis in combination with mass spectrometry, circular dichroism, and nuclear magnetic resonance spectroscopy, we have deduced that the N‐terminal 77 amino acids of HY5 form a premolten globular structure, while amino acids 78–110, which constitute the basic region (BR) of the protein, exist in a molten globule state. Our studies also revealed that the overall structural features of full‐length HY5 are dominated largely by the disordered N‐terminal domain, despite the existence of a bZIP domain at its C‐terminus. We propose that HY5 is a member of the intrinsically unstructured protein (IUP) family, and that HY5 functions as an unstructured protein and benefits from being the same, in vivo. Proteins 2006.

Cnu, a Novel oriC-Binding Protein of Escherichia coli

We have found, using a newly developed genetic method, a protein (named Cnu, for oriC-binding nucleoid-associated) that binds to a specific 26-base-pair sequence (named cnb) in the origin of replication of Escherichia coli, oriC. Cnu is composed of 71 amino acids (8.4 kDa) and shows extensive amino acid identity to a group of proteins belonging to the Hha/YmoA family. Cnu was previously discovered as a protein that, like Hha, complexes with H-NS in vitro. Our in vivo and in vitro assays confirm the results and further suggest that the complex formation with H-NS is involved in Cnu/Hha binding to cnb. Unlike the hns mutants, elimination of either the cnu or hha gene did not disturb the growth rate, origin content, and synchrony of DNA replication initiation of the mutants compared to the wild-type cells. However, the cnu hha double mutant was moderately reduced in origin content. The Cnu/Hha complex with H-NS thus could play a role in optimal activity of oriC.

Structure and function of the regulatory HRDC domain from human Bloom syndrome protein

The helicase and RNaseD C-terminal (HRDC) domain, conserved among members of the RecQ helicase family, regulates helicase activity by virtue of variations in its surface residues. The HRDC domain of Bloom syndrome protein (BLM) is known as a critical determinant of the dissolution function of double Holliday junctions by the BLM–Topoisomerase IIIα complex. In this study, we determined the solution structure of the human BLM HRDC domain and characterized its DNA-binding activity. The BLM HRDC domain consists of five α-helices with a hydrophobic 310-helical loop between helices 1 and 2 and an extended acidic surface comprising residues in helices 3–5. The BLM HRDC domain preferentially binds to ssDNA, though with a markedly low binding affinity (Kd ∼100 μM). NMR chemical shift perturbation studies suggested that the critical DNA-binding residues of the BLM HRDC domain are located in the hydrophobic loop and the N-terminus of helix 2. Interestingly, the isolated BLM HRDC domain had quite different DNA-binding modes between ssDNA and Holliday junctions in electrophoretic mobility shift assay experiments. Based on its surface charge separation and DNA-binding properties, we suggest that the HRDC domain of BLM may be adapted for a unique function among RecQ helicases—that of bridging protein and DNA interactions.