HaJeung Park

Crystal structure of a DNA decamer containing a cis-syn thymine dimer

It is well known that exposure to UV induces DNA damage, which is the first step in mutagenesis and a major cause of skin cancer. Among a variety of photoproducts, cyclobutane-type pyrimidine photodimers (CPD) are the most abundant primary lesion. Despite its biological importance, the precise relationship between the structure and properties of DNA containing CPD has remained to be elucidated. Here, we report the free (unbound) crystal structure of duplex DNA containing a CPD lesion at a resolution of 2.0 Å. Our crystal structure shows that the overall helical axis bends ≈30° toward the major groove and unwinds ≈9°, in remarkable agreement with some previous theoretical and experimental studies. There are also significant differences in local structure compared with standard B-DNA, including pinching of the minor groove at the 3′ side of the CPD lesion, a severe change of the base pair parameter in the 5′ side, and serious widening of both minor and major groves both 3′ and 5′ of the CPD. Overall, the structure of the damaged DNA differs from undamaged DNA to an extent that DNA repair proteins may recognize this conformation, and the various components of the replicational and transcriptional machinery may be interfered with due to the perturbed local and global structure.

The 1.5 A crystal structure of human receptor for advanced glycation endproducts (RAGE) ectodomains reveals unique features determining ligand binding.

Interaction of the pattern recognition receptor, RAGE with key ligands such as advanced glycation end products (AGE), S100 proteins, amyloid β, and HMGB1 has been linked to diabetic complications, inflammatory and neurodegenerative disorders, and cancer. To help answer the question of how a single receptor can recognize and respond to a diverse set of ligands we have investigated the structure and binding properties of the first two extracellular domains of human RAGE, which are implicated in various ligand binding and subsequent signaling events. The 1.5-Å crystal structure reveals an elongated molecule with a large basic patch and a large hydrophobic patch, both highly conserved. Isothermal titration calorimetry (ITC) and deletion experiments indicate S100B recognition by RAGE is an entropically driven process involving hydrophobic interaction that is dependent on Ca2+ and on residues in the C′D loop (residues 54–67) of domain 1. In contrast, competition experiments using gel shift assays suggest that RAGE interaction with AGE is driven by the recognition of negative charges on AGE-proteins. We also demonstrate that RAGE can bind to dsDNA and dsRNA. These findings reveal versatile structural features of RAGE that help explain its ability to recognize of multiple ligands.

The 1.4 angstrom crystal structure of the human oxidized low density lipoprotein receptor lox-1.

The lectin-like oxidized low density lipoprotein receptor-1 (Lox-1) mediates the recognition and internalization of oxidatively modified low density lipoprotein by vascular endothelial cells. This interaction results in a number of pro-atherogenic cellular responses that probably play a significant role in the pathology of atherosclerosis. The 1.4 Å crystal structure of the extracellular C-type lectin-like domain of human Lox-1 reveals a heart-shaped homodimer with a ridge of six basic amino acids extending diagonally across the apolar top of Lox-1, a central hydrophobic tunnel that extends through the entire molecule, and an electrostatically neutral patch of 12 charged residues that resides next to the tunnel at each opening. Based on the arrangement of critical binding residues on the Lox-1 structure, we propose a binding mode for the recognition of modified low density lipoprotein and other Lox-1 ligands.

Induction and reversal of myotonic dystrophy type 1 pre-mRNA splicing defects by small molecules

The ability to control pre-mRNA splicing with small molecules could facilitate the development of therapeutics or cell-based circuits that control gene function. Myotonic dystrophy type 1 (DM1) is caused by the dysregulation of alternative pre-mRNA splicing due to sequestration of muscleblind-like 1 protein (MBNL1) by expanded, non-coding r(CUG) repeats (r(CUG)exp). Here we report two small molecules that induce or ameliorate alternative splicing dysregulation. The thiophene-containing small molecule (1) inhibits the interaction of MBNL1 with its natural pre-mRNA substrates. Compound (2), a substituted naphthyridine, binds r(CUG)exp and displaces MBNL1. Structural models show that 1 binds MBNL1 in the Zn-finger domain and that 2 interacts with UU loops in r(CUG)exp. This study provides a structural framework for small molecules that target MBNL1 by mimicking r(CUG)exp and shows that targeting MBNL1 causes dysregulation of alternative splicing, suggesting that MBNL1 is thus not a suitable therapeutic target for the treatment of DM1.

Identification of Maillard reaction products on peanut allergens that influence binding to the receptor for advanced glycation end products.

Recent immunological data demonstrated that dendritic cells preferentially recognize advanced glycation end product (AGE)‐modified proteins, upregulate expression of the receptor for AGE (RAGE), and consequently bias the immune response toward allergy.

Pharmacological repression of PPARγ promotes osteogenesis

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) is the master regulator of adipogenesis and the pharmacological target of the thiazolidinedione (TZD) class of insulin sensitizers. Activation of PPARγ by TZDs promotes adipogenesis at the expense of osteoblast formation, contributing to their associated adverse effects on bone. Recently we reported the development of PPARγ antagonist SR1664, designed to block the obesity induced phosphorylation of serine 273 (S273) in the absence of classical agonism, to derive insulin sensitizing efficacy with improved therapeutic index. Here we identify the structural mechanism by which SR1664 actively antagonizes PPARγ, and extend these findings to develop the inverse agonist SR2595. Treatment of isolated bone marrow derived mesenchymal stem cells (MSCs) with SR2595 promotes induction of osteogenic differentiation. Together these results identify the structural determinants of ligand mediated PPARγ repression, and suggest a therapeutic approach to promote bone formation.

Structural studies of Streptococcus pneumoniae EPSP synthase in unliganded state, tetrahedral intermediate-bound state and S3P-GLP-bound state.

The shikimate pathway synthesizes aromatic amino acids and other essential metabolites that are necessary for bacteria, plants and fungi to survive. This pathway is not present in vertebrates and therefore represents an attractive target for antibacterial agents. We have successfully crystallized and solved the structure of unliganded, inhibitor‐liganded and tetrahedral intermediate (TI)‐liganded forms of Streptococcus pneumoniae EPSP synthase. The overall topology of the S. pneumoniae EPSP synthase is similar to that of the Escherichia coli EPSP synthase. In addition, the majority of residues responsible for ligand binding were conserved between the two proteins. TI‐liganded structure provides absolute configuration of the C‐2 atom from the F‐PEP moiety of the enzyme‐bound intermediate and also defines key residues responsible for the enzyme reaction. Comparison of the unliganded state and substrate‐bound state of the enzyme provides insights into the structural mechanisms involved in dynamic events of ligand binding, domain movement and closure. This structural study of the pathogenic bacteria S. pneumoniae EPSP synthase with inhibitor and TI will provide invaluable information for the design of new‐generation antibiotics.

The rickettsia surface cell antigen 4 applies mimicry to bind to and activate vinculin.

Background: Rickettsiae infect the host cell by co-opting the actin cytoskeleton. Results: The Rickettsiae invasin sca4 harbors two α-helices that bind and activate vinculin; the binding mode of one of these α-helices is unique. Conclusion: Rickettsiae use mimicry of talin to subvert vinculin functions. Significance: The unique nature of the sca4-vinculin interaction suggests it can be targeted by small molecules. Pathogenic Rickettsia species cause high morbidity and mortality, especially R. prowazekii, the causative agent of typhus. Like many intracellular pathogens, Rickettsia exploit the cytoskeleton to enter and spread within the host cell. Here we report that the cell surface antigen sca4 of Rickettsia co-localizes with vinculin in cells at sites of focal adhesions in sca4-transfected cells and that sca4 binds to and activates vinculin through two vinculin binding sites (VBSs) that are conserved across all Rickettsia. Remarkably, this occurs through molecular mimicry of the vinculin-talin interaction that is also seen with the IpaA invasin of the intracellular pathogen Shigella, where binding of these VBSs to the vinculin seven-helix bundle head domain (Vh1) displaces intramolecular interactions with the vinculin tail domain that normally clamp vinculin in an inactive state. Finally, the vinculin·sca4-VBS crystal structures reveal that vinculin adopts a new conformation when bound to the C-terminal VBS of sca4. Collectively, our data define the mechanism by which sca4 activates vinculin and interacts with the actin cytoskeleton, and they suggest important roles for vinculin in Rickettsia pathogenesis.

Novel Vinculin Binding Site of the IpaA Invasin of Shigella

Internalization of Shigella into host epithelial cells, where the bacteria replicates and spreads to neighboring cells, requires a type 3 secretion system (T3SS) effector coined IpaA. IpaA binds directly to and activates the cytoskeletal protein vinculin after injection in the host cell cytosol, and this was previously thought to be directed by two amphipathic α-helical vinculin-binding sites (VBS) found in the C-terminal tail domain of IpaA. Here, we report a third VBS, IpaA-VBS3, that is located N-terminal to the other two VBSs of IpaA and show that one IpaA molecule can bind up to three vinculin molecules. Biochemical in vitro Shigella invasion assays and the 1.6 Å crystal structure of the vinculin·IpaA-VBS3 complex showed that IpaA-VBS3 is functionally redundant with the other two IpaA-VBSs in cell invasion and in activating the latent F-actin binding functions of vinculin. Multiple VBSs in IpaA are reminiscent of talin, which harbors 11 VBSs. However, most of the talin VBSs have low affinity and are buried in helix bundles, whereas all three of the VBSs of IpaA are high affinity, readily available, and in close proximity to each other in the IpaA structure. Although deletion of IpaA-VBS3 has no detectable effects on Shigella invasion of epithelial cells, deletion of all three VBSs impaired bacterial invasion to levels found in an ipaA null mutant strain. Thus, IpaA-directed mimicry of talin in activating vinculin occurs through three high affinity VBSs that are essential for Shigella pathogenesis.

Mechanism of Aldolase Control of Sorting Nexin 9 Function in Endocytosis

Sorting nexin 9 (SNX9) functions in a complex with the GTPase dynamin-2 at clathrin-coated pits, where it provokes fission of vesicles to complete endocytosis. Here the SNX9·dynamin-2 complex binds to clathrin and adapter protein complex 2 (AP-2) that line these pits, and this occurs through interactions of the low complexity domain (LC4) of SNX9 with AP-2. Intriguingly, localization of the SNX9·dynamin-2 complex to clathrin-coated pits is blocked by interactions with the abundant glycolytic enzyme aldolase, which also binds to the LC4 domain of SNX9. The crystal structure of the LC4 motif of human SNX9 in complex with aldolase explains the biochemistry and biology of this interaction, where SNX9 binds near the active site of aldolase via residues 165–171 that are also required for the interactions of SNX9 with AP-2. Accordingly, SNX9 binding to aldolase is structurally precluded by the binding of substrate to the active site. Interactions of SNX9 with aldolase are far more extensive and differ from those of the actin-nucleating factor WASP with aldolase, indicating considerable plasticity in mechanisms that direct the functions of the aldolase as a scaffold protein.