Search tips
Search criteria

Results 1-14 (14)

Clipboard (0)

Select a Filter Below

more »
Year of Publication
Document Types
1.  Structure of the Myotonic Dystrophy Type 2 RNA and Designed Small Molecules That Reduce Toxicity 
ACS chemical biology  2013;9(2):538-550.
Myotonic dystrophy type 2 (DM2) is an untreatable neuromuscular disorder caused by a r(CCUG) expansion (r(CCUG)exp) that folds into an extended hairpin with periodically repeating 2×2 nucleotide internal loops (5’CCUG/3’GUCC). We designed multivalent compounds that improve DM2-associated defects using information about RNA-small molecule interactions. We also report the first crystal structure of r(CCUG)exp refined to 2.35 Å. Structural analysis of the three 5’CCUG/3’GUCC repeat internal loops (L) reveals that the CU pairs in L1 are each stabilized by one hydrogen bond and a water-mediated hydrogen bond while CU pairs in L2 and L3 are stabilized by two hydrogen bonds. Molecular dynamics (MD) simulations reveal that the CU pairs are dynamic and stabilized by Na+ and water molecules. MD simulations of the binding of the small molecule to r(CCUG) repeats reveal that the lowest free energy binding mode occurs via the major groove, in which one C residue is unstacked and the cross-strand nucleotides are displaced. Moreover, we modeled the binding of our dimeric compound to two 5’CCUG/3’GUCC motifs, which shows that the scaffold on which the RNA-binding modules are displayed provides an optimal distance to span two adjacent loops.
PMCID: PMC3944380  PMID: 24341895
2.  Crystal structure of UbiX, an aromatic acid decarboxylase from the psychrophilic bacterium Colwellia psychrerythraea that undergoes FMN-induced conformational changes 
Scientific Reports  2015;5:8196.
The ubiX gene of Colwellia psychrerythraea strain 34H encodes a 3-octaprenyl-4-hydroxybenzoate carboxylase (CpsUbiX, UniProtKB code: Q489U8) that is involved in the third step of the ubiquinone biosynthesis pathway and harbors a flavin mononucleotide (FMN) as a potential cofactor. Here, we report the crystal structures of two forms of CpsUbiX: an FMN-bound wild type form and an FMN-unbound V47S mutant form. CpsUbiX is a dodecameric enzyme, and each monomer possesses a typical Rossmann-fold structure. The FMN-binding domain of UbiX is composed of three neighboring subunits. The highly conserved Gly15, Ser41, Val47, and Tyr171 residues play important roles in FMN binding. Structural comparison of the FMN-bound wild type form with the FMN-free form reveals a significant conformational difference in the C-terminal loop region (comprising residues 170–176 and 195–206). Subsequent computational modeling and liposome binding assay both suggest that the conformational flexibility observed in the C-terminal loops plays an important role in substrate and lipid bindings. The crystal structures presented in this work provide structural framework and insights into the catalytic mechanism of CpsUbiX.
PMCID: PMC4316190  PMID: 25645665
3.  Structural Basis and Biological Consequences for JNK2/3 Isoform Selective Aminopyrazoles 
Scientific Reports  2015;5:8047.
Three JNK isoforms, JNK1, JNK2, and JNK3 have been reported and unique biological function has been ascribed to each. It is unknown if selective inhibition of these isoforms would confer therapeutic or safety benefit. To probe JNK isoform function we designed JNK2/3 inhibitors that have >30-fold selectivity over JNK1. Utilizing site-directed mutagenesis and x-ray crystallography we identified L144 in JNK3 as a key residue for selectivity. To test whether JNK2/3 selective inhibitors protect human dopaminergic neurons against neurotoxin-induced mitochondrial dysfunction, we monitored reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP). The results showed that JNK2/3 selective inhibitors protected against 6-hydroxydopamine-induced ROS generation and MMP depolarization. These results suggest that it was possible to develop JNK2/3 selective inhibitors and that residues in hydrophobic pocket I were responsible for selectivity. Moreover, the findings also suggest that inhibition of JNK2/3 likely contributed to protecting mitochondrial function and prevented ultimate cell death.
PMCID: PMC4306959  PMID: 25623238
4.  Identifcation of Maillard reaction product on peanut allergens that influence binding to the receptor for advanced glycation end products 
Allergy  2013;68(12):1546-1554.
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 towards allergy.
Peanut extract was characterized by mass spectrometry (MS) to elucidate the specific residues and specific AGE modifications found in raw and roasted peanuts and on rAra h 1 that was artificially glycated by incubation with glucose or xylose. The binding of the RAGE-V1C1 domain to peanut allergens was assessed by PAGE and Western analysis with anti-Ara h 1, 2, and 3 antibodies. IgE binding to rAra h 1 was also assessed using the same methods.
AGE modifications were found on Ara h 1 and Ara h 3 in both raw and roasted peanut extract. No AGE modifications were found on Ara h 2. MS and Western blot analysis demonstrated RAGE binds selectively to Ara h 1 and Ara h 3 derived from peanut extract whereas the analysis failed to demonstrate Ara h 2 binding to RAGE. rAra h 1 with no AGE modifications did not bind RAGE, however after AGE modification with xylose, rAra h 1 bound to RAGE.
AGE modifications to Ara h 1 and Ara h 3 can be found in both raw and roasted peanuts. RAGE was demonstrated to selectively interact with AGE modified rAra h 1. If sensitization to peanut allergens occurs in dendritic cells via RAGE interactions, these cells are likely interacting with modified Ara h 1, and Ara h 3, and not Ara h 2.
PMCID: PMC3915869  PMID: 24266677
5.  Molecular Mechanisms of Host Cytoskeletal Rearrangements by Shigella Invasins 
Pathogen-induced reorganization of the host cell cytoskeleton is a common strategy utilized in host cell invasion by many facultative intracellular bacteria, such as Shigella, Listeria, enteroinvasive E. coli and Salmonella. Shigella is an enteroinvasive intracellular pathogen that preferentially infects human epithelial cells and causes bacillary dysentery. Invasion of Shigella into intestinal epithelial cells requires extensive remodeling of the actin cytoskeleton with the aid of pathogenic effector proteins injected into the host cell by the activity of the type III secretion system. These so-called Shigella invasins, including IpaA, IpaC, IpgB1, IpgB2 and IpgD, modulate the actin-regulatory system in a concerted manner to guarantee efficient entry of the bacteria into host cells.
PMCID: PMC4227214  PMID: 25310650
actin; bacillary dysentery; bacterial proteins; invasin; review; Shigella
6.  A Small Molecule Bidentate-Binding Dual Inhibitor Probe of the LRRK2 and JNK Kinases 
ACS chemical biology  2013;8(8):1747-1754.
Both JNK and LRRK2 are associated with Parkinson’s disease (PD). Here we report a reasonably selective and potent kinase inhibitor (compound 6) that bound to both JNK and LRRK2 (a dual inhibitor). A bidentate-binding strategy that simultaneously utilized the ATP hinge binding and a unique protein surface site outside of the ATP pocket was applied to the design and identification of this kind of inhibitor. Compound 6 was a potent JNK3 and modest LRRK2 dual inhibitor with an enzyme IC50 value of 12 nM and 99 nM (LRRK2-G2019S), respectively. 6 also exhibited good cell potency, inhibited LRRK2:G2019S induced mitochondrial dysfunction in SHSY5Y cells, and was demonstrated to be reasonably selective against a panel of 116 kinases from representative kinase families. Design of such a probe molecule may help enable testing if dual JNK and LRRK2 inhibitions have added or synergistic efficacy in protecting against neurodegeneration in PD.
PMCID: PMC3759981  PMID: 23751758
Dual Inhibitor; Bidentate-Binding kinase inhibitors; JNK3; LRRK2; Parkinson’s Disease; PD
7.  Induction and Reversal of Myotonic Dystrophy Type 1 Pre-mRNA Splicing Defects by Small Molecules 
Nature communications  2013;4:2044.
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.
PMCID: PMC3710115  PMID: 23806903
8.  Crystallization and preliminary X-ray crystallographic analysis of the human kindlin-2 PH domain 
The PH domain of human kindlin-2 was expressed, purified and crystallized. A complete X-ray diffraction data set was obtained at 2.8 Å resolution.
Kindlins contribute to the correct assembly of integrin-containing focal adhesion sites through their direct interaction with the cytoplasmic tail of β-­integrin. The FERM domain of kindlins has a unique subdomain organization: the F2 subdomain harbours a centrally located pleckstrin homology (PH) domain that is thought to be involved in the membrane targeting of kindlins. FERM domains are found in a number of cytoskeletal proteins that mediate the interaction between integrins and cytosolic proteins. In the present study, the PH domain of human kindlin-2 was subcloned, solubly expressed in Escherichia coli and crystallized using the hanging-drop vapour-diffusion method. A diffraction data set was collected at 2.8 Å resolution using synchrotron radiation on BL-4A at the Pohang Accelerator Laboratory (Pohang, Republic of Korea).
PMCID: PMC3107146  PMID: 21636915
focal adhesion; kindlin; pleckstrin homology domain; talin
9.  Myotonic Dystrophy Type 1 RNA Crystal Structures Reveal Heterogeneous 1×1 Nucleotide UU Internal Loop Conformations⊥ 
Biochemistry  2011;50(45):9928-9935.
RNA internal loops often display a variety of conformations in solution. Herein, we visualize conformational heterogeneity in the context of the 5′CUG/3′GUC repeat motif present in the RNA that causes myotonic dystrophy type 1 (DM1). Specifically, two crystal structures are disclosed of a model DM1 triplet repeating construct, 5′r(UUGGGC(CUG)3GUCC)2, refined to 2.20 Å and 1.52 Å resolution. Here, differences in orientation of the 5′ dangling UU end between the two structures induce changes in the backbone groove width, which reveals that non-canonical 1×1 nucleotide UU internal loops can display an ensemble of pairing conformations. In the 2.20 Å structure, CUGa, the 5′UU forms one hydrogen-bonded pairs with a 5′UU of a neighboring helix in the unit cell to form a pseudo-infinite helix. The central 1×1 nucleotide UU internal loop has no hydrogen bonds, while the terminal 1×1 nucleotide UU internal loops each form a one hydrogen-bonded pair. In the 1.52 Å structure, CUGb, the 5′ UU dangling end is tucked into the major groove of the duplex. While the canonical paired bases show no change in base pairing, in CUGb the terminal 1×1 nucleotide UU internal loops form now two hydrogen-bonded pairs. Thus, the shift in major groove induced by the 5′UU dangling end alters non-canonical base patterns. Collectively, these structures indicate that 1×1 nucleotide UU internal loops in DM1 may sample multiple conformations in vivo. This observation has implications for the recognition of this RNA, and other repeating transcripts, by protein and small molecule ligands.
PMCID: PMC3218087  PMID: 21988728
10.  Dramatic improvement of crystal quality for low-­temperature-grown rabbit muscle aldolase 
Rabbit muscle aldolase (RMA) was crystallized in complex with the low-complexity domain (LC4) of sorting nexin 9. Monoclinic crystals were obtained at room temperature that displayed large mosaicity and poor X-ray diffraction. However, orthorhombic RMA–LC4 crystals grown at 277 K under similar conditions exhibited low mosaicity, allowing data collection to 2.2 Å Bragg spacing and structure determination.
Rabbit muscle aldolase (RMA) was crystallized in complex with the low-complexity domain (LC4) of sorting nexin 9. Monoclinic crystals were obtained at room temperature that displayed large mosaicity and poor X-ray diffraction. However, orthorhombic RMA–LC4 crystals grown at 277 K under similar conditions exhibited low mosaicity, allowing data collection to 2.2 Å Bragg spacing and structure determination. It was concluded that the improvement of crystal quality as indicated by the higher resolution of the new RMA–LC4 complex crystals was a consequence of the introduction of new lattice contacts at lower temperature. The lattice contacts corresponded to an increased number of interactions between high-entropy side chains that mitigate the lattice strain incurred upon cryocooling and accompanying mosaic spread increases. The thermodynamically unfavorable immobilization of high-entropy side chains used in lattice formation was compensated by an entropic increase in the bulk-solvent content owing to the greater solvent content of the crystal lattice.
PMCID: PMC2864701  PMID: 20445268
rabbit muscle aldolase; improvement of crystal quality; low-complexity domain; sorting nexin 9
11.  TonB Interacts with Nonreceptor Proteins in the Outer Membrane of Escherichia coli 
Journal of Bacteriology  2002;184(6):1640-1648.
The Escherichia coli TonB protein serves to couple the cytoplasmic membrane proton motive force to active transport of iron-siderophore complexes and vitamin B12 across the outer membrane. Consistent with this role, TonB has been demonstrated to participate in strong interactions with both the cytoplasmic and outer membranes. The cytoplasmic membrane determinants for that interaction have been previously characterized in some detail. Here we begin to examine the nature of TonB interactions with the outer membrane. Although the presence of the siderophore enterochelin (also known as enterobactin) greatly enhanced detectable cross-linking between TonB and the outer membrane receptor, FepA, the absence of enterochelin did not prevent the localization of TonB to the outer membrane. Furthermore, the absence of FepA or indeed of all the iron-responsive outer membrane receptors did not alter this association of TonB with the outer membrane. This suggested that TonB interactions with the outer membrane were not limited to the TonB-dependent outer membrane receptors. Hydrolysis of the murein layer with lysozyme did not alter the distribution of TonB, suggesting that peptidoglycan was not responsible for the outer membrane association of TonB. Conversely, the interaction of TonB with the outer membrane was disrupted by the addition of 4 M NaCl, suggesting that these interactions were proteinaceous. Subsequently, two additional contacts of TonB with the outer membrane proteins Lpp and, putatively, OmpA were identified by in vivo cross-linking. These contacts corresponded to the 43-kDa and part of the 77-kDa TonB-specific complexes described previously. Surprisingly, mutations in these proteins individually did not appear to affect TonB phenotypes. These results suggest that there may be multiple redundant sites where TonB can interact with the outer membrane prior to transducing energy to the outer membrane receptors.
PMCID: PMC134908  PMID: 11872715
12.  A dynamic structural model of expanded RNA CAG repeats: A refined X-ray structure and computational investigations using molecular dynamics and umbrella sampling simulations 
One class of functionally important RNA is repeating transcripts that cause disease through various mechanisms. For example, expanded r(CAG) repeats can cause Huntington’s and other disease through translation of toxic proteins. Herein, crystal structure of r[5ʹUUGGGC(CAG)3GUCC]2, a model of CAG expanded transcripts, refined to 1.65 Å resolution is disclosed that show both anti-anti and syn-anti orientations for 1×1 nucleotide AA internal loops. Molecular dynamics (MD) simulations using Amber force field in explicit solvent were run for over 500 ns on model systems r(5ʹGCGCAGCGC)2 (MS1) and r(5ʹCCGCAGCGG)2 (MS2). In these MD simulations, both anti-anti and syn-anti AA base pairs appear to be stable. While anti-anti AA base pairs were dynamic and sampled multiple anti-anti conformations, no syn-anti↔anti-anti transformations were observed. Umbrella sampling simulations were run on MS2, and a 2D free energy surface was created to extract transformation pathways. In addition, over 800 ns explicit solvent MD simulation was run on r[5ʹGGGC(CAG)3GUCC]2, which closely represents the refined crystal structure. One of the terminal AA base pairs (syn-anti conformation), transformed to anti-anti conformation. The pathway followed in this transformation was the one predicted by umbrella sampling simulations. Further analysis showed a binding pocket near AA base pairs in syn-anti conformations. Computational results combined with the refined crystal structure show that global minimum conformation of 1×1 nucleotide AA internal loops in r(CAG) repeats is anti-anti but can adopt syn-anti depending on the environment. These results are important to understand RNA dynamic-function relationships and develop small molecules that target RNA dynamic ensembles.
PMCID: PMC3625063  PMID: 23441937
Triplet disease; Huntington’s disease; X-RAY structure of CAG repeat RNA construct; RNA loop dynamics; molecular dynamics simulations; umbrella sampling
13.  Discovery of potent and selective covalent inhibitors of JNK 
Chemistry & biology  2012;19(1):140-154.
The mitogen activated kinases JNK1/2/3 are key enzymes in signaling modules that transduce and integrate extracellular stimuli into coordinated cellular response. Here we report the discovery of the first irreversible inhibitors of JNK1/2/3. We describe two JNK3 co-crystal structures at 2.60 and 2.97 Å resolutions that show the compounds form covalent bonds with a conserved cysteine residue. JNK-IN-8 is a selective JNK inhibitor that inhibits phosphorylation of c-Jun, a direct substrate of JNK kinase, in cells exposed to sub-micromolar drug in a manner that depends on covalent modification of the conserved cysteine residue. Extensive biochemical, cellular and pathway-based profiling establish the selectivity of JNK-IN-8 for JNK and suggest that the compound will be broadly useful as a pharmacological probe of JNK-dependent signal transduction. Potential lead compounds have also been identified for kinases including IRAK1, PIK3C3, PIP4K2C, and PIP5K3.
PMCID: PMC3270411  PMID: 22284361

Results 1-14 (14)