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author:("Gan, jinhua")
1.  Meclofenamic acid selectively inhibits FTO demethylation of m6A over ALKBH5 
Nucleic Acids Research  2014;43(1):373-384.
Two human demethylases, the fat mass and obesity-associated (FTO) enzyme and ALKBH5, oxidatively demethylate abundant N6-methyladenosine (m6A) residues in mRNA. Achieving a method for selective inhibition of FTO over ALKBH5 remains a challenge, however. Here, we have identified meclofenamic acid (MA) as a highly selective inhibitor of FTO. MA is a non-steroidal, anti-inflammatory drug that mechanistic studies indicate competes with FTO binding for the m6A-containing nucleic acid. The structure of FTO/MA has revealed much about the inhibitory function of FTO. Our newfound understanding, revealed herein, of the part of the nucleotide recognition lid (NRL) in FTO, for example, has helped elucidate the principles behind the selectivity of FTO over ALKBH5. Treatment of HeLa cells with the ethyl ester form of MA (MA2) has led to elevated levels of m6A modification in mRNA. Our collective results highlight the development of functional probes of the FTO enzyme that will (i) enable future biological studies and (ii) pave the way for the rational design of potent and specific inhibitors of FTO for use in medicine.
doi:10.1093/nar/gku1276
PMCID: PMC4288171  PMID: 25452335
2.  Synthesis of 6-Se-Guanosine RNAs for Structural Study 
Organic letters  2013;15(15):3934-3937.
6-Se-guanosine phosphoramidite and RNAs have been synthesized by selenium substitution of the 6-oxygen atom and it is revealed that the Se-derivatization is relatively stable and that bulge and wobble structures can better accommodate a large Se atom than duplex. This Se-modification is useful in structural study of RNAs and their protein complexes.
doi:10.1021/ol401698n
PMCID: PMC3763942  PMID: 23859218
3.  Structure-Based DNA-Targeting Strategies with Small Molecule Ligands for Drug Discovery 
Medicinal research reviews  2013;33(5):1119-1173.
Nucleic acids are the molecular targets of many clinical anticancer drugs. However, compared with proteins, nucleic acids have traditionally attracted much less attention as drug targets in structure-based drug design, partially because limited structural information of nucleic acids complexed with potential drugs is available. Over the past several years, enormous progresses in nucleic acid crystallization, heavy-atom derivatization, phasing, and structural biology have been made. Many complicated nucleic acid structures have been determined, providing new insights into the molecular functions and interactions of nucleic acids, especially DNAs complexed with small molecule ligands. Thus, opportunities have been created to further discover nucleic acid-targeting drugs for disease treatments. This review focuses on the structure studies of DNAs complexed with small molecule ligands for discovering lead compounds, drug candidates, and/or therapeutics.
doi:10.1002/med.21278
PMCID: PMC3954796  PMID: 23633219
nucleic acids; structure-based drug discovery; small molecule ligands; modification and derivatization; structures of DNA–ligand complexes
4.  Structural insights of non-canonical U•U pair and Hoogsteen interaction probed with Se atom 
Nucleic Acids Research  2013;41(22):10476-10487.
Unlike DNA, in addition to the 2′-OH group, uracil nucleobase and its modifications play essential roles in structure and function diversities of non-coding RNAs. Non-canonical U•U base pair is ubiquitous in non-coding RNAs, which are highly diversified. However, it is not completely clear how uracil plays the diversifing roles. To investigate and compare the uracil in U-A and U•U base pairs, we have decided to probe them with a selenium atom by synthesizing the novel 4-Se-uridine (SeU) phosphoramidite and Se-nucleobase-modified RNAs (SeU-RNAs), where the exo-4-oxygen of uracil is replaced by selenium. Our crystal structure studies of U-A and U•U pairs reveal that the native and Se-derivatized structures are virtually identical, and both U-A and U•U pairs can accommodate large Se atoms. Our thermostability and crystal structure studies indicate that the weakened H-bonding in U-A pair may be compensated by the base stacking, and that the stacking of the trans-Hoogsteen U•U pairs may stabilize RNA duplex and its junction. Our result confirms that the hydrogen bond (O4…H-C5) of the Hoogsteen pair is weak. Using the Se atom probe, our Se-functionalization studies reveal more insights into the U•U interaction and U-participation in structure and function diversification of nucleic acids.
doi:10.1093/nar/gkt799
PMCID: PMC3905866  PMID: 24013566
5.  Hydrogen bond formation between the naturally modified nucleobase and phosphate backbone 
Nucleic Acids Research  2012;40(16):8111-8118.
Natural RNAs, especially tRNAs, are extensively modified to tailor structure and function diversities. Uracil is the most modified nucleobase among all natural nucleobases. Interestingly, >76% of uracil modifications are located on its 5-position. We have investigated the natural 5-methoxy (5-O-CH3) modification of uracil in the context of A-form oligonucleotide duplex. Our X-ray crystal structure indicates first a H-bond formation between the uracil 5-O-CH3 and its 5′-phosphate. This novel H-bond is not observed when the oxygen of 5-O-CH3 is replaced with a larger atom (selenium or sulfur). The 5-O-CH3 modification does not cause significant structure and stability alterations. Moreover, our computational study is consistent with the experimental observation. The investigation on the uracil 5-position demonstrates the importance of this RNA modification at the atomic level. Our finding suggests a general interaction between the nucleobase and backbone and reveals a plausible function of the tRNA 5-O-CH3 modification, which might potentially rigidify the local conformation and facilitates translation.
doi:10.1093/nar/gks426
PMCID: PMC3439885  PMID: 22641848
6.  Novel RNA base pair with higher specificity using single selenium atom 
Nucleic Acids Research  2012;40(11):5171-5179.
Specificity of nucleobase pairing provides essential foundation for genetic information storage, replication, transcription and translation in all living organisms. However, the wobble base pairs, where U in RNA (or T in DNA) pairs with G instead of A, might compromise the high specificity of the base pairing. The U/G wobble pairing is ubiquitous in RNA, especially in non-coding RNA. In order to increase U/A pairing specificity, we have hypothesized to discriminate against U/G wobble pair by tailoring the steric and electronic effects at the 2-exo position of uridine and replacing the 2-exo oxygen with a selenium atom. We report here the first synthesis of the 2-Se-U-RNAs as well as the 2-Se-uridine (SeU) phosphoramidite. Our biophysical and structural studies of the SeU-RNAs indicate that this single atom replacement can indeed create a novel U/A base pair with higher specificity than the natural one. We reveal that the SeU/A pair maintains a structure virtually identical to the native U/A base pair, while discriminating against U/G wobble pair. This oxygen replacement with selenium offers a unique chemical strategy to enhance the base pairing specificity at the atomic level.
doi:10.1093/nar/gks010
PMCID: PMC3367167  PMID: 22323523
7.  Crystal structure of an isolated, unglycosylated antibody CH2 domain 
The CH2 (CH3 for IgM and IgE) domain of an antibody plays an important role in mediating effector functions and preserving antibody stability. It is the only domain in human immunoglobulins (Igs) which is involved in weak interchain protein-protein interactions with another CH2 domain solely through sugar moieties. The N-linked glycosylation at Asn297 is conserved for mammalian IgGs as well as homologous regions of other antibody isotypes. To examine the structural details of the CH2 domain in the absence of glycosylation and other antibody domains, we determined the crystal structure of an isolated, unglycosylated antibody γ1 CH2 domain at 1.7 Å, and compared it with the corresponding CH2 structures from intact Fc, IgG and Fc receptor complexes. Furthermore, we studied the oligomeric state of the protein in solution using size exclusion chromatography. The results suggested that the unglycosylated human antibody CH2 domain is a monomer and its structure is similar to that found in the intact Fc, IgG and Fc receptor complex structures. However, we observed certain structural variations along the Fc receptor binding sites. Owing to the small size, stability and non-immunogenic Ig template, the CH2 domain structure could be useful for the development of antibody domains exerting some effector functions and/or antigen specificity if made by protein design, and, as a robust scaffold in protein engineering applications.
doi:10.1107/S0907444908025274
PMCID: PMC2596763  PMID: 18931413
antibody; immunoglobulin; unglycosylated CH2, IgG; Fc; CH2 domain
8.  Structure of RapA, a Swi2/Snf2 Protein That Recycles RNA Polymerase during Transcription 
Structure (London, England : 1993)  2008;16(9):1417-1427.
Summary
RapA, as abundant as σ70 in the cell, is an RNA polymerase (RNAP)-associated Swi2/Snf2 protein with ATPase activity. It stimulates RNAP recycling during transcription. Here, we report the first structure of RapA, which is also the first full-length structure for the entire Swi2/Snf2 family. RapA contains seven domains, two of which exhibit novel protein folds. Our model of RapA in complex with ATP and double-stranded (ds) DNA suggests that RapA may bind to and translocate on dsDNA. Our kinetic template-switching assay shows that RapA facilitates the release of sequestered RNAP from a posttranscrption/posttermination complex (PTC) for transcription reinitiation. Our in vitro competition experiment indicates that RapA binds to core RNAP only but is readily displaceable by σ70. RapA is likely another general transcription factor, the structure of which provides a framework for future studies of this bacterial Swi2/Snf2 protein and its important roles in RNAP recycling during transcription.
doi:10.1016/j.str.2008.06.012
PMCID: PMC2607195  PMID: 18786404
9.  Structure of an isolated unglycosylated antibody CH2 domain 
The crystal structure of an isolated unglycosylated antibody CH2 domain has been determined at 1.7 Å resolution.
The CH2 (CH3 for IgM and IgE) domain of an antibody plays an important role in mediating effector functions and preserving antibody stability. It is the only domain in human immunoglobulins (Igs) which is involved in weak interchain protein–protein interactions with another CH2 domain solely through sugar moieties. The N-linked glycosylation at Asn297 is conserved in mammalian IgGs as well as in homologous regions of other antibody isotypes. To examine the structural details of the CH2 domain in the absence of glycosylation and other antibody domains, the crystal structure of an isolated unglycosylated antibody γ1 CH2 domain was determined at 1.7 Å resolution and compared with corresponding CH2 structures from intact Fc, IgG and Fc receptor complexes. Furthermore, the oligomeric state of the protein in solution was studied using size-exclusion chromatography. The results suggested that the unglycosylated human antibody CH2 domain is a monomer and that its structure is similar to that found in the intact Fc, IgG and Fc receptor complex structures. However, certain structural variations were observed in the Fc receptor-binding sites. Owing to its small size, stability and non-immunogenic Ig template, the CH2-domain structure could be useful for the development by protein design of antibody domains exerting effector functions and/or antigen specificity and as a robust scaffold in protein-engineering applications.
doi:10.1107/S0907444908025274
PMCID: PMC2596763  PMID: 18931413
antibodies; immunoglobulins; unglycosylated CH2; IgG; Fc; CH2 domains
10.  Structural Basis for the Aldolase and Epimerase Activities of Staphylococcus aureus Dihydroneopterin Aldolase 
Journal of molecular biology  2007;368(1):161-169.
Dihydroneopterin aldolase (DHNA) catalyzes the conversion of 7,8-dihydroneopterin (DHNP) to 6-hydroxymethyl-7,8-dihydropterin (HP) and also the epimerization of DHNP to 7,8-dihydromonopterin (DHMP). Although crystal structures of the enzyme from several microorganisms have been reported, no structural information is available about the critical interactions between DHNA and the trihydroxypropyl moiety of the substrate, which undergoes bond cleavage and formation. Here, we present the structures of Staphylococcus aureus DHNA (SaDHNA) in complex with neopterin (NP, an analog of DHNP) and with monapterin (MP, an analog of DHMP), filling the gap in the structural analysis of the enzyme. In combination with previously reported SaDHNA structures in its ligand-free form (PDB entry 1DHN) and in complex with HP (PDB entry 2DHN), four snapshots for the catalytic center assembly along the reaction pathway can be derived, advancing our knowledge about the molecular mechanism of SaDHNA-catalyzed reactions. An additional step appears to be necessary for the epimerization of DHMP to DHNP. Three active site residues (E22, K100, and Y54) function coordinately during catalysis: together, they organize the catalytic center assembly, and individually, each plays a central role at different stages of the catalytic cycle.
doi:10.1016/j.jmb.2007.02.009
PMCID: PMC1885205  PMID: 17331536
aldolase; dihydroneopterin aldolase; dihydroneopterin; dihydromonapterin; pterin

Results 1-10 (10)