51 research outputs found
Crystal structure and mutational analysis of human uracil-DNA glycosylase: Structural basis for specificity and catalysis
AbstractCrystal structures of the DNA repair enzyme human uracil-DNA glycosylase (UDG), combined with mutational analysis, reveal the structural basis for the specificity of the enzyme. Within the classic α/ÎČ fold of UDG, sequence-conserved residues form a positively charged, active-site groove the width of duplex DNA, at the C-terminal edge of the central four-stranded parallel ÎČ sheet. In the UDG-6-aminouracil complex, uracil binds at the base of the groove within a rigid preformed pocket that confers selectivity for uracil over other bases by shape complementarity and by main chain and Asn-204 side chain hydrogen bonds. Main chain nitrogen atoms are positioned to stabilize the oxyanion intermediate generated by His-268 acting via nucleophilic attack or general base mechanisms. Specific binding of uracil flipped out from a DNA duplex provides a structural mechanism for damaged base recognition
Fragment- and structure-based drug discovery for developing therapeutic agents targeting the DNA Damage Response
Cancer will directly affect the lives of over one-third of the population. The DNA Damage Response (DDR) is an intricate system involving damage recognition, cell cycle regulation, DNA repair, and ultimately cell fate determination, playing a central role in cancer etiology and therapy. Two primary therapeutic approaches involving DDR targeting include: combinatorial treatments employing anticancer genotoxic agents; and synthetic lethality, exploiting a sporadic DDR defect as a mechanism for cancer-specific therapy. Whereas, many DDR proteins have proven âundruggableâ, Fragment- and Structure-Based Drug Discovery (FBDD, SBDD) have advanced therapeutic agent identification and development. FBDD has led to 4 (with âŒ50 more drugs under preclinical and clinical development), while SBDD is estimated to have contributed to the development of >200, FDA-approved medicines. Protein X-ray crystallography-based fragment library screening, especially for elusive or âundruggableâ targets, allows for simultaneous generation of hits plus details of protein-ligand interactions and binding sites (orthosteric or allosteric) that inform chemical tractability, downstream biology, and intellectual property. Using a novel high-throughput crystallography-based fragment library screening platform, we screened five diverse proteins, yielding hit rates of âŒ2â8% and crystal structures from âŒ1.8 to 3.2 Ă
. We consider current FBDD/SBDD methods and some exemplary results of efforts to design inhibitors against the DDR nucleases meiotic recombination 11 (MRE11, a.k.a., MRE11A), apurinic/apyrimidinic endonuclease 1 (APE1, a.k.a., APEX1), and flap endonuclease 1 (FEN1)
XPD Helicase Structures and Activities: Insights into the Cancer and Aging Phenotypes from XPD Mutations
Mutations in XPD helicase, required for nucleotide excision repair (NER) as part of the transcription/repair complex TFIIH, cause three distinct phenotypes: cancer-prone xeroderma pigmentosum (XP), or aging disorders Cockayne syndrome (CS), and trichothiodystrophy (TTD). To clarify molecular differences underlying these diseases, we determined crystal structures of the XPD catalytic core from Sulfolobus acidocaldarius and measured mutant enzyme activities. Substrate-binding grooves separate adjacent Rad51/RecA-like helicase domains (HD1, HD2) and an arch formed by 4FeS and Arch domains. XP mutations map along the HD1 ATP-binding edge and HD2 DNA-binding channel and impair helicase activity essential for NER. XP/CS mutations both impair helicase activity and likely affect HD2 functional movement. TTD mutants lose or retain helicase activity but map to sites in all four domains expected to cause framework defects impacting TFIIH integrity. These results provide a foundation for understanding disease consequences of mutations in XPD and related 4Fe-4S helicases including FancJ
The James Webb Space Telescope Mission
Twenty-six years ago a small committee report, building on earlier studies,
expounded a compelling and poetic vision for the future of astronomy, calling
for an infrared-optimized space telescope with an aperture of at least .
With the support of their governments in the US, Europe, and Canada, 20,000
people realized that vision as the James Webb Space Telescope. A
generation of astronomers will celebrate their accomplishments for the life of
the mission, potentially as long as 20 years, and beyond. This report and the
scientific discoveries that follow are extended thank-you notes to the 20,000
team members. The telescope is working perfectly, with much better image
quality than expected. In this and accompanying papers, we give a brief
history, describe the observatory, outline its objectives and current observing
program, and discuss the inventions and people who made it possible. We cite
detailed reports on the design and the measured performance on orbit.Comment: Accepted by PASP for the special issue on The James Webb Space
Telescope Overview, 29 pages, 4 figure
Structural basis for endothelial nitric oxide synthase binding to calmodulin
The enzyme nitric oxide synthase (NOS) is exquisitely regulated in vivo by the Ca(2+) sensor protein calmodulin (CaM) to control production of NO, a key signaling molecule and cytotoxin. The differential activation of NOS isozymes by CaM has remained enigmatic, despite extensive research. Here, the crystal lographic structure of Ca(2+)-loaded CaM bound to a 20 residue peptide comprising the endothelial NOS (eNOS) CaM-binding region establishes their individual conformations and intermolecular interactions, and suggests the basis for isozyme-specific differences. The α-helical eNOS peptide binds in an antiparallel orientation to CaM through extensive hydrophobic interactions. Unique NOS interactions occur with: (i) the CaM flexible central linker, explaining its importance in NOS activation; and (ii) the CaM C-terminus, explaining the NOS-specific requirement for a bulky, hydrophobic residue at position 144. This binding mode expands mechanisms for CaM-mediated activation, explains eNOS deactivation by Thr495 phosphorylation, and implicates specific hydrophobic residues in the Ca(2+) independence of inducible NOS
Active and alkylated human AGT structures: a novel zinc site, inhibitor and extrahelical base binding
Human O(6)âalkylguanine-DNA alkyltransferase (AGT), which directly reverses endogenous alkylation at the O(6)-position of guanine, confers resistance to alkylation chemotherapies and is therefore an active anticancer drug target. Crystal structures of active human AGT and its biologically and therapeutically relevant methylated and benzylated product complexes reveal an unexpected zinc-stabilized helical bridge joining a two-domain α/ÎČ structure. An asparagine hinge couples the active site motif to a helixâturnâhelix (HTH) motif implicated in DNA binding. The reactive cysteine environment, its position within a groove adjacent to the alkyl-binding cavity and mutational analyses characterize DNA-damage recognition and inhibitor specificity, support a structure-based dealkylation mechanism and suggest a molecular basis for destabilization of the alkylated protein. These results support damaged nucleotide flipping facilitated by an arginine finger within the HTH motif to stabilize the extrahelical O(6)âalkylguanine without the protein conformational change originally proposed from the empty Ada structure. Cysteine alkylation sterically shifts the HTH recognition helix to evidently mechanistically couple release of repaired DNA to an opening of the protein fold to promote the biological turnover of the alkylated protein
Structures of the N^Ï-Hydroxy-l-Arginine Complex of Inducible Nitric Oxide Synthase Oxygenase Dimer with Active and Inactive Pterins
Nitric oxide synthases (NOSs) catalyze two mechanistically distinct, tetrahydrobiopterin (H4B)-dependent, heme-based oxidations that first convert l-arginine (l-Arg) to N^Ï-hydroxy-l-arginine (NHA) and then NHA to l-citrulline and nitric oxide. Structures of the murine inducible NOS oxygenase domain (iNOS_(ox)) complexed with NHA indicate that NHA and l-Arg both bind with the same conformation adjacent to the heme iron and neither interacts directly with it nor with H_4B. Steric restriction of dioxygen binding to the heme in the NHA complex suggests either small conformational adjustments in the ternary complex or a concerted reaction of dioxygen with NHA and the heme iron. Interactions of the NHA hydroxyl with active center ÎČ-structure and the heme ring polarize and distort the hydroxyguanidinium to increase substrate reactivity. Steric constraints in the active center rule against superoxo-iron accepting a hydrogen atom from the NHA hydroxyl in their initial reaction, but support an Fe(III)-peroxo-NHA radical conjugate as an intermediate. However, our structures do not exclude an oxo-iron intermediate participating in either l-Arg or NHA oxidation. Identical binding modes for active H_4B, the inactive quinonoid-dihydrobiopterin (q-H_2B), and inactive 4-amino-H_4B indicate that conformational differences cannot explain pterin inactivity. Different redox and/or protonation states of q-H_2B and 4-amino-H_4B relative to H_4B likely affect their ability to electronically influence the heme and/or undergo redox reactions during NOS catalysis. On the basis of these structures, we propose a testable mechanism where neutral H_4B transfers both an electron and a 3,4-amide proton to the heme during the first step of NO synthesis
Structures of the N^Ï-Hydroxy-l-Arginine Complex of Inducible Nitric Oxide Synthase Oxygenase Dimer with Active and Inactive Pterins
Nitric oxide synthases (NOSs) catalyze two mechanistically distinct, tetrahydrobiopterin (H4B)-dependent, heme-based oxidations that first convert l-arginine (l-Arg) to N^Ï-hydroxy-l-arginine (NHA) and then NHA to l-citrulline and nitric oxide. Structures of the murine inducible NOS oxygenase domain (iNOS_(ox)) complexed with NHA indicate that NHA and l-Arg both bind with the same conformation adjacent to the heme iron and neither interacts directly with it nor with H_4B. Steric restriction of dioxygen binding to the heme in the NHA complex suggests either small conformational adjustments in the ternary complex or a concerted reaction of dioxygen with NHA and the heme iron. Interactions of the NHA hydroxyl with active center ÎČ-structure and the heme ring polarize and distort the hydroxyguanidinium to increase substrate reactivity. Steric constraints in the active center rule against superoxo-iron accepting a hydrogen atom from the NHA hydroxyl in their initial reaction, but support an Fe(III)-peroxo-NHA radical conjugate as an intermediate. However, our structures do not exclude an oxo-iron intermediate participating in either l-Arg or NHA oxidation. Identical binding modes for active H_4B, the inactive quinonoid-dihydrobiopterin (q-H_2B), and inactive 4-amino-H_4B indicate that conformational differences cannot explain pterin inactivity. Different redox and/or protonation states of q-H_2B and 4-amino-H_4B relative to H_4B likely affect their ability to electronically influence the heme and/or undergo redox reactions during NOS catalysis. On the basis of these structures, we propose a testable mechanism where neutral H_4B transfers both an electron and a 3,4-amide proton to the heme during the first step of NO synthesis
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