30 research outputs found
Novel complex MAD phasing and RNase H structural insights using selenium oligonucleotides
The crystal structures of proteinānucleic acid complexes are commonly determined using selenium-derivatized proteins via MAD or SAD phasing. Here, the first proteinānucleic acid complex structure determined using selenium-derivatized nucleic acids is reported. The RNase HāRNA/DNA complex is used as an example to demonstrate the proof of principle. The high-resolution crystal structure indicates that this selenium replacement results in a local subtle unwinding of the RNA/DNA substrate duplex, thereby shifting the RNA scissile phosphate closer to the transition state of the enzyme-catalyzed reaction. It was also observed that the scissile phosphate forms a hydrogen bond to the water nucleophile and helps to position the water molecule in the structure. Consistently, it was discovered that the substitution of a single O atom by a Se atom in a guide DNA sequence can largely accelerate RNase H catalysis. These structural and catalytic studies shed new light on the guide-dependent RNA cleavage
Derivatization of DNAs with selenium at 6-position of guanine for function and crystal structure studies
To investigate nucleic acid base pairing and stacking via atom-specific mutagenesis and crystallography, we have synthesized for the first time the 6-Se-deoxyguanosine phosphoramidite and incorporated it into DNAs via solid-phase synthesis with a coupling yield over 97%. We found that the UV absorption of the Se-DNAs red-shifts over 100 nm to 360 nm (Īµ = 2.3 Ć 104 Mā1 cmā1), the Se-DNAs are yellow colored, and this Se modification is relatively stable in water and at elevated temperature. Moreover, we successfully crystallized a ternary complex of the Se-G-DNA, RNA and RNase H. The crystal structure determination and analysis reveal that the overall structures of the native and Se-modified nucleic acid duplexes are very similar, the selenium atom participates in a Se-mediated hydrogen bond (Se ā¦ HāN), and the SeG and C form a base pair similar to the natural GāC pair though the Se-modification causes the base-pair to shift (approximately 0.3 Ć
). Our biophysical and structural studies provide new insights into the nucleic acid flexibility, duplex recognition and stability. Furthermore, this novel selenium modification of nucleic acids can be used to investigate chemogenetics and structure of nucleic acids and their protein complexes
Productive reorientation of a bound oxime reactivator revealed in room temperature X-ray structures of native and VX-inhibited human acetylcholinesterase.
A new crystal form of human acetylcholinesterase for exploratory room-temperature crystallography studies.
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A new crystal form of human acetylcholinesterase for exploratory room-temperature crystallography studies
Structure-guided design of novel pharmacologically active molecules relies at least in part on functionally relevant accuracy of macromolecular structures for template based drug design. Currently, about 95% of all macromolecular X-ray structures available in the PDB (Protein Data Bank) were obtained from diffraction experiments at low, cryogenic temperatures. However, it is known that functionally relevant conformations of both macromolecules and pharmacological ligands can differ at higher, physiological temperatures. We describe in this article development and properties of new human acetylcholinesterase (AChE) crystals of space group P31 and a new unit cell, amenable for room-temperature X-ray diffraction studies. We co-crystallized hAChE in P31 unit cell with the reversible inhibitor 9-aminoacridine that binds at the base of the active center gorge in addition to inhibitors that span the full length of the gorge, donepezil (Aricept, E2020) and AChE specific inhibitor BW284c51. Their new low temperature P31 space group structures appear similar to those previously obtained in the different P3121 unit cell. Successful solution of the new room temperature 3.2 Ć
resolution structure of BW284c51*hAChE complex from large P31 crystals enables us to proceed with studying room temperature structures of lower affinity complexes, such as oxime reactivators bound to hAChE, where temperature-related conformational diversity could be expected in both oxime and hAChE, which could lead to better informed structure-based design under conditions approaching physiological temperature
Mannobiose Binding Induces Changes in Hydrogen Bonding and Protonation States of Acidic Residues in Concanavalin A As Revealed by Neutron Crystallography
Plant lectins are
carbohydrate-binding proteins with various biomedical
applications. Concanavalin A (Con A) holds promise in treating cancerous
tumors. To better understand the Con A carbohydrate binding specificity,
we obtained a room-temperature neutron structure of this legume lectin
in complex with a disaccharide ManĪ±1ā2Man, mannobiose.
The neutron structure afforded direct visualization of the hydrogen
bonding between the protein and ligand, showing that the ligand is
able to alter both protonation states and interactions for residues
located close to and distant from the binding site. An unprecedented
low-barrier hydrogen bond was observed forming between the carboxylic
side chains of Asp28 and Glu8, with the D atom positioned equidistant
from the oxygen atoms having an OĀ·Ā·Ā·DĀ·Ā·Ā·O
angle of 101.5Ā°
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Productive reorientation of a bound oxime reactivator revealed in room temperature X-ray structures of native and VX-inhibited human acetylcholinesterase
Exposure to organophosphorus compounds (OPs) may be fatal if untreated, and a clear and present danger posed by nerve agent OPs has become palpable in recent years. OPs inactivate acetylcholinesterase (AChE) by covalently modifying its catalytic serine. Inhibited AChE cannot hydrolyze the neurotransmitter acetylcholine leading to its build-up at the cholinergic synapses and creating an acute cholinergic crisis. Current antidotes, including oxime reactivators that attack the OP-AChE conjugate to free the active enzyme, are inefficient. Better reactivators are sought, but their design is hampered by a conformationally rigid portrait of AChE extracted exclusively from 100K X-ray crystallography and scarcity of structural knowledge on human AChE (hAChE). Here, we present room temperature X-ray structures of native and VX-phosphonylated hAChE with an imidazole-based oxime reactivator, RS-170B. We discovered that inhibition with VX triggers substantial conformational changes in bound RS-170B from a "nonproductive" pose (the reactive aldoxime group points away from the VX-bound serine) in the reactivator-only complex to a "semi-productive" orientation in the VX-modified complex. This observation, supported by concurrent molecular simulations, suggested that the narrow active-site gorge of hAChE may be significantly more dynamic than previously thought, allowing RS-170B to reorient inside the gorge. Furthermore, we found that small molecules can bind in the choline-binding site hindering approach to the phosphorous of VX-bound serine. Our results provide structural and mechanistic perspectives on the reactivation of OP-inhibited hAChE and demonstrate that structural studies at physiologically relevant temperatures can deliver previously overlooked insights applicable for designing next-generation antidotes
Room Temperature Neutron Crystallography of Drug Resistant HIV1 Protease Uncovers Limitations of Xray Structural Analysis at 100 K
Insights into the Phosphoryl Transfer Catalyzed by cAMP-Dependent Protein Kinase: An Xāray Crystallographic Study of Complexes with Various Metals and Peptide Substrate SP20
X-ray
structures of several ternary substrate and product complexes
of the catalytic subunit of cAMP-dependent protein kinase (PKAc) have
been determined with different bound metal ions. In the PKAc complexes,
Mg<sup>2+</sup>, Ca<sup>2+</sup>, Sr<sup>2+</sup>, and Ba<sup>2+</sup> metal ions could bind to the active site and facilitate the phosphoryl
transfer reaction. ATP and a substrate peptide (SP20) were modified,
and the reaction products ADP and the phosphorylated peptide were
found trapped in the enzyme active site. Finally, we determined the
structure of a pseudo-Michaelis complex containing Mg<sup>2+</sup>, nonhydrolyzable AMP-PCP (Ī²,Ī³-methyleneadenosine 5ā²-triphosphate)
and SP20. The product structures together with the pseudo-Michaelis
complex provide snapshots of different stages of the phosphorylation
reaction. Comparison of these structures reveals conformational, coordination,
and hydrogen bonding changes that might occur during the reaction
and shed new light on its mechanism, roles of metals, and active site
residues