Novel complex MAD phasing and RNase H structural insights using selenium oligonucleotides

The crystal structures of protein–nucleic acid complexes are commonly determined using selenium-derivatized proteins via MAD or SAD phasing. Here, the first protein–nucleic acid complex structure determined using selenium-derivatized nucleic acids is reported. The RNase H–RNA/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 (ε = 2.3 × 104 M−1 cm−1), 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 … H–N), and the SeG and C form a base pair similar to the natural G–C pair though the Se-modification causes the base-pair to shift (approximately 0.3 Å). 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

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 Å 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 Manα1–2Man, 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 O···D···O angle of 101.5°

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

Insights into the Phosphoryl Transfer Catalyzed by cAMP-Dependent Protein Kinase: An X‑ray 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, Mg²⁺, Ca²⁺, Sr²⁺, and Ba²⁺ 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 Mg²⁺, nonhydrolyzable AMP-PCP (β,γ-methyleneadenosine 5′-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