14 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
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°
Neutron Diffraction Reveals Hydrogen Bonds Critical for cGMP-Selective Activation: Insights for cGMP-Dependent Protein Kinase Agonist Design
High selectivity of cyclic-nucleotide
binding (CNB) domains for
cAMP and cGMP are required for segregating signaling pathways; however,
the mechanism of selectivity remains unclear. To investigate the mechanism
of high selectivity in cGMP-dependent protein kinase (PKG), we determined
a room-temperature joint X-ray/neutron (XN) structure of PKG IĪ²
CNB-B, a domain 200-fold selective for cGMP over cAMP, bound to cGMP
(2.2 Ć
), and a low-temperature X-ray structure of CNB-B with
cAMP (1.3 Ć
). The XN structure directly describes the hydrogen
bonding interactions that modulate high selectivity for cGMP, while
the structure with cAMP reveals that all these contacts are disrupted,
explaining its low affinity for cAMP
Genetically encoded fragment-based discovery of glycopeptide ligands for carbohydrate-binding proteins
We describe an approach to accelerate the search for competitive inhibitors for carbohydrate-recognition domains (CRDs). Genetically encoded fragment-based-discovery (GE-FBD) uses selection of phagedisplayed glycopeptides to dock a glycan fragment at the CRD and guide selection of Synergistic peptide motifs adjacent to the CRD. Starting from concanavalin A (ConA), a mannose (Man)-binding protein, as a bait, we narrowed a library of 10(8) glycopeptides to 86 leads that share a consensus motif, Man-WYD. Validation of synthetic leads yielded Man-WYDLF that exhibited 40 50-fold enhancement in affinity over methyl alpha-D-mannopyranoside (MeMan). Lectin array Suggested specificity: Man-WYD derivative bound only to 3 out of 17 proteins-ConA, LcH, and PSA-that bind to Man. An X-ray structure of ConA.:Man-WYD proved that the trimannoside core and Man-WYD exhibit identical CRD docking; but their extra-CRD binding modes are significantly. different. Still, they have comparable affinity and selectivity for various Man-binding proteins. The intriguing observation provides new insight into functional mimicry :of carbohydrates by peptide ligands. GE-FBD may provide an alternative to rapidly search for competitive inhibitors for lectins
Genetically encoded fragment-based discovery of glycopeptide ligands for carbohydrate-binding proteins
We describe an approach to accelerate the search for competitive inhibitors for carbohydrate-recognition domains (CRDs). Genetically encoded fragment-based-discovery (GE-FBD) uses selection of phagedisplayed glycopeptides to dock a glycan fragment at the CRD and guide selection of Synergistic peptide motifs adjacent to the CRD. Starting from concanavalin A (ConA), a mannose (Man)-binding protein, as a bait, we narrowed a library of 10(8) glycopeptides to 86 leads that share a consensus motif, Man-WYD. Validation of synthetic leads yielded Man-WYDLF that exhibited 40 50-fold enhancement in affinity over methyl alpha-D-mannopyranoside (MeMan). Lectin array Suggested specificity: Man-WYD derivative bound only to 3 out of 17 proteins-ConA, LcH, and PSA-that bind to Man. An X-ray structure of ConA.:Man-WYD proved that the trimannoside core and Man-WYD exhibit identical CRD docking; but their extra-CRD binding modes are significantly. different. Still, they have comparable affinity and selectivity for various Man-binding proteins. The intriguing observation provides new insight into functional mimicry :of carbohydrates by peptide ligands. GE-FBD may provide an alternative to rapidly search for competitive inhibitors for lectins