Defining the Specificity of Carbohydrate-Protein Interactions by Quantifying Functional Group Contributions.

Protein-carbohydrate interactions are significant in a wide range of biological processes, disruption of which has been implicated in many different diseases. The capability of glycan-binding proteins (GBPs) to specifically bind to the corresponding glycans allows GBPs to be utilized in glycan biomarker detection or conversely to serve as targets for therapeutic intervention. However, understanding the structural origins of GBP specificity has proven to be challenging due to their typically low binding affinities (mM) and their potential to display broad or complex specificities. Here we perform molecular dynamics (MD) simulations and post-MD energy analyses with the Poisson-Boltzmann and generalized Born solvent models (MM-PB/GBSA) of the Erythrina cristagalli lectin (ECL) with its known ligands, and with new cocrystal structures reported herein. While each MM-PB/GBSA parametrization resulted in different estimates of the desolvation free energy, general trends emerged that permit us to define GBP binding preferences in terms of ligand substructure specificity. Additionally, we have further decomposed the theoretical interaction energies into contributions made between chemically relevant functional groups. Based on these contributions, the functional groups in each ligand can be assembled into a pharmacophore comprised of groups that are either critical for binding, or enhance binding, or are noninteracting. It is revealed that the pharmacophore for ECL consists of the galactopyranose (Gal) ring atoms along with C6 and the O3 and O4 hydroxyl groups. This approach provides a convenient method for identifying and quantifying the glycan pharmacophore and provides a novel method for interpreting glycan specificity that is independent of residue-level glycan nomenclature. A pharmacophore approach to defining specificity is readily transferable to molecular design software and, therefore, may be particularly useful in designing therapeutics (glycomimetics) that target GBPs.

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°.

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 phage-displayed 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 α-d-mannopyranoside (MeMan). Lectin array suggested specificity: Man-WYD derivative bound only to 3 out of 17 proteins­ConA, LcH, and PSA­that 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.

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 Iß CNB-B, a domain 200-fold selective for cGMP over cAMP, bound to cGMP (2.2 Å), and a low-temperature X-ray structure of CNB-B with cAMP (1.3 Å). 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.

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 (epsilon = 2.3 x 10(4) 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 (Se)G 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 A). 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.