Structural basis for specific recognition of core fucosylation in N-glycans by Pholiota squarrosa lectin (PhoSL).

Fucosylation of the N-glycan core via the α1-6 linkage (core fucosylation) is detected in specific types of cancers and related diseases, and thereby serves for a relevant biomarker. The lectin from a mushroom Pholiota squarrosa (PhoSL) shows a clear specificity to core fucosylation, without recognizing those with other types of fucosylation, such as the H type via the α1-2 linkage or the Lewis type via the α1-3 or α1-4 linkage. Here we determined the crystal structure of the PhoSL trimer in complex with a disaccharide fucose(α1-6)N-acetylglucosamine (GlcNAc). In the three sugar-binding pockets of PhoSL, extensive hydrophobic and hydrogen-bonding contacts were formed with the fucose moiety. In contrast, the GlcNAc moiety showed only a few hydrophobic and hydrogen-bonding contacts. To elucidate the mechanism for the specificity, we performed molecular dynamics simulations on this disaccharide and a trisaccharide fucose(α1-6)[GlcNAc(ß1-4)]GlcNAc in complex with PhoSL. It was observed that the GlcNAc corresponding to the outer one of the N-glycan core entered the sugar-binding pocket with the N-acetyl group placed stably at the bottom, forming extensive hydrophobic and hydrogen-bonding interactions. In addition, these glycans adopted unstressed favorable conformations when bound to PhoSL. In contrast, H- and Lewis-types of fucosylated trisaccharides adopting favorable conformations caused inevitable steric hindrance with the steep edge of the binding pocket, when docked with PhoSL. Therefore, the specificity to core fucosylation of PhoSL was achieved by a combination of these preferential and exclusive mechanisms.

Is it possible for short peptide composed of positively- and negatively-charged "hydrophilic" amino acid residue-clusters to form metastable "hydrophobic" packing?

We theoretically and experimentally analyzed a conformational ensemble of a small, characteristic polypeptide consisting of positively- and negatively-charged amino acid residue clusters, (Lys)9(Glu)9(Lys)9, designed based on the supercoiled DNA-recognition (SDR) domain, with the capability of preferentially binding to supercoiled DNA. Advanced molecular dynamics (MD) simulations coupled with a generalized ensemble technique revealed that substantial amounts of ordered, helical structures were present at the boundaries of the Lys and Glu segments in the obtained conformational ensemble. In fact, the helical content of the peptide calculated from our MD simulations was consistent with that estimated from our experimental analysis employing circular dichroism (CD) spectroscopy. The statistical analysis of the structural ensemble revealed the metastable hydrophobic contact clusters, which were stabilized by closely cohesive residue contacts, formed through "hybrid" hydrophobic (methylene groups) and electrostatic (salt bridges) residue contacts. Both short-range and long-range residue contacts were involved in the metastable hydrophobic clusters, constituting the aforementioned local helical conformations and the compact entire structures, respectively. A significant helical propensity was also found in the (Lys)n and (Glu)m boundaries of other conventional protein structures deposited in the Protein Data Bank (PDB), thus indicating the generality of this conformational trend that has been identified herein.

Revisiting antibody modeling assessment for CDR-H3 loop.

The antigen-binding site of antibodies, also known as complementarity-determining region (CDR), has hypervariable sequence properties. In particular, the third CDR loop of the heavy chain, CDR-H3, has such variability in its sequence, length, and conformation that ordinary modeling techniques cannot build a high-quality structure. At Stage 2 of the Second Antibody Modeling Assessment (AMA-II) held in 2013, the model structures of the CDR-H3 loops were submitted by the seven modelers and were critically assessed. After our participation in AMA-II, we rebuilt one of the long CDR-H3 loops with 13 residues (A52 antibody) by a more precise method, using enhanced conformational sampling with the explicit water model, as compared to our previous method employed at AMA-II. The current stable models obtained from the free energy landscape at 300 K include structures similar to the X-ray crystal structures. Those models were not built in our previous work at AMA-II. The current free energy landscape suggested that the CDR-H3 loop structures in the crystal are not stable in solution, but they are stabilized by the crystal packing effect.