3BVT: Golgi Mannosidase Ii D204a Catalytic Nucleophile Mutant Complex With Methyl (alpha-d-mannopyranosyl)-(1->3)-s-alpha-d-mannopyranoside

Citation:
Abstract
Inhibition of Golgi alpha-mannosidase II (GMII), which acts late in the N-glycan processing pathway, provides a route to blocking cancer-induced changes in cell surface oligosaccharide structures. To probe the substrate requirements of GMII, oligosaccharides were synthesized that contained an alpha(1,3)- or alpha(1,6)-linked 1-thiomannoside. Surprisingly, these oligosaccharides were not observed in X-ray crystal structures of native Drosophila GMII (dGMII). However, a mutant enzyme in which the catalytic nucleophilic aspartate was changed to alanine (D204A) allowed visualization of soaked oligosaccharides and led to the identification of the binding site for the alpha(1,3)-linked mannoside of the natural substrate. These studies also indicate that the conformational change of the bound mannoside to a high-energy B 2,5 conformation is facilitated by steric hindrance from, and the formation of strong hydrogen bonds to, Asp204. The observation that 1-thio-linked mannosides are not well tolerated by the catalytic site of dGMII led to the synthesis of a pentasaccharide containing the alpha(1,6)-linked Man of the natural substrate and the beta(1,2)-linked GlcNAc moiety proposed to be accommodated by the extended binding site of the enzyme. A cocrystal structure of this compound with the D204A enzyme revealed the molecular interactions with the beta(1,2)-linked GlcNAc. The structure is consistent with the approximately 80-fold preference of dGMII for the cleavage of substrates containing a nonreducing beta(1,2)-linked GlcNAc. By contrast, the lysosomal mannosidase lacks an equivalent GlcNAc binding site and kinetic analysis indicates oligomannoside substrates without non-reducing-terminal GlcNAc modifications are preferred, suggesting that selective inhibitors for GMII could exploit the additional binding specificity of the GlcNAc binding site.
PDB ID: 3BVTDownload
MMDB ID: 65234
PDB Deposition Date: 2008/1/7
Updated in MMDB: 2017/11
Experimental Method:
x-ray diffraction
Resolution: 1.3  Å
Source Organism:
Similar Structures:
Biological Unit for 3BVT: monomeric; determined by author and by software (PISA)
Molecular Components in 3BVT
Label Count Molecule
Protein (1 molecule)
1
Alpha-mannosidase 2(Gene symbol: alpha-Man-IIa)
Molecule annotation
Chemicals (7 molecules)
1
1
2
1
3
1
4
1
5
1
6
2
* Click molecule labels to explore molecular sequence information.

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