Solution structure of Alg13: the sugar donor subunit of a yeast N-acetylglucosamine transferase

Abstract

The solution structure of Alg13, the glycosyl donor-binding domain of an important bipartite glycosyltransferase in the yeast Saccharomyces cerevisiae, is presented. This glycosyltransferase is unusual in that it is active only in the presence of a binding partner, Alg14. Alg13 is found to adopt a unique topology among glycosyltransferases. Rather than the conventional Rossmann fold found in all GT-B enzymes, the N-terminal half of the protein is a Rossmann-like fold with a mixed parallel and antiparallel beta sheet. The Rossmann fold of the C-terminal half of Alg13 is conserved. However, although conventional GT-B enzymes usually possess three helices at the C terminus, only two helices are present in Alg13. Titration of Alg13 with both UDP-GlcNAc, the native glycosyl donor, and a paramagnetic mimic, UDP-TEMPO, shows that the interaction of Alg13 with the sugar donor is primarily through the residues in the C-terminal half of the protein.

Figure 1

Predicted secondary structural elements and experimentally determined secondary structural elements. Prediction is done using secondary prediction function of mGenthreader (Jones, 1999). Experimental secondary structure is determined using back bone dihedral angles predicted using TALOS and chemical shifts.

Figure 2

Annotated HSQC spectrum of deuterated Alg13. Each assigned peak is labeled with the residue number and one letter residue name.

Figure 3

A) Backbone ensemble of 10 lowest energy Alg13 structures from 50 structures calculated using XPLOR-NIH. Residues 30 to 220 are shown. The back bone is colored based on secondary structure. B) Schematic ribbon diagram of the Alg13 structure.

Figure 4

A) Schematic illustration of the predicted topology of Alg13, The numbering of the element is according to the scheme from Figure 1. Note that the predicted helix after β2 gave rise to both α3 and β3. B) Schematic illustration of the experimentally determined topology of Alg13.

Figure 5

Rotational correlation time measurements of Alg13. The C-terminus is considerably more dynamic than the rest of the protein.

Figure 6

A) Chemical shift changes produced by 2.6 mM UDP-GlcNAc. The two horizontal lines on the graph represent standard deviations in chemical shift changes. B) Mapping of UDP-GlcNAc-generated chemical shift changes on Alg13 structure. Residues with perturbation bigger than two standard deviations are colored red. This includes residues 35, 39, 123, 126, 143, 144, 145, 147, 148, 150, 155, 158, 159, 160, 166, 168 and 169. Side chains of possible carbohydrate-interacting residues (residues 165 to 158) are shown in green. C) Mapping of UDP-TEMPO-generated signal dispersion on Alg13 structure. Residues 41, 124 to 126, 139, 146, 150, 161 to 169, 171, 174, 175 and 213 to 220 are peaks that have disappeared in the presence of TEMPO and reappeared after reduction and are colored in blue. Residues 35 to 39, 141, 143, 144, 145, 147, 148 and 160 are peaks that have disappeared in the presence of TEMPO but failed to reappear after reduction of TEMPO and are colored in red. Side chains of possible carbohydrate-interacting residues (residues 165 to 158) are shown in green.