Oligosialic Acid Binding Sites

In soaking and co-crystallization experiments of endoNF variants with oligosialic acid of different length, two binding sites have been identified, bl and b2, that are located in the p-barrel domain and the p-prism of the stalk domain, respectively (Table 3). In the mutant endoNF-R647A, a pentamer and a tetramer of Sia are bound to bl and b2, respectively spheres in Fig. 2c [106]). 

Polysialic acid has been reported to undergo conformational changes in solution. Several left-handed helical epitopes with different pitches have been calculated and their relative occurrence in solution has been discussed [131, 132], Polysiahc acid bound to the cell surface adopts a similar average epitope as in solution [133], A sialic acid decamer is bound in an extended helix to the monoclonal antibody 735 [134], In contrast, a sialic acid pentamer has been described to bind endoNF in a compressed helical epitope [135] which has also been measured for polysialic acid in solution [132] and on the cell surface [133], Therefore, polysialic acid selectively undergoes conformational changes upon binding to endosiahdase or monoclonal antibody. 

In the crystal structure of the mutant endoNF-R647A the two bound sialo-oligomers have been described to bind in two different left-handed helical conformations [106]. The pentamer in the bl site shows the compressed conformation with a helical pitch of 2.5 A per monomer. In contrast, the tetramer in the b2 

7 A per monomer. The average hehcal parameters of the two epitopes perfectly fit those measured previously by NMR [131, 135]. The two different epitopes may reflect important requirements for a processive degradation of polysialic acid ([105, 106] see Sect. 10). 

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The crystal structure of the catalytic part of endoNF (amino acids 246-910) has been solved in three studies [12,106,113] (Table 3). First, the protein structure has been solved in its apo state and with bound substrate after soaking of sialic acid trimer. By this means, two oligosialic acid binding sites have been identified [12] (see Fig. 2c and Sect. 8). To allow identification of longer sialic acid oligomers bound to endoNF, two active site mutants have been created upon structural information from the first structure [106]. Finally, an endoNF structure has been solved at the high resolution of 0.98 A [113]. 

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