Mapping substrate interactions of the human membrane-associated neuraminidase, NEU3, using STD NMR.
Related Articles Mapping substrate interactions of the human membrane-associated neuraminidase, NEU3, using STD NMR.
Glycobiology. 2014 Oct 6;
Authors: Albohy A, Richards MR, Cairo CW
Abstract
Saturation transfer difference (STD) NMR is a powerful technique which can be used to investigate interactions between proteins and their substrates. The method identifies specific sites of interaction found on a small molecule ligand when in complex with a protein. The ability of STD NMR to provide specific insight into binding interactions in the absence of other structural data is an attractive feature for its use with membrane proteins. We chose to employ STD NMR in our ongoing investigations of the human membrane-associated neuraminidase NEU3 and its interaction with glycolipid substrates (e.g. GM3). In order to identify critical substrate-enzyme interactions, we performed STD NMR with a catalytically inactive form of the enzyme, NEU3(Y370F), containing an N-terminal MBP affinity tag. In the absence of crystallographic data on the enzyme, these data represent a critical experimental test of proposed homology models, as well as valuable new structural data. To aid interpretation of the STD NMR data, we compared the results with molecular dynamics (MD) simulations of the enzyme-substrate complexes. We find that the homology model is able to predict essential features of the experimental data, including close contact of the hydrophobic aglycone and the Neu5Ac residue with the enzyme. Additionally, the model and STD NMR data agree on the facial recognition of the Gal and Glc residues of the GM3-analog studied. We conclude that the homology model of NEU3 can be used to predict substrate recognition, but our data indicate that unstructured portions of the NEU3 model may require further refinement.
PMID: 25294388 [PubMed - as supplied by publisher]
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