While DNA encodes protein structure, glycans provide a complementary layer of information to protein function. As a prime example of the significance of glycans, the ability of the cell surface receptor CD44 to bind its ligand, hyaluronan, is modulated by N-glycosylation. However, the details of this modulation remain unclear. Based on atomistic simulations and NMR, we provide evidence that CD44 has multiple distinct binding sites for hyaluronan, and that N-glycosylation modulates their respective roles. We find that non-glycosylated CD44 favors the canonical sub-micromolar binding site, while glycosylated CD44 binds hyaluronan with an entirely different micromolar binding site. Our findings show (for the first time) how glycosylation can alter receptor affinity by shielding specific regions of the host protein, thereby promoting weaker binding modes. The mechanism revealed in this work emphasizes the importance of glycosylation in protein function and poses a challenge for protein structure determination where glycosylation is usually neglected.

Computational Physics Laboratory Tampere University PO Box 692 33014 Tampere Finland

Department of Cell Biology Faculty of Science Charles University Vinicna 7 128 00 Prague Czech Republic

Department of Physics University of Helsinki P O Box 64 00014 Helsinki Finland

Institute of Molecular Genetics Czech Academy of Sciences Videnska 1083 142 20 Prague 4 Czech Republic

Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences Flemingovo nam 2 166 10 Prague 6 Czech Republic

MEMPHYS Centre for Biomembrane Physics Odense Denmark

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