Mechanism of binding site conformational switching in the CD44-hyaluronan protein-carbohydrate binding interaction.

The transmembrane protein CD44, which has been implicated in cancer biology and inflammation, mediates cell adhesion through multimeric interactions with the linear extracellular glycosaminoglycan hyaluronan (HA; in megadaltons). Affinity switching of CD44 from a low-affinity state to a high-affinity state is required for normal CD44 physiological function; crystal structures of the CD44 hyaluronan binding domain complexed with HA oligomers point to a conformational rearrangement at a binding site loop, leading to the formation of direct contact between the oligomer and an arginine side chain as a molecular basis for affinity switching. Here, all-atom explicit-solvent molecular dynamics simulations were used to characterize the dynamics and thermodynamics of oligomeric hyaluronan (oHA) and its two crystallographic complexes with the CD44 hyaluronan binding domain: the "A-form," which lacks arginine-HA close contact, and the "B-form," which has direct arginine side-chain-HA contact. From the simulations, the conformational properties of oHA are essentially unaltered in going from the unbound state to either the A-form or the B-form bound state, with the oligomer retaining its flexibility when bound and with only two of the eight monosaccharides in the oligomer maintaining uninterrupted contact with the protein. Biased simulations revealed that altering the backbone conformation of a tyrosine residue in the arginine loop can induce the A-form→B-form conformational transition and that a large free-energy barrier prevents ready interconversion between the two forms, thereby suggesting that the tyrosine backbone forms a molecular switch.