Introduction Hyaluronan (HA) is a ubiquitous high molecular mass glycosaminoglycan composed of a repeating disaccharide. CD44, the major cell surface receptor for HA, has a HA‐binding domain (CD44_HABD) at the N‐terminus of the protein, the 3D structure of which has been determined by both NMR and X‐ray crystallography (Teriete et al. 2004); NMR spectra collected on the protein in complex with HA oligosaccharides has allowed us to predict how they may thread across the interaction surface. Amino acids previously implicated in HA binding include R41, Y42, R78 and Y79 (Peach et al. 1993; Bajorath et al. 1998), which form a cluster on the surface of the Link module‐like region (Teriete et al. 2004), as well as residues in the C‐terminal extension (R150, R154, K158 and R162) (Peach et al. 1993). The position of the putative‐binding residues in the C‐terminal segment, and NMR data, led to the hypothesis of two modes of HA binding (Teriete et al. 2004). Here, this hypothesis is tested by NMR studies of single‐site mutants in the context of the CD44_HABD construct.Materials and methods Four mutants of CD44_HABD were made, each with a single residue substitution (R150A, R154A, K158A and R162A). These constructs were expressed as 15N‐labelled proteins in Escherichia coli, refolded and purified to homogeneity. 1H–15N HSQC spectra were acquired on the mutants in the presence of varying concentrations of HA hexasaccharide (HA6) and compared to the wild‐type construct to determine changes of protein fold and ligand binding.Results The mutants R150A, R154A and K158A have similar HSQC spectra to wild‐type CD44_HABD except for local chemical shift perturbations around the altered residue. Conversely, the R162A mutant has widespread chemical shift differences compared to wild‐type indicating that this mutation disrupts the fold. On binding HA6, the R150A, R154A and K158A mutants all experience shift perturbations similar to that seen with the wild‐type protein.Discussion The interaction of HA6 with wild‐type CD44_HABD has been found to cause a significant conformational change in the protein (Teriete et al. 2004). The results here indicate that this ligand‐induced rearrangement can also occur in the R150A, R154A and R158A mutants. Therefore, these mutations do not seem to affect the binding of CD44 to HA6. It has been shown previously that the R162A mutation has reduced affinity for HA compared to wild‐type protein (Peach et al. 1993). This loss of function may be due to the perturbation of the protein fold, and it is possible therefore that R162 does not participate directly in binding. Work is in progress to test the functional activity of these CD44_HABD mutants using ELISA‐like assays and to investigate their chemical shift perturbation in the presence of longer oligosaccharides.