MOLECULAR MODELING OF THE MULTIDOMAIN STRUCTURES OF THE PROTEOGLYCAN BINDING REGION AND THE LINK PROTEIN OF CARTILAGE BY NEUTRON AND SYNCHROTRON X-RAY-SCATTERING

The interaction of proteoglycan monomers with hyaluronate in cartilage is mediated by a globular binding region at the N-terminus of the proteoglycan monomer; this interaction is stabilized by link protein. Sequences show that both the binding region (27% carbohydrate) and the link protein (6% carbohydrate) contain an immunoglobulin (Ig) fold domain and two proteoglycan tandem repeat (PTR) domains. Both proteins were investigated by neutron and synchrotron X-ray solution scattering, in which nonspecific aggregate formation was reduced by the use of citraconylation to modify surface lysine residues. The neutron and X-ray radius of gyration R(G) of native and citraconylated binding region is 5.1 nm, and the cross-sectional R(G) (R(XS)) is 1.9-2.0 nm. No neutron contrast dependence of the R(G), values was observed; however, a large contrast dependence was seen for the R(XS) values which is attributed to the high carbohydrate content of the binding region. The neutron R(G) for citraconylated link protein is 2.9 nm, its R(XS) is 0.8 nm, and these data are also independent of the neutron contrast. The scattering curves of binding region and link protein were modeled using small spheres. Both protein structures were defined initially by the representation of one domain by a crystal structure for a variable Ig fold and a fixed volume for the two PTR domains calculated from sequence data. The final models showed that the different dimensions and neutron contrast properties of binding region compared to link protein could be attributed to an extended glycosylated C-terminal peptide with extended carbohydrate structures in the binding region. As a further control, these scattering curve models were used to define hydrodynamic sphere models, which were used to compute sedimentation coefficients of 3.4 and 2.6 S for the binding region and link protein, respectively, in good agreement with the experimental values. It is concluded that, under physiological conditions, the three globular domains of link protein and binding region are contained within dimensions of 10 nm X 2.5 nm X 3 nm and that the extended C-terminal peptide of the binding region has a length in the range of 8-17 nm.