Neutron and X-ray solution-scattering studies of the ternary complex between proteoglycan-binding region, link...

Proteoglycan aggregates of cartilage are stabilized by the formation of a ternary complex between the G1 domain at the N-terminus of the proteoglycan monomer (aggrecan), link protein and hyaluronan polysaccharide. Both the G1 domain and link protein contain similar three-domain structures formed from an immunoglobulin fold and two proteoglycan tandem repeats, the arrangement of which had been investigated by neutron and synchrotron X-ray scattering [Perkins, Nealis, Dunham, Hardingham & Muir (1991) Biochemistry 30, 10708-10716]. Here, solution scattering was used to investigate the ternary complexes formed between a proteolytic fragment of proteoglycan monomer containing G1 (termed binding region), link protein and hyaluronan oligosaccharides containing either 34 or 450 saccharide units (HA34 and HA450). The ternary complex with HA34 had a neutron radius of gyration, RG, at infinite contrast not exceeding 5.5 nm. The ternary complex with HA34 had an X-ray cross-sectional radius of gyration Rxs of 2.4 nm and a neutron Rxs at infinite contrast of 2.00 nm. Since both were similar or larger than the Rxs for binding region (X-rays, 2.04 nm; neutrons, 1.84 nm) and link protein (neutrons, 0.8 nm), analyses showed that the cross-sectional mean width of the ternary complex is greater than those in each of the free proteins, i.e. the two proteins associated side-by-side. Similar results were obtained with HA450 complexed with binding region and with both binding region and link protein. This structural model was verified by hydrodynamic simulations of the experimental sedimentation coefficient of 5.5 S, which showed that a compact ternary-complex structure was formed. Although scattering curve simulations using small spheres were limited for the ternary complex with HA34 because of its approximate RG value, the scattering data were compatible with the formation of a compact complex formed by side-by-side contacts between G1 and link protein.