Differences in conformational stability of the two alpha domains of the disease-associated and non-disease-associated subtypes of HLA-B27.

The MHC Class I molecule, HLA-B27, is strongly linked with development of the inflammatory arthritic disease, ankylosing spondylitis (AS); whereas the B2705 subtype shows strong association, B2709 is not associated with disease, even though the two subtypes differ in only a single residue at position 116. Currently, attention is focused on the misfolding propensities of these two subtypes, including studies of disulfide-linked dimers and non-covalently formed high molecular weight (HMW) aggregates. Using mutants retaining only a single cysteine at positions C67 or C164, and using a cysteine-reactive, environment-sensitive, fluorescence probe (acrylodan), we find that within the same overall population of identical single-cysteine HLA-B27 molecules, there exist sub-populations which (a) possess free cysteines which react with acrylodan, (b) form disulfide-linked dimers, and (c) form HMW aggregates. Further, using acrylodan fluorescence, we find (d) that the α1 and α2 domains unfold independently of each other in HMW aggregates, (e) that these two domains of B2709 are less stable to chemical and thermal denaturation than the corresponding domains of B2705, suggesting easier clearance of misfolded molecules in the former, and (f) C67 is much more exposed in B2705 than in B2709, which could potentially explain how B*2705 more easily forms C67-mediated disulfide-bonded dimers.