Quantitative assessment of complex formation of nuclear-receptor accessoryproteins

Like other nuclear receptors, steroid hormone receptors form large protein hetero-complexes in their inactive, ligand-friendly state. Several heat-sho ck proteins, immunophilins and others have been identified as members of th ese highly dynamic complexes. The interaction kinetics and dynamics of hsp9 0, hsp70, p60 (Hop), FKBP52, FKBP51, p48 (Hip) and p23 have been assessed b y a biosensor approach measuring the complex formation in real time. A core chaperone complex has been reconstituted from p60, hsp90 and hsp70. p60 fo rms a molecular bridge between hsp90 and hsp70 with an affinity in the rang e of 10(5) M-1. Dynamics of hsp90-p60 complex formation is modulated by ATP through changes in the co-operativity of interaction. At low protein conce ntrations ATP stabilizes the complex. Binding of p23 to hsp90 did not chang e the affinity of the hsp90-p60 complex and the stabilizing effect of ATP. Saturation of the p48-hsp70 interaction could not be achieved, suggesting m ultiple binding sites. A picture of the protein complex, including stoichio metric coefficients, co-operativity of interaction and equilibrium-binding constants, has been formed.