Thermodynamic analysis of the interaction between YY1 and the AAV P5 promoter initiator element

Background: We previously determined the co-crystal structure of the zinc f inger region of transcription factor YYI (YY1 Delta) bound to the initiator element (Inr) of the adenoassociated virus (AAV) P5 gene promoter [Houbavi y, H.B, et al. (1996) Proc. Natl. Acad. Sci. USA 93, 13577-13582]. Our stru cture explained both binding specificity and the ability of YY1 to support specific, unidirectional transcription initiation. Results: To further understand Inr recognition by YY1, we analyzed the YY1 Delta -Inr interaction by isothermal titration calorimetry (ITC) and used l imited proteolysis, DNase I footprinting and missing nucleoside experiments to show that YY1 Delta and full-length YY1 (YY1WT) have indistinguishable DNA binding properties. Conclusions: YY1 binding occurs at an equilibrium dissociation constant (K- d) Of about 1 muM, and exhibits a large negative heat capacity change (Delt aC(p),). We analyzed the thermodynamic behavior of YY1 Delta in terms of bu ried solvent-accessible surface area resulting from interaction of two rigi d bodies, which could not explain our measured value of DeltaC(p). We must, therefore, postulate conformational changes in YY1 and/or the Inr or quest ion the validity of current DeltaC(p) analysis methods for protein-DNA inte ractions. (C) 2001 Elsevier Science Ltd. All rights reserved.