Calorimetric evidence for allosteric subunit interactions associated with inhibitor binding to band 3 transporter.

Abstract

A calorimetric endotherm occurring at 68 degrees C (the C-transition) has been assigned previously to the integral domain of band 3 and was shown to be shifted to 78 degrees C after covalent binding of 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS). In this study, we correlate the fractional appearance of the shifted C-transition with the fraction of DIDS bound to the band 3 monomer population. Our results show a distinctly nonlinear correlation plot with the appearance of the shifted C-transition lagging behind DIDS labeling of the band 3 monomer population. The lag suggests that both monomers of a band 3 dimer must be labeled by DIDS in order for the shifted C-transition to appear at 78 degrees C, implying that the thermal unfolding of the integral domain of band 3 is modulated by allosteric interactions between subunits. This is the first in situ structural evidence supporting ligand-mediated subunit interactions within a "carrier"-type transporter protein oligomer.