| Abstract
| - The homotetrameric protein transthyretin (TTR) must undergo rate-limiting dissociation to itsconstituent monomers in order to enable partial denaturation that allows the process of amyloidogenesisassociated with human pathology to ensue. The TTR quaternary structure contains two distinct dimerinterfaces, one of which creates the two binding sites for the natural ligand thyroxine. Tetramer dissociationcould proceed through three distinct pathways; scission into dimers along either of the two unique quaternaryinterfaces followed by dimer dissociation represents two possibilities. Alternatively, the tetramer couldlose monomers sequentially. To elucidate the TTR dissociation pathway, we employed two different TTRconstructs, each featuring covalent attachment of proximal subunits. We demonstrate that tethering the Aand B subunits of TTR with a disulfide bond (as well as the symmetrically disposed C and D subunits)allows urea-mediated dissociation of the resulting (TTR-S-S-TTR)2 construct, affording (TTR-S-S-TTR)1retaining a stable 16-stranded β-sheet structure that is equivalent to the dimer not possessing a thyroidbinding site. In contrast, linking the A and C subunits employing a peptide tether (TTR-L-TTR)2 affordsa kinetically stable quaternary structure that does not dissociate or denature in urea. Both tethered constructsand wild-type TTR exhibit analogous stability based on guanidine hydrochloride denaturation curves.The latter denaturant can denature the tetramer, unlike urea, which can only denature monomeric TTR;hence urea requires dissociation to monomers to function. Under native conditions, the (TTR-S-S-TTR)2construct is able to dissociate and incorporate subunits from labeled WT TTR homotetramers at a rateequivalent to that exhibited by WT TTR. In contrast, the (TTR-L-TTR)2 construct is unable to exchangeany subunits, even after 180 h. All of the data presented herein and elsewhere demonstrate that the pathwayof TTR tetramer dissociation occurs by scission of the tetramer along the crystallographic C2 axis affordingAB and CD dimers that rapidly dissociate into monomers. Determination of the mechanism of dissociationprovides an explanation for why small molecules that bind at the AB/CD dimer−dimer interface imposekinetic stabilization upon TTR and disease-associated variants thereof.
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