NMR Measurements Reveal the Structural Basis of Transthyretin Destabilization by Pathogenic Mutations

Abstract

Inherited mutations of transthyretin (TTR) destabilize its structure, leading to aggregation and familial amyloid disease. Although numerous crystal structures of wild-type (WT) and mutant TTRs have been determined, they have failed to yield a comprehensive structural explanation for destabilization by pathogenic mutations. To identify structural and dynamic variations that are not readily observed in the crystal structures, we used NMR to study WT TTR and three kinetically and/or thermodynamically destabilized pathogenic variants (V30M, L55P, and V122I). Sequence-corrected chemical shifts reveal important structural differences between WT and mutant TTR. The L55P mutation linked to aggressive early onset cardiomyopathy and polyneuropathy induces substantial structural perturbations in both the DAGH and CBEF β-sheets, whereas the V30M polyneuropathy-linked substitution perturbs primarily the CBEF sheet. In both variants, the structural perturbations propagate across the entire width of the β-sheets from the site of mutation. Structural changes caused by the V122I cardiomyopathy-associated mutation are restricted to the immediate vicinity of the mutation site, directly perturbing the subunit interfaces. NMR relaxation dispersion measurements show that WT TTR and the three pathogenic variants undergo millisecond time scale conformational fluctuations to populate a common excited state with an altered structure in the subunit interfaces. The excited state is most highly populated in L55P. The combined application of chemical shift analysis and relaxation dispersion to these pathogenic variants reveals differences in ground state structure and in the population of a transient excited state that potentially facilitates tetramer dissociation, providing new insights into the molecular mechanism by which mutations promote TTR amyloidosis.

Binding of Monovalent and Bivalent Ligands by Transthyretin Causes Different Short- and Long-Distance Conformational Changes

“…The NMR backbone chemical shift assignment of wild type TTR has previously been reported under non-physiological pH and temperature conditions 16 and a new assignment at neutral pH has recently been deposited in the bmrb databank. 17…”

“…L55 is located opposite to K48; A120 is involved in the intermolecular pairing between strands H and H′; and V121 forms an intramolecular H-bond with T106 of the facing strand G. Evidence from 19 F NMR also suggests that A120, together with F87, may additionally be critical for TTR stabilization as shown by the A120L TTR variant favoring a mis-packed state. 17…”

Mechanisms of Transthyretin Inhibition of IAPP Amyloid Formation

“…Since the monomeric concentration of both Y69H, as well as F64S, is very low under native conditions this strongly suggests a partial exposure of the TTR polypeptide that normally is buried within its native fold. The occurrence of such partial unfolding is supported by previous studies using nuclear magnetic resonance (NMR) where a different surface exposure between different tetrameric variants of TTR has been shown using both Hydrogen-Deuterium (H/D) exchange [ 75 ] and relaxation dispersion experiments [ 76 , 77 ]. The exposure of normally hidden parts of TTR has also been shown using the conformational specific antibody MAB (39–44) , which normally only binds to unfolded TTR or its amyloid fold but where a strong reactivity is obtained to the tetrameric form of Y78F [ 78 ].…”

The Structural Understanding of Transthyretin Misfolding and the Inspired Drug Approaches for the Treatment of Heart Failure Associated With Transthyretin Amyloidosis

“…The acidification of solution triggers the flexible loop region between A and B to interact with A strand, leading to conformational changes occurring in the inner core domain ( Lim et al, 2016 ). The impact of single amino acid mutation on the stability of native TTR tetramer is attributed to a propagation of structural perturbation from the mutation site to the entire inner/outer ß -sheet domains ( Leach et al, 2018 ). The transmission electron micrographs showed that the TTR amyloid aggregates are rigid and unbranched fibrils with several micrometers in length and 70 to 130 Å in diameter ( Serpell et al, 1995 ).…”