An Engineered Transthyretin Monomer that Is Nonamyloidogenic, Unless It Is Partially Denatured

Abstract

Transthyretin (TTR) is a soluble human plasma protein that can be converted into amyloid by acid-mediated dissociation of the homotetramer into monomers. The pH required for disassembly also results in tertiary structural changes within the monomeric subunits. To understand whether these tertiary structural changes are required for amyloidogenicity, we created the Phe87Met/Leu110Met TTR variant (M-TTR) that is monomeric according to analytical ultracentrifugation and gel filtration analyses and nonamyloidogenic at neutral pH. Results from far-and near-UV circular dichroism spectroscopy, onedimensional proton NMR spectroscopy, and X-ray crystallography, as well as the ability of M-TTR to form a complex with retinol binding protein, indicate that M-TTR forms a tertiary structure at pH 7 that is very similar if not identical to that found within the tetramer. Reducing the pH results in tertiary structural changes within the M-TTR monomer, rendering it amyloidogenic, demonstrating the requirement for partial denaturation. M-TTR exhibits stability toward acid and urea denaturation that is nearly identical to that characterizing wild-type (WT) TTR at low concentrations (0.01-0.1 mg/mL), where monomeric WT TTR is significantly populated at intermediate urea concentrations prior to the tertiary structural transition. However, the kinetics of denaturation and fibril formation are much faster for M-TTR than for tetrameric WT TTR, particularly at near-physiological concentrations, because of the barrier associated with the tetramer to folded monomer preequilibrium. These results demonstrate that the tetramer to folded monomer transition is insufficient for fibril formation; further tertiary structural changes within the monomer are required.

Quantification of the Thermodynamically Linked Quaternary and Tertiary Structural Stabilities of Transthyretin and Its Disease-Associated Variants: The Relationship between Stability and Amyloidosis

“…Monomeric TTR Variant (M-TTR)-M-TTR was purified according to the above procedure, with slight modifications to the ammonium fractionation step: A 25-90% ammonium sulfate pellet was obtained, resuspended, and dialyzed in 3,500 MWCO Snakeskin dialysis tubing (Pierce Biotechnology, Rockford, Illinois). The typical yield is 40-60 mg of M-TTR from 2 L of cell culture; M-TTR thus obtained is >95% monomeric, as previously reported (19,23).…”

“…Generally, rate-limiting tetramer dissociation enables monomer misfolding that is required for TTR to form amyloid (5,(16)(17)(18)(19)(20)(21)(22), by a very efficient downhill polymerization process (23).…”

“…The thermodynamic linkage of the TTR tetramer dissociation and monomer unfolding equilibria make it challenging to extract thermodynamic data from the urea denaturation curves (7). How disease-associated mutations influence quaternary and tertiary structural stability and how folding and tetramerization energetics correlate with disease phenotypes is some of the valuable information sought herein.Generally, rate-limiting tetramer dissociation enables monomer misfolding that is required for TTR to form amyloid (5,(16)(17)(18)(19)(20)(21)(22), by a very efficient downhill polymerization process (23).Most of the disease-associated TTR mutations form normal tetrameric structures based on crystallographic analysis (24) and function normally, including transporting and binding thyroid hormone and holo retinol binding protein in the plasma and cerebrospinal fluid (CSF). Thus, it is the propensity of these variants to misfold, not their inability to fold and function, that results in disease through a gain of toxic function mechanism(s), the details of which remain elusive but appear to be associated with TTR aggregation (7,17,18,25,26).…”

The Sequence-Dependent Unfolding Pathway Plays a Critical Role in the Amyloidogenicity of Transthyretin

“…This intermediate is captured on the unfolding pathways of both V30M and L55P mutants, but not on the pathway of WT-TTR. This observation is supported by the fact that the wild-type monomer is nonamyloidogenic unless the pH is reduced (23), whereas the V30M and L55P mutants can form amyloid protofibrils under physiological conditions after a long incubation (40,60). The wild-type monomeric intermediate which was capable of amyloid deposits at low pH was found to be predominantly a -sheet structure with conformational rearrangements in the C strand-loop-D strand region without a large exposed hydrophobic surface area (16).…”

“…Moreover, single-point mutations could affect every stage of amyloid formation. Studies of the thermodynamic stability and dissociation kinetics of tetramers have shown that single-point mutations affect the native state stability and/or dissociation barrier (12,22), but dissociation alone is not sufficient for amyloid formation (23). Further tertiary structural changes within a monomer that lead to the monomeric intermediate state are required.…”